oxidative stress tolerance as a component of the tissue
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Oxidative stress tolerance as a component
of the tissue tolerance mechanism in
wheat and barley
by
Haiyang Wang
School of Land and Food
MSc Huazhong Agricultural University China
BSc Henan Agricultural University China
Submitted in fulfilment of the requirement for the Degree of Doctor of
Philosophy
University of Tasmania
August 2018
Preliminaries
i
Declarations and statements
Declaration of originality
This thesis contains no material which has been accepted for a degree or diploma
by the University or any other institution except by way of background information
and duly acknowledged in the thesis and to the best of my knowledge and belief
no material previously published or written by another person except where due
acknowledgement is made in the text of the thesis nor does the thesis contain any
material that infringes copyright
Authority of access
This thesis is not to be made available for loan or copying for two years following
the date this statement was signed Following that time the thesis may be made
available for loan and limited copying and communication in accordance with the
Copyright Act 1968
Statement regarding published work contained in thesis
The publishers of the papers comprising Chapters 3 to 6 hold the copyright for that
content and access to the material should be sought from the respective journals
The remaining non-published content of the thesis may be made available for loan
and limited copying and communication in accordance with the Copyright Act
1968
Haiyang Wang
University of Tasmania
August 2018
Preliminaries
ii
Statement of co-authorship
The following people and institutions contributed to the publication of work
undertaken as part of this thesis
Candidate Haiyang Wang University of Tasmania
Author 1 Sergey Shabala University of Tasmania
Author 2 Lana Shabala University of Tasmania
Author 3 Meixue Zhou University of Tasmania
Author details and their roles
Paper 1 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Published in International Journal of Molecular Sciences (2015) 19 702 Located
in chapter 3
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
Paper 2 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Submitted to Plant Methods Located in chapter 6
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
We the undersigned agree with the above stated ldquoproportion of work undertakenrdquo
for each of the above published (or submitted) peer-reviewed manuscripts
contributing to this thesis
Preliminaries
iii
Signed
Sergey Shabala Holger Meinke
Supervisor Director
Tasmanian Institute of Agriculture Tasmanian Institute of Agriculture
University of Tasmania University of Tasmania
Date 31072018 ____________________
Preliminaries
iv
List of publications
Journal publications
Wang H Shabala L Zhou M Shabala S (2018) Hydrogen peroxide-induced root
Ca2+ and K+ fluxes correlate with salt tolerance in cereals towards the cell-based
phenotyping International Journal of Molecular Sciences 19 702
Wang H Shabala L Zhou M Shabala S Developing a high-throughput
phenotyping method for oxidative stress tolerance in cereal roots Plant Methods
(submitted 12042018)
Manuscripts in preparation
Wang H Shabala L Zhou M Shabala S H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in cereals and QTL identification
regarding this trait
Conference papers
Wang H Shabala L Zhou M Shabala S (Oral presentation) ldquoRevealing the causal
relationship between salinity and oxidative stress tolerance in wheat and barleyrdquo
The XIX International Botanical Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoHigh-throughput
assays for oxidative stress tolerance in cerealsrdquo The XIX International Botanical
Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoRevealing the
causal relationship between salinity and oxidative stress tolerance in wheat and
barleyrdquo Australian Barley Technical Symposium September 2017 Hobart
Tasmania
Wang H Shabala L Zhou L Shabala S (Poster presentation) ldquoDeveloping a
high-throughput phenotyping method for oxidative stress tolerance in cereal
rootsrdquo 10th International Symposium on Root Research July 2018 Jerusalem
Israel
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
References
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Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
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Cuin TA Shabala S (2007) Compatible solutes reduce ROS-induced potassium
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Davenport RJ Munoz-Mayor A Jha D Essah PA Rus A Tester M (2007) The
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Davies JM (2014) Annexin-mediated calcium signalling in plants Plants 3 128-
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Deinlein U Stephan AB Horie T Luo W Xu G Schroeder JI (2014) Plant salt-
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De la Garma JG Fernandez-Garcia N Bardisi E Pallol B Rubio-Asensio JS Bru
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Demidchik V Cuin TA Svistunenko D Smith SJ Miller AJ Shabala S Sokolik
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Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Demidchik V Shabala SN Coutts KB Tester MA Davies JM (2003) Free oxygen
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Demidchik V (2018) ROS-activated ion channels in plants Biophysical
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Demidchik VV Sokolik AI Yurin VM (1997a) Mechanisms of conductance
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Demidchik V Sokolik A Yurin V (1997b) The effect of Cu2+ on ion transport
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Demidchik V Straltsova D Medvedev SS Pozhvanov GA Sokolik A Yurin V
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Dionisio-Sese ML Tobita S (1998) Antioxidant responses of rice seedlings to
salinity stress Plant Sci 135 1ndash9
Dreyer I Uozumi N (2011) Potassium channels in plant cells FEBS J 278 4293-
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203
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128ndash143
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Garcia AB Engler JD Iyer S Gerats T Van Montagu M Caplan AB (1997)
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169
Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
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Gregorio GB Senadhira D Mendoza RD Manigbas NL Roxas JP Guerta CQ
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2014
Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hanin M Ebel C Ngom M Laplaze L Masmoudi K (2016) New insights on plant
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Sci 7 1787
Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Hediye Sekmen A Tuumlrkan İ Takio S (2007) Differential responses of antioxidative
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Hernandez JA Ferrer MA Jimeacutenez A Barcelo AR Sevilla F (2001) Antioxidant
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Horie T Karahara I Katsuhara M (2012) Salinity tolerance mechanisms in
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
Plant Physiol Bioch 43 1089ndash1094
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2939ndash2947
Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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2268
Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Ji H Pardo JM Batelli G Van Oosten MJ Bressan RA Li X (2013) The Salt
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Joo JH Bae YS Lee JS (2001) Role of auxin-induced reactive oxygen species in
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Miah G Rafii MY Ismail MR Puteh AB Rahim HA Asfaliza R Latif MA (2013)
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Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Miller G Schlauch K Tam R Cortes D Torres MA Shulaev V Dangl JL Mittler
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Munns R James RA Lauchli A (2006) Approaches to increasing the salt tolerance
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Munns R Tester M (2008) Mechanisms of salinity tolerance Annu Rev Plant Biol
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Nadeem MA Nawaz MA Shahid MQ Doğan Y Comertpay G Yıldız M
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Navrot N Rouhier N Gelhaye E Jacquot JP (2007) Reactive oxygen species
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Newman IA (2001) Ion transport in roots measurement of fluxes using ion-
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Nieves-Cordones M Aleman F Martinez V Rubio F (2014) K+ uptake in plant
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Noctor G (2006) Metabolic signalling in defence and stress the central roles of
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Noreen Z Ashraf M (2009a) Assessment of variation in antioxidative defense
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Oh DH Dassanayake M Haas JS Kropornika A Wright C drsquoUrzo MP Hong H
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Palmgren MG Nissen P (2011) P-type ATPases Annu Rev Biophys 40 243-266
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Paul JW Qi Y (2016) CRISPRCas9 for plant genome editing accomplishments
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Potocky M Jones MA Bezvoda R Smirnoff N Zarsky V (2007) Reactive oxygen
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Price AH Taylor A Ripley SJ Griffiths A Trewavas AJ Knight MR (1994)
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1310
Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Nat Resour Forum 38 282-295
Qiao B Zhang Q Liu D Wang H Yin J Wang R He M Cui M Shang Z Wang
D Zhu Z (2015) A calcium-binding protein rice annexin OsANN1 enhances
heat stress tolerance by modulating the production of H2O2 J Exp Bot 66
5853-5866
Qiu QS (2012) Plant and yeast NHX antiporters roles in membrane trafficking J
Integr Plant Biol 54 66ndash72
Quan LJ Zhang B Shi WW Li HY (2008) Hydrogen peroxide in plants A
versatile molecule of the reactive oxygen species network J Integr Plant Biol
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Raha S Robinson BH (2000) Mitochondria oxygen free radicals disease and
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Ran FA Hsu PD Lin CY Gootenberg JS Konermann S Trevino AE Scott DA
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Redwan M Spinelli F Marti L Weiland M Palm E Azzarello E Mancuso S (2016)
Potassium fluxes and reactive oxygen species production as potential
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1027
Rengasamy P (2006) World salinization with emphasis on Australia J Exp Bot 57
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Richards SL Laohavisit A Mortimer JC Shabala L Swarbreck SM Shabala S
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in Arabidopsis thaliana roots Plant J 77 136ndash145
Rinerson CI Scully ED Palmer NA Donze-Reiner T Rabara RC Tripathi P Shen
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Rodrigo-Moreno AN Andreacutes-Colaacutes NU Poschenrieder C Gunse B Penarrubia L
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844-855
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Roy SJ Negratildeo S Tester M (2014) Salt resistant crop plants Curr Opin Biotechnol
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Schmidt R Schippers JHM (2015) ROS-mediated redox signaling during cell
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Shabala L Zhang J Pottosin I Bose J Zhu M Fuglsang AT Velarde-Buendia A
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Sci 19 687ndash691
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Smethurst CF Rix K Garnett T Auricht G Bayart A Lane P Wilson SJ Shabala
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Sunarpi Horie T Motoda J Kubo M Yang H Yoda K Horie R Chan WY Leung
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Sun J Dai S Wang R Chen S Li N Zhou X Lu C Shen X Zheng X Hu Z Zhang
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45-51
Suzuki K Yamaji N Costa A Okuma E Kobayashi NI Kashiwagi T Katsuhara
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Szalonek M Sierpien B Rymaszewski W Gieczewska K Garstka M Lichocka M
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Tanou G Molassiotis A Diamantidis G (2009) Induction of reactive oxygen
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Tanveer M Shabala S (2018) Targeting redox regulatory mechanisms for salinity
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Veacutery AA Nieves-Cordones M Daly M Khan I Fizames C Sentenac H (2014)
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Genotypic variations in ion homeostasis photochemical efficiency and
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Wu H Shabala L Liu X Azzarello E Zhou M Pandolfi C Chen ZH Bose J Mancuso
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Wu H Shabala L Zhou M Shabala S (2014) Durum and bread wheat differ in their
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Wu H Shabala L Zhou M Stefano G Pandolfi C Mancuso S Shabala S (2015)
Developing and validating a high-throughput assay for salinity tissue
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Wu H Zhu M Shabala L Zhou M Shabala S (2015) K+ retention in leaf
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Wu J Shang Z Wu J Jiang X Moschou PN Sun W Roubelakis-Angelakis KA
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Xie Y Xu S Han B Wu M Yuan X Han Y Gu Q Xu D Yang Q Shen W (2011)
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Xu H Jiang X Zhan K Cheng X Chen X Pardo JM Cui D (2008) Functional
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Xu R Wang J Li C Johnson P Lu C Zhou M (2012) A single locus is responsible
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Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
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47-57
Yadav D Ahmed I Shukla P Boyidi P Kirti PB (2016) Overexpression of
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Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
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Yamauchi Y Furutera A Seki K Toyoda Y Tanaka K Sugimoto Y (2008)
Malondialdehyde generated from peroxidized linolenic acid causes protein
modification in heat-stressed plants Plant Physiol Bioch 46 786ndash793
Yancey PH (2005) Organic osmolytes as compatible metabolic and counteracting
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2830
Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
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Yazici EY Deveci H (2010) Factors affecting decomposition of hydrogen
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Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
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Yokoi S Quintero FJ Cubero B Ruiz MT Bressan RA Hasegawa PM Pardo JM
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Yue SU Zhang W Li FL Guo YL Liu TL Huang H (2000) Identification and
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Yue Y Zhang M Zhang J Duan L Li Z (2012) SOS1 gene overexpression
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Zeng H Xu L Singh A Wang H Du L Poovaiah BW (2015) Involvement of
calmodulin and calmodulin-like proteins in plant responses to abiotic stresses
Front Plant Sci 6 600
Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
Muniz-Murguia J Pottosin II (2011) Polyamines interact with hydroxyl
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Zhang F Li S Yang S Wang L Guo W (2015) Overexpression of a cotton annexin
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Zhang G Sun Y Li Y Dong Y Huang X Yu Y Wang J Wang X Wang X Kang
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Zhang HX Hodson JN Williams JP Blumwald E (2001) Engineering salt-tolerant
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12836
Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
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Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
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Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
Biol 6 441-445
Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
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Zhu M Zhou M Shabala L Shabala S (2017) Physiological and molecular
mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
i
Declarations and statements
Declaration of originality
This thesis contains no material which has been accepted for a degree or diploma
by the University or any other institution except by way of background information
and duly acknowledged in the thesis and to the best of my knowledge and belief
no material previously published or written by another person except where due
acknowledgement is made in the text of the thesis nor does the thesis contain any
material that infringes copyright
Authority of access
This thesis is not to be made available for loan or copying for two years following
the date this statement was signed Following that time the thesis may be made
available for loan and limited copying and communication in accordance with the
Copyright Act 1968
Statement regarding published work contained in thesis
The publishers of the papers comprising Chapters 3 to 6 hold the copyright for that
content and access to the material should be sought from the respective journals
The remaining non-published content of the thesis may be made available for loan
and limited copying and communication in accordance with the Copyright Act
1968
Haiyang Wang
University of Tasmania
August 2018
Preliminaries
ii
Statement of co-authorship
The following people and institutions contributed to the publication of work
undertaken as part of this thesis
Candidate Haiyang Wang University of Tasmania
Author 1 Sergey Shabala University of Tasmania
Author 2 Lana Shabala University of Tasmania
Author 3 Meixue Zhou University of Tasmania
Author details and their roles
Paper 1 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Published in International Journal of Molecular Sciences (2015) 19 702 Located
in chapter 3
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
Paper 2 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Submitted to Plant Methods Located in chapter 6
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
We the undersigned agree with the above stated ldquoproportion of work undertakenrdquo
for each of the above published (or submitted) peer-reviewed manuscripts
contributing to this thesis
Preliminaries
iii
Signed
Sergey Shabala Holger Meinke
Supervisor Director
Tasmanian Institute of Agriculture Tasmanian Institute of Agriculture
University of Tasmania University of Tasmania
Date 31072018 ____________________
Preliminaries
iv
List of publications
Journal publications
Wang H Shabala L Zhou M Shabala S (2018) Hydrogen peroxide-induced root
Ca2+ and K+ fluxes correlate with salt tolerance in cereals towards the cell-based
phenotyping International Journal of Molecular Sciences 19 702
Wang H Shabala L Zhou M Shabala S Developing a high-throughput
phenotyping method for oxidative stress tolerance in cereal roots Plant Methods
(submitted 12042018)
Manuscripts in preparation
Wang H Shabala L Zhou M Shabala S H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in cereals and QTL identification
regarding this trait
Conference papers
Wang H Shabala L Zhou M Shabala S (Oral presentation) ldquoRevealing the causal
relationship between salinity and oxidative stress tolerance in wheat and barleyrdquo
The XIX International Botanical Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoHigh-throughput
assays for oxidative stress tolerance in cerealsrdquo The XIX International Botanical
Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoRevealing the
causal relationship between salinity and oxidative stress tolerance in wheat and
barleyrdquo Australian Barley Technical Symposium September 2017 Hobart
Tasmania
Wang H Shabala L Zhou L Shabala S (Poster presentation) ldquoDeveloping a
high-throughput phenotyping method for oxidative stress tolerance in cereal
rootsrdquo 10th International Symposium on Root Research July 2018 Jerusalem
Israel
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
References
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253 245ndash256
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Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Demidchik V Shabala SN Coutts KB Tester MA Davies JM (2003) Free oxygen
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants where
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Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-
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Fuchs S Grill E Meskiene I Schweighofer A (2013) Type 2C protein phosphatases
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Garcia AB Engler JD Iyer S Gerats T Van Montagu M Caplan AB (1997)
Effects of osmoprotectants upon NaCl stress in rice Plant Physiol 115 159-
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Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Garciadeblas B Senn ME Banuelos MA Rodriguez-Navarro A (2003) Sodium
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Gaymard F Pilot G Lacombe B Bouchez D Bruneau D Boucherez J Michaux-
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Genc Y Oldach K Taylor J Lyons GH (2016) Uncoupling of sodium and chloride
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Gierth M Maumlser P (2007) Potassium transporters in plants - involvement in K+
acquisition redistribution and homeostasis FEBS Lett 581 2348-2356
Gill MB Zeng F Shabala L Zhang G Fan Y Shabala S Zhou M (2017) Cell-
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gobert A Isayenkov S Voelker C Czempinski K Maathuis FJM (2007) The two-
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Gobert A Park G Amtmann A Sanders D Maathuis FJM (2006) Arabidopsis
thaliana Cyclic Nucleotide Gated Channel 3 forms a non-selective ion
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800
Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
response of antioxidative enzymes of chloroplasts and mitochondria to long
term NaCl stress of pea plants Free Radical Res 31 11-18
Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
using modem technologies Procedia Earth Planet Sci 15 507ndash512
Gregorio GB Senadhira D Mendoza RD Manigbas NL Roxas JP Guerta CQ
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stresses in rice Field Crop Res 76 91ndash101
Grondin A Rodrigues O Verdoucq L Merlot S Leonhardt N Maurel C (2015)
Aquaporins contribute to ABA-triggered stomatal closure through OST1-
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2014
Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hanin M Ebel C Ngom M Laplaze L Masmoudi K (2016) New insights on plant
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Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Hernandez JA Ferrer MA Jimeacutenez A Barcelo AR Sevilla F (2001) Antioxidant
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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potassium transport evidence from electron probe analysis Plant Physiol 48
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
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Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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2939ndash2947
Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Nieves-Cordones M Aleman F Martinez V Rubio F (2014) K+ uptake in plant
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Oh DH Dassanayake M Haas JS Kropornika A Wright C drsquoUrzo MP Hong H
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Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Raha S Robinson BH (2000) Mitochondria oxygen free radicals disease and
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Rengasamy P (2006) World salinization with emphasis on Australia J Exp Bot 57
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Rhoads DM Umbach AL Subbaiah CC Siedow JN (2006) Mitochondrial reactive
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Richards SL Laohavisit A Mortimer JC Shabala L Swarbreck SM Shabala S
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Rinerson CI Scully ED Palmer NA Donze-Reiner T Rabara RC Tripathi P Shen
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Rizhsky L Hallak-Herr E Van Breusegem F Rachmilevitch S Barr JE Rodermel S
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Rodrigo-Moreno AN Poschenrieder C Shabala S (2013b) Transition metals a
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Rodrıguez AA Grunberg KA Taleisnik EL (2002) Reactive oxygen species in the
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Plant Mol Biol 38 919-927
Rodriacuteguez-Rosales MP Gaacutelvez FJ Huertas R Aranda MN Baghour M Cagnac O
Venema K (2009) Plant NHX cationproton antiporters Plant Signal Behav
4 265-276
Roy SJ Negratildeo S Tester M (2014) Salt resistant crop plants Curr Opin Biotechnol
26 115ndash124
Ruan CJ da Silva JAT Mopper S Qin P Lutts S (2010) Halophyte improvement
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121
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8
Schmidt R Schippers JHM (2015) ROS-mediated redox signaling during cell
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Schroeder JI (2003) Knockout of the guard cell K+ out channel and stomatal
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122
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environment FEMS Microbiol Rev 30 472-486
Shabala L Zhang J Pottosin I Bose J Zhu M Fuglsang AT Velarde-Buendia A
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2445-2458
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Sci 19 687ndash691
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Plantarum 133 651-669
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839-853
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plant roots current knowledge and hypothesis Plant Sci 241 109ndash119
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Sun J Dai S Wang R Chen S Li N Zhou X Lu C Shen X Zheng X Hu Z Zhang
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Suzuki K Yamaji N Costa A Okuma E Kobayashi NI Kashiwagi T Katsuhara
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Wu H Shabala L Zhou M Stefano G Pandolfi C Mancuso S Shabala S (2015)
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Wu H Zhu M Shabala L Zhou M Shabala S (2015) K+ retention in leaf
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Wu J Shang Z Wu J Jiang X Moschou PN Sun W Roubelakis-Angelakis KA
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Xu H Jiang X Zhan K Cheng X Chen X Pardo JM Cui D (2008) Functional
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Xu R Wang J Li C Johnson P Lu C Zhou M (2012) A single locus is responsible
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Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
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Yadav D Ahmed I Shukla P Boyidi P Kirti PB (2016) Overexpression of
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Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
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Yamauchi Y Furutera A Seki K Toyoda Y Tanaka K Sugimoto Y (2008)
Malondialdehyde generated from peroxidized linolenic acid causes protein
modification in heat-stressed plants Plant Physiol Bioch 46 786ndash793
Yancey PH (2005) Organic osmolytes as compatible metabolic and counteracting
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2830
Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
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Yazici EY Deveci H (2010) Factors affecting decomposition of hydrogen
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Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
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Yokoi S Quintero FJ Cubero B Ruiz MT Bressan RA Hasegawa PM Pardo JM
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Na+H+ antiporters in the salt stress response Plant J 30 529ndash539
Yue SU Zhang W Li FL Guo YL Liu TL Huang H (2000) Identification and
genetic mapping of four novel genes that regulate leaf development in
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Yue Y Zhang M Zhang J Duan L Li Z (2012) SOS1 gene overexpression
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Zeng H Xu L Singh A Wang H Du L Poovaiah BW (2015) Involvement of
calmodulin and calmodulin-like proteins in plant responses to abiotic stresses
Front Plant Sci 6 600
Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
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Zhang F Li S Yang S Wang L Guo W (2015) Overexpression of a cotton annexin
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Zhang G Sun Y Li Y Dong Y Huang X Yu Y Wang J Wang X Wang X Kang
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Zhang HX Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate
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Zhang HX Hodson JN Williams JP Blumwald E (2001) Engineering salt-tolerant
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12836
Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
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Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
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Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
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Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
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Zhu M Zhou M Shabala L Shabala S (2017) Physiological and molecular
mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
ii
Statement of co-authorship
The following people and institutions contributed to the publication of work
undertaken as part of this thesis
Candidate Haiyang Wang University of Tasmania
Author 1 Sergey Shabala University of Tasmania
Author 2 Lana Shabala University of Tasmania
Author 3 Meixue Zhou University of Tasmania
Author details and their roles
Paper 1 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Published in International Journal of Molecular Sciences (2015) 19 702 Located
in chapter 3
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
Paper 2 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Submitted to Plant Methods Located in chapter 6
Candidate contributed to 80 to the planning execution and preparation of the
work for the paper Author 1 author 2 and author 3 contributed to the conception
and design of the research project and drafted significant parts of the paper
We the undersigned agree with the above stated ldquoproportion of work undertakenrdquo
for each of the above published (or submitted) peer-reviewed manuscripts
contributing to this thesis
Preliminaries
iii
Signed
Sergey Shabala Holger Meinke
Supervisor Director
Tasmanian Institute of Agriculture Tasmanian Institute of Agriculture
University of Tasmania University of Tasmania
Date 31072018 ____________________
Preliminaries
iv
List of publications
Journal publications
Wang H Shabala L Zhou M Shabala S (2018) Hydrogen peroxide-induced root
Ca2+ and K+ fluxes correlate with salt tolerance in cereals towards the cell-based
phenotyping International Journal of Molecular Sciences 19 702
Wang H Shabala L Zhou M Shabala S Developing a high-throughput
phenotyping method for oxidative stress tolerance in cereal roots Plant Methods
(submitted 12042018)
Manuscripts in preparation
Wang H Shabala L Zhou M Shabala S H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in cereals and QTL identification
regarding this trait
Conference papers
Wang H Shabala L Zhou M Shabala S (Oral presentation) ldquoRevealing the causal
relationship between salinity and oxidative stress tolerance in wheat and barleyrdquo
The XIX International Botanical Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoHigh-throughput
assays for oxidative stress tolerance in cerealsrdquo The XIX International Botanical
Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoRevealing the
causal relationship between salinity and oxidative stress tolerance in wheat and
barleyrdquo Australian Barley Technical Symposium September 2017 Hobart
Tasmania
Wang H Shabala L Zhou L Shabala S (Poster presentation) ldquoDeveloping a
high-throughput phenotyping method for oxidative stress tolerance in cereal
rootsrdquo 10th International Symposium on Root Research July 2018 Jerusalem
Israel
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
References
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Baacutenfi B Tirone F Durussel I Knisz J Moskwa P Molnaacuter GZ Krause KH Cox
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Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant
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Barragan V Leidi EO Andres Z Rubio L De Luca A Fernandez JA Cubero B
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Becker D Hoth S Ache P Wenkel S Roelfsema MR Meyerhoff O HartungW
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Byrt CS Xu B Krishnan M Lightfoot DJ Athman A Jacobs AK Watson-Haigh
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Castelli SL Grunberg K Muntildeoz N Griffa S Colomba EL Ribotta A Biderbost E
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Cheeseman JM (2006) Hydrogen peroxide concentrations in leaves under natural
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Chen TH Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic
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Chen Z Cuin TA Zhou M Twomey A Naidu BP Shiabala S (2007a) Compatible
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Chen Z Newman I Zhou M Mendham N Zhang G Shabala S (2005) Screening
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Cell Environ 28 1230ndash1246
Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
Potassium and sodium relations in salinised barley tissues as a basis of
differential salt tolerance Funct Plant Biol 34 150ndash162
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Collard BCY Mackill DJ (2008) Marker-assisted selection an approach for
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Cuin TA Betts SA Chalmandrier R Shabala S (2008) A roots ability to retain K+
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Cuin TA Bose J Stefano G Jha D Tester M Mancuso S Shabala S (2011)
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Cuin TA Shabala S (2007) Compatible solutes reduce ROS-induced potassium
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Cuin TA Shabala S (2008) Compatible solutes mitigate damaging effects of salt
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Signal Behav 3 207-208
Cuin TA Tian Y Betts SA Chalmandrier R Shabala S (2009) Ionic relations and
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Davenport RJ Munoz-Mayor A Jha D Essah PA Rus A Tester M (2007) The
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Dbira S Al Hassan M Gramazio P Ferchichi A Vicente O Prohens J Boscaiu M
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Deinlein U Stephan AB Horie T Luo W Xu G Schroeder JI (2014) Plant salt-
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De la Garma JG Fernandez-Garcia N Bardisi E Pallol B Rubio-Asensio JS Bru
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Demidchik V Cuin TA Svistunenko D Smith SJ Miller AJ Shabala S Sokolik
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Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Demidchik V Shabala SN Coutts KB Tester MA Davies JM (2003) Free oxygen
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Demidchik V Straltsova D Medvedev SS Pozhvanov GA Sokolik A Yurin V
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-
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Fry SC Miller JG Dumville JC (2002) A proposed role for copper ions in cell wall
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Garcia AB Engler JD Iyer S Gerats T Van Montagu M Caplan AB (1997)
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Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Garciadeblas B Senn ME Banuelos MA Rodriguez-Navarro A (2003) Sodium
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Genc Y Oldach K Taylor J Lyons GH (2016) Uncoupling of sodium and chloride
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Gierth M Maumlser P (2007) Potassium transporters in plants - involvement in K+
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Gill MB Zeng F Shabala L Zhang G Fan Y Shabala S Zhou M (2017) Cell-
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gobert A Isayenkov S Voelker C Czempinski K Maathuis FJM (2007) The two-
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Gobert A Park G Amtmann A Sanders D Maathuis FJM (2006) Arabidopsis
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
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Gregorio GB Senadhira D Mendoza RD Manigbas NL Roxas JP Guerta CQ
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Grondin A Rodrigues O Verdoucq L Merlot S Leonhardt N Maurel C (2015)
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Guo B Wei Y Xu R Lin S Luan H Lv C Zhang X Song X Xu R (2016)
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Gupta B Huang BR (2014) Mechanism of salinity tolerance in plants
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Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hanin M Ebel C Ngom M Laplaze L Masmoudi K (2016) New insights on plant
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Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Venkateswarlu B Shanker A Shanker C Maheswari M Eds
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Hediye Sekmen A Tuumlrkan İ Takio S (2007) Differential responses of antioxidative
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Hepler PK (2005) Calcium a central regulator of plant growth and development
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Hernandez JA Ferrer MA Jimeacutenez A Barcelo AR Sevilla F (2001) Antioxidant
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Horie T Hauser F Schroeder JI (2009) HKT transporter-mediated salinity
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Horie T Karahara I Katsuhara M (2012) Salinity tolerance mechanisms in
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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salt tolerance in durum wheat Plant Physiol 142 1718ndash1727
Humble GD Raschke K (1971) Stomatal opening quantitatively related to
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447-453
Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Inoue H Kudo T Kamada H Kimura M Yamaguchi I Hamamoto H (2005)
Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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Isayenkov S Isner JC Maathuis FJM (2010) Vacuolar ion channels roles in plant
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2939ndash2947
Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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2268
Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
Overexpression of TaNHX3 a vacuolar Na+H+ antiporter gene in wheat
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Malzahn A Lowder L Qi Y (2017) Plant genome editing with TALEN and
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Plant Physiol 173 91ndash111
Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Miller G Shulaev V Mittler R (2008) Reactive oxygen signaling and abiotic stress
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Nieves-Cordones M Aleman F Martinez V Rubio F (2014) K+ uptake in plant
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Oh DH Dassanayake M Haas JS Kropornika A Wright C drsquoUrzo MP Hong H
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Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Rengasamy P (2006) World salinization with emphasis on Australia J Exp Bot 57
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Richards SL Laohavisit A Mortimer JC Shabala L Swarbreck SM Shabala S
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Rodrıguez AA Grunberg KA Taleisnik EL (2002) Reactive oxygen species in the
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Roy SJ Negratildeo S Tester M (2014) Salt resistant crop plants Curr Opin Biotechnol
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Schmidt R Schippers JHM (2015) ROS-mediated redox signaling during cell
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Senthil‐Kumar M Srikanthbabu V Mohan Raju B Shivaprakash N Udayakumar
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Shabala S Cuin TA (2008) Potassium transport and plant salt tolerance Physiol
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Shabala S Wu HH Bose J (2015) Salt stress sensing and early signalling events in
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Shahbaz M Ashraf M (2013) Improving salinity tolerance in cereals Crit Rev
Plant Sci 32 237ndash249
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Shalata A Mittova V Volokita M Guy M Tal M (2001) Response of the cultivated
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Sharma P Jha AB Dubey RS Pessarakli M (2012) Reactive oxygen species
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Shewry PR (2009) Wheat J Exp Bot 60 1537-1553
References
124
Shi H Lee BH Wu SJ Zhu JK (2003) Overexpression of a plasma membrane
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Sitnicka D Figurska K Orzechowski S (2010) Functional analysis of genes Adv
Cell Bio 2 1-6
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Smethurst CF Rix K Garnett T Auricht G Bayart A Lane P Wilson SJ Shabala
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Sunarpi Horie T Motoda J Kubo M Yang H Yoda K Horie R Chan WY Leung
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Sun J Dai S Wang R Chen S Li N Zhou X Lu C Shen X Zheng X Hu Z Zhang
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References
125
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Suzuki N Mittler R (2006) Reactive oxygen species and temperature stresses a
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45-51
Suzuki K Yamaji N Costa A Okuma E Kobayashi NI Kashiwagi T Katsuhara
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Szalonek M Sierpien B Rymaszewski W Gieczewska K Garstka M Lichocka M
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Tanou G Molassiotis A Diamantidis G (2009) Induction of reactive oxygen
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Tanveer M Shabala S (2018) Targeting redox regulatory mechanisms for salinity
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234
Tester M Davenport R (2003) Na+ tolerance and Na+ transport in higher plants
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Tran D El-Maarouf-Bouteau H Rossi M Biligui B Briand J Kawano T Mancuso
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Veacutery AA Nieves-Cordones M Daly M Khan I Fizames C Sentenac H (2014)
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Wang N Qi HK Su GL Yang J Zhou H Xu QH Huang Q Yan GT (2016)
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Watanabe S Matsumoto M Hakomori Y Takagi H Shimada H Sakamoto A
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Wu H Shabala L Liu X Azzarello E Zhou M Pandolfi C Chen ZH Bose J Mancuso
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Wu H Shabala L Zhou M Shabala S (2014) Durum and bread wheat differ in their
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Wu H Shabala L Zhou M Stefano G Pandolfi C Mancuso S Shabala S (2015)
Developing and validating a high-throughput assay for salinity tissue
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Wu H Zhu M Shabala L Zhou M Shabala S (2015) K+ retention in leaf
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Wu J Shang Z Wu J Jiang X Moschou PN Sun W Roubelakis-Angelakis KA
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membrane hyperpolarization-activated Ca2+-permeable channels and pollen
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Xia X Zhou Y Shi K Zhou J Foyer CH Yu J (2015) Interplay between reactive
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Xie Y Xu S Han B Wu M Yuan X Han Y Gu Q Xu D Yang Q Shen W (2011)
Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1
and the regulatory role of RbohD-derived reactive oxygen species synthesis
Plant J 66 280ndash292
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Xie Y Mao Y Zhang W Lai D Wang Q Shen W (2014) Reactive oxygen species-
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Xue ZY Zhi DY Xue GP Zhang H Zhao YX Xia GM (2004) Enhanced salt
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and a reduced level of leaf Na+ Plant Sci 167 849-859
Xu H Jiang X Zhan K Cheng X Chen X Pardo JM Cui D (2008) Functional
characterization of a wheat plasma membrane Na+H+ antiporter in yeast
Arch Biochem Biophys 473 8ndash15
Xu R Wang J Li C Johnson P Lu C Zhou M (2012) A single locus is responsible
for salinity tolerance in a Chinese landrace barley (Hordeum vulgare L)
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Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
water alleviates salt stress in rice during seed germination Plant Soil 370
47-57
Yadav D Ahmed I Shukla P Boyidi P Kirti PB (2016) Overexpression of
Arabidopsis AnnAt8 alleviates abiotic stress in transgenic Arabidopsis and
tobacco Plants 5 18
Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
and opportunities Trends Plant Sci 10 615-620
Yamauchi Y Furutera A Seki K Toyoda Y Tanaka K Sugimoto Y (2008)
Malondialdehyde generated from peroxidized linolenic acid causes protein
modification in heat-stressed plants Plant Physiol Bioch 46 786ndash793
Yancey PH (2005) Organic osmolytes as compatible metabolic and counteracting
cytoprotectants in high osmolarity and other stresses J Exp Biol 208 2819-
2830
Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
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tolerance in transgenic Arabidopsis Mol Plant 2 22-31
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Yan J Tsuichihara N Etoh T Iwai S (2007) Reactive oxygen species and nitric
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30 1320-1325
Yazici EY Deveci H (2010) Factors affecting decomposition of hydrogen
peroxide In Proceedings of the XIIth International Mineral Processing
Symposium Cappadocia Turkey 6ndash10
Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
transgenic maize with improved salt tolerance by the introduction of AtNHX1
gene Acta Bot Sin 46 854-861
Yokoi S Quintero FJ Cubero B Ruiz MT Bressan RA Hasegawa PM Pardo JM
(2002) Differential expression and function of Arabidopsis thaliana NHX
Na+H+ antiporters in the salt stress response Plant J 30 529ndash539
Yue SU Zhang W Li FL Guo YL Liu TL Huang H (2000) Identification and
genetic mapping of four novel genes that regulate leaf development in
Arabidopsis Cell Res 10 325-335
Yue Y Zhang M Zhang J Duan L Li Z (2012) SOS1 gene overexpression
increased salt tolerance in transgenic tobacco by maintaining a higher K+Na+
ratio J Plant Physiol 169 255-261
Zeng H Xu L Singh A Wang H Du L Poovaiah BW (2015) Involvement of
calmodulin and calmodulin-like proteins in plant responses to abiotic stresses
Front Plant Sci 6 600
Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
Muniz-Murguia J Pottosin II (2011) Polyamines interact with hydroxyl
radicals in activating Ca2+ and K+ transport across the root epidermal plasma
membranes Plant Physiol 157 2167-2180
Zhang F Li S Yang S Wang L Guo W (2015) Overexpression of a cotton annexin
gene GhAnn1 enhances drought and salt stress tolerance in transgenic cotton
Plant Mol Biol 87 47-67
References
131
Zhang G Sun Y Li Y Dong Y Huang X Yu Y Wang J Wang X Wang X Kang
Z (2013) Characterization of a wheat C2 domain protein encoding gene
regulated by stripe rust and abiotic stresses Biol Plantarum 57 701-710
Zhang HX Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate
salt in foliage but not in fruit Nat Biotechnol 19 765-768
Zhang HX Hodson JN Williams JP Blumwald E (2001) Engineering salt-tolerant
Brassica plants characterization of yield and seed oil quality in transgenic
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12836
Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
crop plants J Exp Bot 50 291ndash302
Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
major QTL for abiotic stress tolerance in barley and implications for barley
breeding Planta 245 283-295
Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
Biol 6 441-445
Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
stomatal characteristics with salinity stress tolerance in contrasting barley
accessions Funct Plant Biol 42 252ndash263
Zhu M Zhou M Shabala L Shabala S (2017) Physiological and molecular
mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
iii
Signed
Sergey Shabala Holger Meinke
Supervisor Director
Tasmanian Institute of Agriculture Tasmanian Institute of Agriculture
University of Tasmania University of Tasmania
Date 31072018 ____________________
Preliminaries
iv
List of publications
Journal publications
Wang H Shabala L Zhou M Shabala S (2018) Hydrogen peroxide-induced root
Ca2+ and K+ fluxes correlate with salt tolerance in cereals towards the cell-based
phenotyping International Journal of Molecular Sciences 19 702
Wang H Shabala L Zhou M Shabala S Developing a high-throughput
phenotyping method for oxidative stress tolerance in cereal roots Plant Methods
(submitted 12042018)
Manuscripts in preparation
Wang H Shabala L Zhou M Shabala S H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in cereals and QTL identification
regarding this trait
Conference papers
Wang H Shabala L Zhou M Shabala S (Oral presentation) ldquoRevealing the causal
relationship between salinity and oxidative stress tolerance in wheat and barleyrdquo
The XIX International Botanical Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoHigh-throughput
assays for oxidative stress tolerance in cerealsrdquo The XIX International Botanical
Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoRevealing the
causal relationship between salinity and oxidative stress tolerance in wheat and
barleyrdquo Australian Barley Technical Symposium September 2017 Hobart
Tasmania
Wang H Shabala L Zhou L Shabala S (Poster presentation) ldquoDeveloping a
high-throughput phenotyping method for oxidative stress tolerance in cereal
rootsrdquo 10th International Symposium on Root Research July 2018 Jerusalem
Israel
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
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GORK a delayed outward rectifier expressed in guard cells of Arabidopsis
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Adem GD Roy SJ Zhou M Bowman JP Shabala S (2014) Evaluating contribution
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Aharon GS Apse MP Duan SL Hua XJ Blumwald E (2003) Characterization of
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Ahmad P Jaleel CA Salem MA Nabi G Sharma S (2010) Roles of enzymatic and
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Alfocea FP Balibrea ME Alarcon JJ Bolarin MC (2000) Composition of xylem
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Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants Plant
Physiol 107 1049ndash1054
Agarwal PK Jha B (2010) Transcription factors in plants and ABA dependent and
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Amtmann A Fischer M Marsh EL Stefanovic A Sanders D Schachtman DP
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Amtmann A Sanders D (1998) Mechanisms of Na+ uptake by plant cells Adv Bot
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Anjum NA Sofo A Scopa A Roychoudhury A Gill SS Iqbal M Lukatkin AS
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Anschuumltz U Becker D Shabala S (2014) Going beyond nutrition regulation of
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Apel K Hirt H (2004) Reactive oxygen species metabolism oxidative stress and
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Apse MP Aharon GS Snedden WA Blumwald E (1999) Salt tolerance conferred
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Asada K (1993) Molecular mechanism of production and scavenging of active
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Asada K (2006) Production and scavenging of reactive oxygen species in
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Ashraf M Ali Q (2008) Relative membrane permeability and activities of some
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Azooz MM Ismail AM Elhamd MA (2009) Growth lipid peroxidation and
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Baik BK Ullrich SE (2008) Barley for food characteristics improvement and
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Baacutenfi B Tirone F Durussel I Knisz J Moskwa P Molnaacuter GZ Krause KH Cox
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Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant
Mol Biol 69 473ndash488
Barragan V Leidi EO Andres Z Rubio L De Luca A Fernandez JA Cubero B
Pardo JM (2012) Ion exchangers NHX1 and NHX2 mediate active potassium
uptake into vacuoles to regulate cell turgor and stomatal function in
Arabidopsis Plant Cell 24 1127ndash1142
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Bassil E Ohto MA Esumi T Tajima H Zhu Z Cagnac O Belmonte M Peleg Z
Yamaguchi T Blumwald E (2011a) The Arabidopsis intracellular Na+H+
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plant growth and development Plant Cell 23 224ndash239
Bassil E Tajima H Liang YC Ohto M Ushijima K Nakano R Esumi T Coku A
Belmonte M Blumwald E (2011b) The Arabidopsis Na+H+ antiporters
NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth
flower development and reproduction Plant Cell 23 3482ndash3497
Baucher M Peacuterez-Morga D El Jaziri M (2012) Insight into plant annexin function
From shoot to root signaling Plant Signal Behav 7 524ndash528
Bauwe H Hagemann M Fernie AR (2010) Photorespiration players partners and
origin Trends Plant Sci 15 330ndash336
Baxter A Mittler R Suzuki N (2014) ROS as key players in plant stress signalling J
Exp Bot 65 1229ndash1240
Becker D Hoth S Ache P Wenkel S Roelfsema MR Meyerhoff O HartungW
Hedrich R (2003) Regulation of the ABA-sensitive Arabidopsis potassium
channel gene GORK in response to water stress FEBS Lett 554 119ndash126
Ben-Ari G Lavi U (2012) Marker-assisted selection in plant breeding In Plant
Biotechnology and Agriculture pp 163-184
Berthomieu P Coneacutejeacutero G Nublat A BrackenburyWJ Lambert C Savio C
Uozumi N Oiki S Yamada K Cellier F Gosti F (2003) Functional analysis
of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is
crucial for salt tolerance EMBO J 22 2004ndash2014
Bethke PC Jones RL (2001) Cell death of barley aleurone protoplasts is mediated
by reactive oxygen species Plant J 25 19-29
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57 101-107
Biswal B Joshi PN Raval MK Biswal UC (2011) Photosynthesis a global sensor
of environmental stress in green plants stress signalling and adaptation Curr
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Bita C Gerats T (2013) Plant tolerance to high temperature in a changing
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Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
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Cuin TA Bose J Stefano G Jha D Tester M Mancuso S Shabala S (2011)
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Cuin TA Shabala S (2008) Compatible solutes mitigate damaging effects of salt
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Cuin TA Tian Y Betts SA Chalmandrier R Shabala S (2009) Ionic relations and
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Dbira S Al Hassan M Gramazio P Ferchichi A Vicente O Prohens J Boscaiu M
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Deinlein U Stephan AB Horie T Luo W Xu G Schroeder JI (2014) Plant salt-
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De la Garma JG Fernandez-Garcia N Bardisi E Pallol B Rubio-Asensio JS Bru
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Demidchik V Cuin TA Svistunenko D Smith SJ Miller AJ Shabala S Sokolik
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Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
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Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Inoue H Kudo T Kamada H Kimura M Yamaguchi I Hamamoto H (2005)
Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Jiang J Ma S Ye N Jiang M Cao J Zhang J (2017) WRKY transcription factors
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Ji H Pardo JM Batelli G Van Oosten MJ Bressan RA Li X (2013) The Salt
Overly Sensitive (SOS) pathway established and emerging roles Mol Plant
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Jin Q Zhu K Cui W Xie Y Han BI Shen W (2013) Hydrogen gas acts as a novel
bioactive molecule in enhancing plant tolerance to paraquat‐induced
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Joo JH Bae YS Lee JS (2001) Role of auxin-induced reactive oxygen species in
root gravitropism Plant Physiol 126 1055ndash1060
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Karuppanapandian T Moon JC Kim C Manoharan K Kim W (2011) Reactive
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kim TH Boumlhmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
Overexpression of TaNHX3 a vacuolar Na+H+ antiporter gene in wheat
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Maksimović JD Zhang J Zeng F Živanović BD Shabala L Zhou M Shabala S
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Malzahn A Lowder L Qi Y (2017) Plant genome editing with TALEN and
CRISPR Cell Biosci 7 21
Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Richards SL Laohavisit A Mortimer JC Shabala L Swarbreck SM Shabala S
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Rodrıguez AA Grunberg KA Taleisnik EL (2002) Reactive oxygen species in the
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Schmidt R Schippers JHM (2015) ROS-mediated redox signaling during cell
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Wang M Zheng Q Shen Q Guo S (2013) The critical role of potassium in plant
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Wang F Chen ZH Liu X Colmer TD Shabala L Salih A Zhou M Shabala S
(2016) Revealing the roles of GORK channels and NADPH oxidase in
acclimation to hypoxia in Arabidopsis J Expl Bot 68 3191-3204
Wang N Qi HK Su GL Yang J Zhou H Xu QH Huang Q Yan GT (2016)
Genotypic variations in ion homeostasis photochemical efficiency and
antioxidant capacity adjustment to salinity in cotton (Gossypium hirsutum L)
Soil Sci Plant Nutr 62 240ndash246
Wang R Jing W Xiao L Jin Y Shen L Zhang W (2015) The rice high-affinity
potassium transporter11 is involved in salt tolerance and regulated by an
MYB-type transcription factor Plant Physiol 168 1076ndash1090
Wang Y Chen Z Zhang B Hills A Blatt MR (2013) PYRPYLRCAR abscisic
acid receptors regulate K+ and Clminus channels through reactive oxygen species-
mediated activation of Ca2+ channels at the plasma membrane of intact
Arabidopsis guard cells Plant Physiol 163 566ndash577
Wani SH Singh NB Haribhushan A Mir JI (2013) Compatible solute engineering
in plants for abiotic stress tolerance - role of glycine betaine Curr Genom 14
157ndash165
Ward JM Schroeder JI (1994) Calcium-activated K+ channels and calcium-induced
calcium release by slow vacuolar ion channels in guard-cell vacuoles
implicated in the control of stomatal closure Plant Cell 6 669-683
Watanabe S Matsumoto M Hakomori Y Takagi H Shimada H Sakamoto A
(2014) The purine metabolite allantoin enhances abiotic stress tolerance
through synergistic activation of abscisic acid metabolism Plant Cell
Environ 37 1022ndash1036
Wegner LH Raschke K (1994) Ion channels in the xylem parenchyma of barley
roots (a procedure to isolate protoplasts from this tissue and a patch-clamp
exploration of salt passageways into xylem vessels Plant Physiol 105 799-
813
References
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Weis AE Simms EL Hochberg ME (2000) Will plant vigor and tolerance be
genetically correlated Effects of intrinsic growth rate and self-limitation on
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White PJ (1999) The molecular mechanism of sodium influx to root cells Trends
Plant Sci 4 245-246
Wu H Shabala L Liu X Azzarello E Zhou M Pandolfi C Chen ZH Bose J Mancuso
S Shabala S (2015) Linking salinity stress tolerance with tissue-specific Na+
sequestration in wheat roots Front Plant Sci 6 71
Wu H Shabala L Zhou M Shabala S (2014) Durum and bread wheat differ in their
ability to retain potassium in leaf mesophyll implications for salinity stress
tolerance Plant Cell Physiol 55 1749ndash1762
Wu H Shabala L Zhou M Stefano G Pandolfi C Mancuso S Shabala S (2015)
Developing and validating a high-throughput assay for salinity tissue
tolerance in wheat and barley Planta 242 847-857
Wu H Zhu M Shabala L Zhou M Shabala S (2015) K+ retention in leaf
mesophyll an overlooked component of salinity tolerance mechanism a case
study for barley J Integr Plant Biol 57 171ndash185
Wu J Shang Z Wu J Jiang X Moschou PN Sun W Roubelakis-Angelakis KA
Zhang S (2010) Spermidine oxidase-derived H2O2 regulates pollen plasma
membrane hyperpolarization-activated Ca2+-permeable channels and pollen
tube growth Plant J 63 1042ndash1053
Xia X Zhou Y Shi K Zhou J Foyer CH Yu J (2015) Interplay between reactive
oxygen species and hormones in the control of plant development and stress
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Xie Y Xu S Han B Wu M Yuan X Han Y Gu Q Xu D Yang Q Shen W (2011)
Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1
and the regulatory role of RbohD-derived reactive oxygen species synthesis
Plant J 66 280ndash292
References
129
Xie Y Mao Y Zhang W Lai D Wang Q Shen W (2014) Reactive oxygen species-
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Xue ZY Zhi DY Xue GP Zhang H Zhao YX Xia GM (2004) Enhanced salt
tolerance of transgenic wheat (Tritivum aestivum L) expressing a vacuolar
Na+H+ antiporter gene with improved grain yields in saline soils in the field
and a reduced level of leaf Na+ Plant Sci 167 849-859
Xu H Jiang X Zhan K Cheng X Chen X Pardo JM Cui D (2008) Functional
characterization of a wheat plasma membrane Na+H+ antiporter in yeast
Arch Biochem Biophys 473 8ndash15
Xu R Wang J Li C Johnson P Lu C Zhou M (2012) A single locus is responsible
for salinity tolerance in a Chinese landrace barley (Hordeum vulgare L)
PLoS One 7e43079
Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
water alleviates salt stress in rice during seed germination Plant Soil 370
47-57
Yadav D Ahmed I Shukla P Boyidi P Kirti PB (2016) Overexpression of
Arabidopsis AnnAt8 alleviates abiotic stress in transgenic Arabidopsis and
tobacco Plants 5 18
Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
and opportunities Trends Plant Sci 10 615-620
Yamauchi Y Furutera A Seki K Toyoda Y Tanaka K Sugimoto Y (2008)
Malondialdehyde generated from peroxidized linolenic acid causes protein
modification in heat-stressed plants Plant Physiol Bioch 46 786ndash793
Yancey PH (2005) Organic osmolytes as compatible metabolic and counteracting
cytoprotectants in high osmolarity and other stresses J Exp Biol 208 2819-
2830
Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
(2009) Overexpression of SOS (Salt Overly Sensitive) genes increases salt
tolerance in transgenic Arabidopsis Mol Plant 2 22-31
References
130
Yan J Tsuichihara N Etoh T Iwai S (2007) Reactive oxygen species and nitric
oxide are involved in ABA inhibition of stomatal opening Plant Cell Environ
30 1320-1325
Yazici EY Deveci H (2010) Factors affecting decomposition of hydrogen
peroxide In Proceedings of the XIIth International Mineral Processing
Symposium Cappadocia Turkey 6ndash10
Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
transgenic maize with improved salt tolerance by the introduction of AtNHX1
gene Acta Bot Sin 46 854-861
Yokoi S Quintero FJ Cubero B Ruiz MT Bressan RA Hasegawa PM Pardo JM
(2002) Differential expression and function of Arabidopsis thaliana NHX
Na+H+ antiporters in the salt stress response Plant J 30 529ndash539
Yue SU Zhang W Li FL Guo YL Liu TL Huang H (2000) Identification and
genetic mapping of four novel genes that regulate leaf development in
Arabidopsis Cell Res 10 325-335
Yue Y Zhang M Zhang J Duan L Li Z (2012) SOS1 gene overexpression
increased salt tolerance in transgenic tobacco by maintaining a higher K+Na+
ratio J Plant Physiol 169 255-261
Zeng H Xu L Singh A Wang H Du L Poovaiah BW (2015) Involvement of
calmodulin and calmodulin-like proteins in plant responses to abiotic stresses
Front Plant Sci 6 600
Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
Muniz-Murguia J Pottosin II (2011) Polyamines interact with hydroxyl
radicals in activating Ca2+ and K+ transport across the root epidermal plasma
membranes Plant Physiol 157 2167-2180
Zhang F Li S Yang S Wang L Guo W (2015) Overexpression of a cotton annexin
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Plant Mol Biol 87 47-67
References
131
Zhang G Sun Y Li Y Dong Y Huang X Yu Y Wang J Wang X Wang X Kang
Z (2013) Characterization of a wheat C2 domain protein encoding gene
regulated by stripe rust and abiotic stresses Biol Plantarum 57 701-710
Zhang HX Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate
salt in foliage but not in fruit Nat Biotechnol 19 765-768
Zhang HX Hodson JN Williams JP Blumwald E (2001) Engineering salt-tolerant
Brassica plants characterization of yield and seed oil quality in transgenic
plants with increased vacuolar sodium accumulation P Natl A Sci 98 12832-
12836
Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
crop plants J Exp Bot 50 291ndash302
Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
major QTL for abiotic stress tolerance in barley and implications for barley
breeding Planta 245 283-295
Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
Biol 6 441-445
Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
stomatal characteristics with salinity stress tolerance in contrasting barley
accessions Funct Plant Biol 42 252ndash263
Zhu M Zhou M Shabala L Shabala S (2017) Physiological and molecular
mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
iv
List of publications
Journal publications
Wang H Shabala L Zhou M Shabala S (2018) Hydrogen peroxide-induced root
Ca2+ and K+ fluxes correlate with salt tolerance in cereals towards the cell-based
phenotyping International Journal of Molecular Sciences 19 702
Wang H Shabala L Zhou M Shabala S Developing a high-throughput
phenotyping method for oxidative stress tolerance in cereal roots Plant Methods
(submitted 12042018)
Manuscripts in preparation
Wang H Shabala L Zhou M Shabala S H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in cereals and QTL identification
regarding this trait
Conference papers
Wang H Shabala L Zhou M Shabala S (Oral presentation) ldquoRevealing the causal
relationship between salinity and oxidative stress tolerance in wheat and barleyrdquo
The XIX International Botanical Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoHigh-throughput
assays for oxidative stress tolerance in cerealsrdquo The XIX International Botanical
Congress July 2017 Shenzhen China
Wang H Shabala L Zhou M Shabala S (Poster presentation) ldquoRevealing the
causal relationship between salinity and oxidative stress tolerance in wheat and
barleyrdquo Australian Barley Technical Symposium September 2017 Hobart
Tasmania
Wang H Shabala L Zhou L Shabala S (Poster presentation) ldquoDeveloping a
high-throughput phenotyping method for oxidative stress tolerance in cereal
rootsrdquo 10th International Symposium on Root Research July 2018 Jerusalem
Israel
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
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Bonales-Alatorre E Shabala S Chen ZH Pottosin I (2013) Reduced tonoplast fast-
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Bose J Pottosin II Shabala SS Palmgren MG Shabala S (2011) Calcium efflux
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Butcher K Wick AF DeSutter T Chatterjee A Harmon J (2016) Soil salinity a
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Byrt CS Xu B Krishnan M Lightfoot DJ Athman A Jacobs AK Watson-Haigh
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Case RM Eisner D Gurney A Jones O Muallem S Verkhratsky A (2007)
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Cheeseman JM (2006) Hydrogen peroxide concentrations in leaves under natural
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Chen Z Newman I Zhou M Mendham N Zhang G Shabala S (2005) Screening
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Cell Environ 28 1230ndash1246
Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
Potassium and sodium relations in salinised barley tissues as a basis of
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Collard BCY Mackill DJ (2008) Marker-assisted selection an approach for
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Cotsaftis O Plett D Shirley N Tester M Hrmova M (2012) A two-staged model
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Cuin TA Betts SA Chalmandrier R Shabala S (2008) A roots ability to retain K+
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Cuin TA Bose J Stefano G Jha D Tester M Mancuso S Shabala S (2011)
Assessing the role of root plasma membrane and tonoplast Na+H+
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Cuin TA Shabala S (2007) Compatible solutes reduce ROS-induced potassium
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Cuin TA Shabala S (2008) Compatible solutes mitigate damaging effects of salt
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Signal Behav 3 207-208
Cuin TA Tian Y Betts SA Chalmandrier R Shabala S (2009) Ionic relations and
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Davenport RJ Munoz-Mayor A Jha D Essah PA Rus A Tester M (2007) The
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Dbira S Al Hassan M Gramazio P Ferchichi A Vicente O Prohens J Boscaiu M
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Deinlein U Stephan AB Horie T Luo W Xu G Schroeder JI (2014) Plant salt-
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De la Garma JG Fernandez-Garcia N Bardisi E Pallol B Rubio-Asensio JS Bru
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Del Rio D Stewart AJ Pellegrini N (2005) A review of recent studies on
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Demidchik V (2015) Mechanisms of oxidative stress in plants from classical
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Demidchik V Cuin TA Svistunenko D Smith SJ Miller AJ Shabala S Sokolik
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175 387ndash404
Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Demidchik V Shabala SN Coutts KB Tester MA Davies JM (2003) Free oxygen
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Demidchik V Straltsova D Medvedev SS Pozhvanov GA Sokolik A Yurin V
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Dreyer I Uozumi N (2011) Potassium channels in plant cells FEBS J 278 4293-
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Ellouzi H Ben Hamed K Cela J Munne-Bosch S Abdelly C (2011) Early effects
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Fan Y Zhu M Shabala S Li C Johnson P Zhou M (2014) Antioxidant activity in
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Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-
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Feki K Quintero FJ Pardo JM Masmoudi K (2011) Regulation of durum wheat
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Flowers TJ Flowers SA (2005) Why does salinity pose such a difficult problem for
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Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants where
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Foreman J Demidchik V Bothwell JHF Mylona P Miedema H Torres MA
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Reactive oxygen species produced by NADPH oxidase regulate plant cell
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Foyer CH Noctor G (2003) Redox sensing and signalling associated with reactive
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Foyer CH Noctor G (2009) Redox regulation in photosynthetic organisms
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Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-
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Fry SC Miller JG Dumville JC (2002) A proposed role for copper ions in cell wall
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Fuchs S Grill E Meskiene I Schweighofer A (2013) Type 2C protein phosphatases
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Fukuda A Chiba K Maeda M Nakamura A Maeshima M Tanaka Y (2004a)
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Fukuda A Nakamura A Tagiri A Tanaka H Miyao A Hirochika H Tanaka Y
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Garcia AB Engler JD Iyer S Gerats T Van Montagu M Caplan AB (1997)
Effects of osmoprotectants upon NaCl stress in rice Plant Physiol 115 159-
169
Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Garciadeblas B Senn ME Banuelos MA Rodriguez-Navarro A (2003) Sodium
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Gaymard F Pilot G Lacombe B Bouchez D Bruneau D Boucherez J Michaux-
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Genc Y Oldach K Taylor J Lyons GH (2016) Uncoupling of sodium and chloride
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Gierth M Maumlser P (2007) Potassium transporters in plants - involvement in K+
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Gill MB Zeng F Shabala L Zhang G Fan Y Shabala S Zhou M (2017) Cell-
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gobert A Isayenkov S Voelker C Czempinski K Maathuis FJM (2007) The two-
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Gobert A Park G Amtmann A Sanders D Maathuis FJM (2006) Arabidopsis
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
using modem technologies Procedia Earth Planet Sci 15 507ndash512
Gregorio GB Senadhira D Mendoza RD Manigbas NL Roxas JP Guerta CQ
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Grondin A Rodrigues O Verdoucq L Merlot S Leonhardt N Maurel C (2015)
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Gupta B Huang BR (2014) Mechanism of salinity tolerance in plants
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Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hanin M Ebel C Ngom M Laplaze L Masmoudi K (2016) New insights on plant
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Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Hediye Sekmen A Tuumlrkan İ Takio S (2007) Differential responses of antioxidative
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Horie T Karahara I Katsuhara M (2012) Salinity tolerance mechanisms in
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Inoue H Kudo T Kamada H Kimura M Yamaguchi I Hamamoto H (2005)
Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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Isayenkov S Isner JC Maathuis FJM (2010) Vacuolar ion channels roles in plant
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Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Ji H Pardo JM Batelli G Van Oosten MJ Bressan RA Li X (2013) The Salt
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Joo JH Bae YS Lee JS (2001) Role of auxin-induced reactive oxygen species in
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Malho R Liu Q Monteiro D Rato C Camacho L Dinis A (2006) Signalling
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Malzahn A Lowder L Qi Y (2017) Plant genome editing with TALEN and
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McBrien DCH Hassall KA (1965) Loss of cell potassium by Chlorella vulgaris
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Miah G Rafii MY Ismail MR Puteh AB Rahim HA Asfaliza R Latif MA (2013)
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Michard E Simon AA Tavares B Wudick MM Feijoacute JA (2017) Signaling with
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Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Miller G Schlauch K Tam R Cortes D Torres MA Shulaev V Dangl JL Mittler
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Miller G Shulaev V Mittler R (2008) Reactive oxygen signaling and abiotic stress
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Miller G Suzuki N Ciftci-Yilmaz S Mittler R (2010) Reactive oxygen species
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Mittler R (2002) Oxidative stress antioxidants and stress tolerance Trends Plant
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Mittler R (2017) ROS are good Trends Plant Sci 22 11ndash19
Mittler R Vanderauwera S Gollery M Van Breusegem F (2004) Reactive oxygen
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Mittler R Vanderauwera S Suzuki N Miller G Tognetti VB Vandepoele K
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Mittova V Guy M Tal M Volokita M (2002) Response of the cultivated tomato
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Moslashller IM (2001) Plant mitochondria and oxidative stress electron transport
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Moslashller IM Jensen PE Hansson A (2007) Oxidative modifications to cellular
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Moslashller IM Sweetlove LJ (2010) ROS signallingndashspecificity is required Trends
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Moslashller IS Gilliham M Deepa J Mayo GM Roy SJ Coates JC Haseloff J Tester
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Munns R James RA Lauchli A (2006) Approaches to increasing the salt tolerance
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Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
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Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
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Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
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Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
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ratio J Plant Physiol 169 255-261
Zeng H Xu L Singh A Wang H Du L Poovaiah BW (2015) Involvement of
calmodulin and calmodulin-like proteins in plant responses to abiotic stresses
Front Plant Sci 6 600
Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
Muniz-Murguia J Pottosin II (2011) Polyamines interact with hydroxyl
radicals in activating Ca2+ and K+ transport across the root epidermal plasma
membranes Plant Physiol 157 2167-2180
Zhang F Li S Yang S Wang L Guo W (2015) Overexpression of a cotton annexin
gene GhAnn1 enhances drought and salt stress tolerance in transgenic cotton
Plant Mol Biol 87 47-67
References
131
Zhang G Sun Y Li Y Dong Y Huang X Yu Y Wang J Wang X Wang X Kang
Z (2013) Characterization of a wheat C2 domain protein encoding gene
regulated by stripe rust and abiotic stresses Biol Plantarum 57 701-710
Zhang HX Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate
salt in foliage but not in fruit Nat Biotechnol 19 765-768
Zhang HX Hodson JN Williams JP Blumwald E (2001) Engineering salt-tolerant
Brassica plants characterization of yield and seed oil quality in transgenic
plants with increased vacuolar sodium accumulation P Natl A Sci 98 12832-
12836
Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
crop plants J Exp Bot 50 291ndash302
Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
major QTL for abiotic stress tolerance in barley and implications for barley
breeding Planta 245 283-295
Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
Biol 6 441-445
Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
stomatal characteristics with salinity stress tolerance in contrasting barley
accessions Funct Plant Biol 42 252ndash263
Zhu M Zhou M Shabala L Shabala S (2017) Physiological and molecular
mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
v
Acknowledgements
Four years ago I was enrolled as a PhD candidate in University of Tasmania
Here at this special moment with completion of my PhD study I would like to
express my sincere thanks to UTAS and Grain Research and Development
Corporation (GRDC) for their great financial support during my candidature
At the same time I am very glad and lucky to be a member in Sergey Shabalarsquos
Plant Physiology lab with the dedicated supervision by Prof Sergey Shabala Prof
Meixue Zhou and Dr Lana Shabala As my primary supervisor Prof Sergey
Shabala showed his omnipotence in solving any problems I met during my PhD
study He also enlightened me with his wide knowledge and professionalism in
papers writing My co-supervisor Prof Meixue Zhou and Dr Lana Shabala also
helped me a lot both of them were very kind-hearted in guiding my study on all
aspects during the past years I am really appreciated for the great help and
instructions from AProf Zhonghua Chen with the genetic analysis work Many
thanks to all of them
I also would like to thank sincerely all my current (Juan Liu Ping Yun Dr
Tracey Cuin Ali Kiani-Pouya Amarah Batool Babar Shahzad Fatemeh Rasouli
Joseph Hartley Hassan Dhshan Justin Direen Mohsin Tanveer Muhammad Gill
Dr Nadia Bazihizina Tetsuya Ishikawa Widad Al-Shawi and Hasanuzzaman
Hasan) and former (Dr Nana Su Dr Qi Wu Dr Yuan Huang Dr Min Yu Dr
Xuewen Li Dr Yun Fan Dr Xin Huang Dr Min Zhu Dr Honghong Wu Dr
Yanling Ma Dr Feifei Wang Dr Xuechen Zhang Dr Maheswari Jayakumar Dr
Jayakumar Bose Dr William Percey Dr Edgar Bonales Shivam Sidana Zhinous
Falakboland and Dr Getnet Adam) lab colleagues for their help I will always
remember them all
Great thanks to my family (mother father sister) Thanks for their
unconditional support and love to me and great concern for my living and studying
during my stay in Australia
Finally special thanks to my beloved idol Mr Kai Wang who appeared in
October 2015 and fulfilled my spiritual life He also gave me a good example of
insisting on his originality and having the right attitude towards his acting career I
will always learn from him and try to be a professional in my research area in the
near future
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
References
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Foreman J Demidchik V Bothwell JHF Mylona P Miedema H Torres MA
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Garcia AB Engler JD Iyer S Gerats T Van Montagu M Caplan AB (1997)
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169
Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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181-192
Garciadeblas B Senn ME Banuelos MA Rodriguez-Navarro A (2003) Sodium
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Gaymard F Pilot G Lacombe B Bouchez D Bruneau D Boucherez J Michaux-
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gobert A Isayenkov S Voelker C Czempinski K Maathuis FJM (2007) The two-
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Gobert A Park G Amtmann A Sanders D Maathuis FJM (2006) Arabidopsis
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
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Gregorio GB Senadhira D Mendoza RD Manigbas NL Roxas JP Guerta CQ
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Grondin A Rodrigues O Verdoucq L Merlot S Leonhardt N Maurel C (2015)
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2014
Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hanin M Ebel C Ngom M Laplaze L Masmoudi K (2016) New insights on plant
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Sci 7 1787
Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Hernandez JA Ferrer MA Jimeacutenez A Barcelo AR Sevilla F (2001) Antioxidant
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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Humble GD Raschke K (1971) Stomatal opening quantitatively related to
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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2939ndash2947
Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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2268
Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
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Ji H Pardo JM Batelli G Van Oosten MJ Bressan RA Li X (2013) The Salt
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Joo JH Bae YS Lee JS (2001) Role of auxin-induced reactive oxygen species in
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Malzahn A Lowder L Qi Y (2017) Plant genome editing with TALEN and
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Munns R James RA Lauchli A (2006) Approaches to increasing the salt tolerance
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Munns R Tester M (2008) Mechanisms of salinity tolerance Annu Rev Plant Biol
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Nadeem MA Nawaz MA Shahid MQ Doğan Y Comertpay G Yıldız M
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Navrot N Rouhier N Gelhaye E Jacquot JP (2007) Reactive oxygen species
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Nieves-Cordones M Aleman F Martinez V Rubio F (2014) K+ uptake in plant
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Oh DH Dassanayake M Haas JS Kropornika A Wright C drsquoUrzo MP Hong H
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Potocky M Jones MA Bezvoda R Smirnoff N Zarsky V (2007) Reactive oxygen
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Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Qiu QS (2012) Plant and yeast NHX antiporters roles in membrane trafficking J
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Raha S Robinson BH (2000) Mitochondria oxygen free radicals disease and
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Rengasamy P (2006) World salinization with emphasis on Australia J Exp Bot 57
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Wu H Shabala L Zhou M Stefano G Pandolfi C Mancuso S Shabala S (2015)
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Wu H Zhu M Shabala L Zhou M Shabala S (2015) K+ retention in leaf
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Wu J Shang Z Wu J Jiang X Moschou PN Sun W Roubelakis-Angelakis KA
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Xu H Jiang X Zhan K Cheng X Chen X Pardo JM Cui D (2008) Functional
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Xu S Zhu S Jiang Y Wang N Wang R Shen W Yang J (2013) Hydrogen-rich
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Yamaguchi T Blumwald E (2005) Developing salt-tolerant crop plants challenges
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Yang Q Chen ZZ Zhou XF Yin HB Li X Xin XF Hong XH Zhu JK Gong Z
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Yin XY Yang AF Zhang KW Zhang JR (2004) Production and analysis of
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Yokoi S Quintero FJ Cubero B Ruiz MT Bressan RA Hasegawa PM Pardo JM
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Yue Y Zhang M Zhang J Duan L Li Z (2012) SOS1 gene overexpression
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Zepeda-Jazo I Velarde-Buendia AM Enriquez-Figueroa R Bose J Shabala S
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Zhang JX Nguyen HT Blum A (1999) Genetic analysis of osmotic adjustment in
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Zhang X Shabala S Koutoulis A Shabala L Zhou M (2017) Meta-analysis of
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Zhu JK (2003) Regulation of ion homeostasis under salt stress Curr Opin Plant
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Zhu M Zhou M Shabala L Shabala S (2015) Linking osmotic adjustment and
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mechanisms mediating xylem Na+ loading in barley in the context of salinity
stress tolerance Plant Cell Environ 40 1009ndash1020
Preliminaries
vi
Table of Contents
Declarations and statements i
Declaration of originality i
Authority of access i
Statement regarding published work contained in thesis i
Statement of co-authorship ii
List of publications iv
Acknowledgements v
List of illustrations and tables xi
List of abbreviation xiv
Abstract xvii
Chapter 1 Literature review 1
11 Salinity as an issue 1
12 Factors contributing to salinity stress tolerance 1
121 Osmotic adjustment 1
122 Root Na+ uptake and efflux 2
123 Vacuolar Na+ sequestration 3
124 Control of xylem Na+ loading 4
125 Na+ retrieval from the shoot 5
126 K+ retention 5
127 Reactive oxygen species (ROS) detoxification 6
13 Oxidative component of salinity stress 6
131 Major types of ROS 6
132 ROS friends and foes 6
133 ROS production in plants under saline conditions 7
134 Mechanisms for ROS detoxification 10
14 ROS control over plant ionic homeostasis salinity stress
context 11
Preliminaries
vii
141 ROS impact on membrane integrity and cellular structures 11
142 ROS control over plant ionic homeostasis 12
143 ROS signalling under stress conditions 16
15 Linking salinity and oxidative stress tolerance 17
151 Genetic variability in oxidative stress tolerance 18
152 Tissue specificity of ROS signalling and tolerance 19
16 Aims and objectives of this study 20
161 Aim of the project 20
162 Outline of chapters 22
Chapter 2 General materials and methods 24
21 Plant materials 24
22 Growth conditions 24
221 Hydroponic system 24
222 Paper rolls 24
23 Microelectrode Ion Flux Estimation (MIFE) 24
231 Ion-selective microelectrodes preparation 24
232 Ion flux measurements 25
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+
fluxes correlate with salt tolerance in cereals towards the
cell-based phenotyping 26
31 Introduction 26
32 Materials and methods 28
321 Plant materials and growth conditions 28
322 K+ and Ca2+ fluxes measurements 29
323 Experimental protocols for microelectrode ion flux estimation (MIFE)
measurements 29
324 Quantifying plant damage index 30
325 Statistical analysis 30
33 Results 30
331 H2O2-induced ion fluxes are dose-dependent 30
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in barley 33
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in wheat 35
Preliminaries
viii
334 Genotypic variation of hydroxyl radical-induced Ca2+ and K+ fluxes in
barley 37
34 Discussion 39
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+ fluxes does
not correlate with salinity stress tolerance in barley 40
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with their overall
salinity stress tolerance but only in mature zone 41
343 Reactive oxygen species (ROS)-induced K+ efflux is accompanied by
an increased Ca2+ uptake 43
344 Implications for breeders 44
Chapter 4 Validating using MIFE technique-measured
H2O2-induced ion fluxes as physiological markers for
salinity stress tolerance breeding in wheat and barley 45
41 Introduction 45
42 Materials and methods 46
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements 46
422 Pharmacological experiments 46
423 Statistical analysis 46
43 Results 47
431 H2O2-induced ions kinetics in mature root zone of cereals 47
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity tolerance in barley 47
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in bread wheat 49
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root zone
correlates with the overall salinity stress tolerance in durum wheat 51
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat in mature
root zone upon oxidative stress 52
436 H2O2-induced ion flux in root mature zone can be prevented by TEA+
Gd3+ and DPI in both barley and wheat 53
44 Discussion 54
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress tolerance 54
442 Barley tends to retain more K+ and acquire less Ca2+ into cytosol in root
mature zone than wheat when subjected to oxidative stress 56
Preliminaries
ix
443 Different identity of ions transport systems in root mature zone upon
oxidative stress between barley and wheat 57
Chapter 5 QTLs for ROS-induced ions fluxes associated
with salinity stress tolerance in barley 59
51 Introduction 59
52 Materials and methods 60
521 Plant material growth conditions and Ca2+ and K+ flux measurements
60
522 QTL analysis 61
523 Genomic analysis of potential genes for salinity tolerance 61
53 Results 62
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment 62
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux 63
533 QTL for KF when using CaF as a covariate 64
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H and 7H
65
54 Discussion 66
541 QTL on 2H and 7H for oxidative stress control both K+ and Ca2+ flux 66
542 Potential genes contribute to oxidative stress tolerance 68
Chapter 6 Developing a high-throughput phenotyping
method for oxidative stress tolerance in cereal roots 71
61 Introduction 71
62 Materials and methods 73
621 Plant materials and growth conditions 73
622 Viability assay 74
623 Root growth assay 75
624 Statistical analysis 76
63 Results 76
631 H2O2 causes loss of the cell viability in a dose-dependent manner 76
632 Genetic variability of root cell viability in response to 10 mM H2O2 77
633 Methodological experiments for cereal screening in root growth upon
oxidative stress 80
Preliminaries
x
634 H2O2ndashinduced changes of root length correlate with the overall salinity
tolerance 81
64 Discussion 82
641 H2O2 causes a loss of the cell viability and decline of growth in barley
roots 82
642 Salt tolerant barley roots possess higher root viability in elongation
zone after long-term ROS exposure 83
643 Evaluating root growth assay screening for oxidative stress tolerance 84
Chapter 7 General discussion and future prospects 86
71 General discussion 86
72 Future prospects 89
References 93
Preliminaries
xi
List of illustrations and tables
Figure 11 ROS production pattern in plantshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
Figure 12 Model of ROS detoxification by Asc-GSH cyclehelliphelliphelliphelliphelliphelliphellip10
Figure 13 Model of ROS detoxification by GPX cyclehelliphelliphelliphelliphelliphelliphelliphelliphellip11
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root
elongationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16
Figure 31 Descriptions of cereal root ion fluxes in response to H2O2 and bullOH in a
single experimenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31
Figure 32 Net K+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
Figure 33 Net Ca2+ fluxes measured from barley variety TX9425 in both root
elongation and mature zone with respective H2O2 concentrations and their
dose-dependencyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip33
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
zone and their correlation between H2O2-induced K+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation and mature
Preliminaries
xii
zone and their correlation between H2O2-induced Ca2+ fluxes and overall
salinity stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip37
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced K+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip38
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 bullOH treatment from both root elongation and mature zone
and their correlation between bullOH-induced Ca2+ fluxes and overall salinity
stress tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip39
Figure 41 Descriptions of net K+ and Ca2+ flux from cereals root mature zone in
response to 10 mM H2O2 in a representative experiment helliphelliphelliphelliphellip47
Figure 42 Genetic variability of oxidative stress tolerance in barleyhelliphelliphelliphellip49
Figure 43 Genetic variability of oxidative stress tolerance in bread wheathelliphellip51
Figure 44 Genetic variability of oxidative stress tolerance in durum wheathellip52
Figure 45 General comparison of H2O2-induced net K+ and Ca2+ fluxes
initialpeak K+ flux and Ca2+ flux values net mean K+ efflux and Ca2+ uptake
values from mature root zone in barley bread wheat and durum
wheathelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Figure 46 Effect of DPI Gd3+ and TEA+ pre-treatment on H2O2-induced net mean
K+ and Ca2+ fluxes from the mature root zone of barley and
wheat helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip54
Figure 51 Frequency distribution for peak K+ flux and peak Ca2+ flux of DH lines
derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
Figure 52 QTLs associated with H2O2-induced peak K+ flux and H2O2-induced
peak Ca2+ fluxhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
Figure 53 Chart view of QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH
line helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
Preliminaries
xiii
Figure 61 Viability staining and fluorescence image acquisitionhelliphelliphelliphelliphellip75
Figure 62 Viability staining of Naso Nijo roots exposed to 0 03 1 3 10 mM
H2O2 for 1 day and 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
Figure 63 Red fluorescence intensity measured from roots of Naso Nijo upon
exposure to various H2O2 concentrations for either one day or three
days helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
Figure 64 Viability staining of root elongation and mature zones of four barley
varieties exposed to 10 mM H2O2 for 3 dayshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
Figure 65 Quantitative red fluorescence intensity from root elongation and mature
zone of five barley varieties exposed to 10 mM H2O2 for 3 dhelliphelliphelliphellip79
Figure 66 Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d and their correlation with the overall salinity
tolerancehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
Table 31 List of barley and wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphellip29
Table 41 List of barley varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip48
Table 42 List of wheat varieties used in this studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip50
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lineshellip62
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariatehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ fluxhelliphelliphelliphelliphellip66
Table 61 Barley varieties used in the studyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
Preliminaries
xiv
List of abbreviation
3Chl Triplet state chlorophyll
1O2 Singlet oxygen
ABA Abscisic acid
AO Antioxidant
APX Ascorbate peroxidase
Asc Ascorbate
BR Brassinosteroid
BSM Basic salt medium
CaLB Calcium-dependent lipid-binding
Cas CRISPR-associated
CAT Catalase
CML Calmodulin like
CNGC Cyclic nucleotide-gated channels
CRISPR Clustered regularly interspaced short palindromic repeats
crRNA CRISPR RNA
CS Compatible solutes
CuA CopperAscorbate
Cys Cysteine
DArT Diversity Array Technology
DH Double haploid
DHAR Dehydroascorbate reductase
DMSP Dimethylsulphoniopropionate
DPI Diphenylene iodonium
DSB Double-stranded break
ER Endoplasmic reticulum
ET Ethylene
ETC Electron transport chain
FAO Food and Agriculture Organization
FDA Fluorescein diacetate
FV Fast vacuolar channel
GA Gibberellin
Gd3+ Gadolinium chloride
GORK Guard cell outward rectifying K+ channel
GPX Glutathione peroxidase
Preliminaries
xv
GR Glutathione reductase
gRNA Guide RNA
GSH Glutathione (reduced form)
GSSG Glutathione (oxidized form)
H2 Hydrogen gas
H2O2 Hydrogen peroxide
HKT High-affinity K+ Transporter
HOObull Perhydroxy radical
IL Introgression line
IM Interval mapping
indel Insertiondeletion
JA Jasmonate
LEA Late-embryogenesis-abundant
LCK1 Low affinity cation transporter
LOD Logarithm of the odds
LOOH Lipid hydroperoxides
MAS Marker assisted selection
MDA Malondialdehyde
MDAR Monodehydroascorbate reductase
MIFE Microelectrode Ion Flux Estimation
MQM Multiple QTL model
Nax1 NA+ EXCLUSION 1
Nax2 NA+ EXCLUSION 2
NHX Na+H+ exchanger
NO Nitric oxide
NSCCs Non-Selective Cation Channels
O2- Superoxide radicals
bullOH Hydroxyl radicals
PCD Programmed Cell Death
PI Propidium iodide
PIP21 Plasma membrane intrinsic protein 21
PM Plasma membrane
POX Peroxidase
PP2C Protein phosphatase 2C family protein
PSI Photosystem I
Preliminaries
xvi
PSII Photosystem II
PUFAs Polyunsaturated fatty acids
QCaF QTLs for H2O2-induced peak Ca2+ flux
QKF QTLs for H2O2-induced peak K+ flux
QTL Quantitative Trait Locus
RBOH Respiratory burst oxidase homologue
RObull Alkoxy radicals
ROS Reactive Oxygen Species
RRL Relative root length
RT-PCR Real-time polymerase chain reaction
SA Salicylic acid
SE Standard error
SKOR Stellar K+ outward rectifier
SL Strigolactone
SODs Superoxide dismutases
SOS Salt Overly Sensitive
SSR Simple Sequence Repeat
SV Slow vacuolar channel
TALENs Transcription activator-like effector nucleases
TEA+ Tetraethylammonium chloride
TFs Transcription factors
tracrRNA Trans-activating crRNA
UQ Ubiquinone
V-ATPase Vacuolar H+-ATPase
VK Vacuolar K+-selective channels
V-PPase Vacuolar H+-PPase
W-W Waterndashwater
ZNFs Zinc finger nucleases
Abstract
xvii
Abstract
Soil salinity is a global issue and a major factor limiting crop production
worldwide One side effect of salinity stress is an overproduction and accumulation
of reactive oxygen species (ROS) causing oxidative stress and leading to severe
cellular damage to plants While the major focus of the salinity-oriented breeding
programs in the last decades was on traits conferring Na+ exclusion or osmotic
adjustment breeding for oxidative stress tolerance has been largely overlooked
ROS are known to activate several different types of ion channels affecting
intracellular ionic homeostasis and thus plantrsquos ability to adapt to adverse
environmental conditions However the molecular identity of many ROS-activated
ion channels remains unexplored and to the best of our knowledge no associated
QTLs have been reported in the literature
This work aimed to fill the above knowledge gaps by evaluating a causal link
between oxidative and salinity stress tolerance The following specific objectives
were addressed
To develop MIFE protocols as a tool for salinity tolerance screening in
cereals
To validate the role of specific ROS in salinity stress tolerance by applying
developed MIFE protocols to a broad range of cereal varieties and establish a causal
relationship between oxidative and salinity stress tolerance in cereals
To map QTLs controlling oxidative stress tolerance in barley
To develop a simple and reliable high-throughput phenotyping method
based on above traits
Working along these lines a range of electrophysiological pharmacological
and imaging experiments were conducted using a broad range of barley and wheat
varieties and barley double haploid (DH) lines
In order to develop the applicable MIFE protocols the causal relationship
between salinity and oxidative stress tolerance in two cereal crops - barley and
wheat - was investigated by measuring the magnitude of ROS-induced net K+ and
Ca2+ fluxes from various root tissues and correlating them with overall whole-plant
responses to salinity No correlation was found between root responses to hydroxyl
radicals and the salinity tolerance However a significant positive correlation was
found for the magnitude of H2O2-induced K+ efflux and Ca2+ uptake in barley and
Abstract
xviii
the overall salinity stress tolerance but only for mature zone and not the root apex
The same trends were found for wheat These results indicate high tissue specificity
of root ion fluxes response to ROS and suggest that measuring the magnitude of
H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool
for cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals
In the next chapter 44 barley and 40 wheat (20 bread wheat and 20 durum
wheat) cultivars contrasting in their salinity tolerance were screened to validate the
above correlation between H2O2-induced ions fluxes and the overall salinity stress
tolerance A strong and negative correlation was reported for all the three cereal
groups indicating the applicability of using the MIFE technique as a reliable
screening tool in cereal breeding programs Pharmacological experiments were
then conducted to explore the molecular identity of H2O2 sensitive Ca2+ and K+
channels in both barley and wheat We showed that both non-selective cation and
K+-selective channels are involved in ROS-induced Ca2+ and K+ flux in barley and
wheat At the same time the ROS generation enzyme NADPH oxidative was also
playing vital role in controlling this process The findings may assist breeders in
identifying possible targets for plant genetic engineering for salinity stress
tolerance
Once the causal association between oxidative and salinity stress has been
established we have mapped QTLs associated with H2O2-induced Ca2+ and K+
fluxes as a proxy for salinity stress tolerance using over 100 DH lines from a cross
between CM72 (salt tolerant) and Gairdner (salt sensitive) Three major QTLs on
2H (QKFCG2H) 5H (QKFCG5H) and 7H (QKFCG7H) were identified to be
responsible for H2O2-induced K+ fluxes while two major QTLs on 2H
(QCaFCG2H) and 7H (QCaFCG7H) were for H2O2-induced Ca2+ fluxes QTL
analysis for H2O2-induced K+ flux by using H2O2-induced Ca2+ flux as covariate
showed that the two QTLs for K+ flux located at 2H and 7H were also controlling
Ca2+ flux while another QTL mapped at 5H was only involved in K+ flux
According to this finding the nearest sequence markers (bpb-8484 on 2H bpb-
5506 on 5H and bpb-3145 on 7H) were selected to identify candidate genes for
salinity tolerance and annotated genes between 6445 and 8095 cM on 2H 4299
and 4838 cM on 5H 11983 and 14086 cM on 7H were deemed to be potential
genes
Abstract
xix
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding the new trait - H2O2-induced Ca2+ and K+ fluxes -
alongside with other (traditional) mechanisms However as the MIFE method has
relatively low throughput capacity finding a suitable proxy will benefit plant
breeders Two high-throughput phenotyping methods - viability assay and root
growth assay - were then tested and assessed In viability staining experiments a
dose-dependent H2O2-triggered loss of root cell viability was observed with salt
sensitive varieties showing significantly more root cell damage In the root growth
assays relative root length (RRL) was measured in plants under different H2O2
concentrations The biggest difference in RRL between contrasting varieties was
observed for 1 mM H2O2 treatment Under these conditions a significant negative
correlation in the reduction in RRL and the overall salinity tolerance was reported
among 11 barley varieties Although both assays showed similar results with that
of MIFE method the root growth assay was way simpler that do not need any
specific skills and training and less time-consuming than MIFE (1 d vs 6 months)
thus can be used as an effective high-throughput phenotyping method
In conclusion this project established a causal link between oxidative and
salinity stress tolerance in both barley and wheat and provided new insights into
fundamental mechanisms conferring salinity stress tolerance in cereals The high
throughput screening protocols were developed and validated and it was H2O2-
induced Ca2+ uptake and K+ efflux from the mature root zone correlated with the
overall salinity stress tolerance with salt-tolerant barley and wheat varieties
possessed greater K+ retention and lesser Ca2+ uptake ability when challenged with
H2O2 The QTL mapping targeting this trait in barley showed three major QTLs for
oxidative stress tolerance conferring salinity stress tolerance The future work
should be focused on pyramiding these QTLs and creating robust salt tolerant
genotypes
Chapter 1 Literature review
1
Chapter 1 Literature review
11 Salinity as an issue
Soil salinity or salinization termed as a soil with high level of soluble salts
occurs all over the world (Rengasamy 2006) It affects approximate 15 (45 out of
230 million hectares) of the worldrsquos agricultural land especially in arid and semi-
arid regions (Munns and Tester 2008) At the same time the consequences of the
global climate change such as rising of seawater level and intrusion of sea salt into
coastal area as well as human activities such as excessive irrigation and land
exploitation are making salinity issue even worse (Horie et al 2012 Ismail and
Horie 2017) The direct impact of soil salinity is that it disturbs cellular metabolism
and plant growth reduces crop production and leads to considerable economic
losses (Schleiff 2008 Shabala et al 2014 Gorji et al 2015) It is estimated that
salinity-caused economic penalties from global agricultural production excesses
US$27 billion per annual this value is ascending on a daily basis (Shabala et al
2015) Furthermore increasing agricultural food production is required to feed the
expanding world population which is unlikely to be simply acquired from the
existing arable land (Shabala 2013) This prompts a need to utilise the salt affected
lands to increase yields To achieve this new traits conferring salinity tolerance
should be discovered and QTLs related to salt tolerance traits should be pyramided
to create salt tolerant crop germplasm
12 Factors contributing to salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms The main components are osmotic adjustment
Na+ exclusion from uptake vacuolar Na+ sequestration control of xylem Na+
loading Na+ retrieval from the shoot K+ retention and ROS detoxification (Munns
and Tester 2008 Shabala et al 2010 Wu et al 2015)
121 Osmotic adjustment
Osmotic adjustment also termed as osmoregulation occurs during the process
of cellular dehydration and plays key role in plants adaptive response to minify the
adverse impact of stress induced by excessive external salts especially during the
Chapter 1 Literature review
2
first phase of salinity stress (Hare et al 1998 Mager et al 2000 Serraj and Sinclair
2002 Shabala and Shabala 2011) It can be achieved by (i) controlling ions fluxes
across membranes from different cellular compartments (ii) accumulating
inorganic ions (eg K+ Na+ and Cl-) (iii) synthesizing a diverse range of organic
osmotica (collectively known as ldquocompatible solutesrdquo) to counteract the osmotic
pressure from external medium (Garcia et al 1997 Serraj and Sinclair 2002
Shabala and Shabala 2011)
Compatible solutes (CS) are low-molecular-weight organic compounds with
high solubility and non-toxic even if they accumulate to high concentration
(Yancey 2005) The ability of plants to accumulate CS has long been taken as a
selection criterion in traditional crop (most of which are glycophytes) breeding
programs to increase osmotic stress tolerance (Ludlow and Muchow 1990 Zhang
et al 1999) Generally these osmoprotectants are identified as (1) amino acids (eg
proline glycine arginine and alanine) (2) non-protein amino acids (eg pipecolic
acid γ-aminobutyric acid ornithine and citrulline) (3) amides (eg glutamine and
asparagine) (4) soluble proteins (eg late-embryogenesis-abundant (LEA) protein)
(5) sugars (eg sucrose glucose trehalose raffinose fructose and fructans) (6)
polyols (or ldquosugar alcoholsrdquo as another name eg mannitol inositol pinitol
sorbitol and glycerol) (7) tertiary sulphonium compounds (eg
dimethylsulphoniopropionate (DMSP)) and (8) quaternary ammonium compounds
(eg glycine betaine β-alanine betaine proline betaine pipecolate betaine
hydroxyproline betaine and choline-O-sulphate) (Slama et al 2015 Parvaiz and
Satyawati 2008)
122 Root Na+ uptake and efflux
There are several major pathways mediating Na+ uptake across plasma
membrane (PM) (i) Non-selective cation channels (NSCCs) (Tyerman and Skerrett
1998 Amtmann and Sanders 1998 White 1999 Demidchik et al 2002) (ii) High
affinity K+ transporter (HKT1) (Laurie et al 2002 Garciadeblas et al 2003) (iii)
Low affinity cation transporter (LCK1) (Schachtman et al 1997 Amtmann et al
2001) which therefore facilitate Na+ uptake However only a small fraction of
absorbed Na+ is accumulated in root tissues indicating that a major bulk of the Na+
is extruded from cytosol to the rhizosphere (Munns 2002) However unlike animals
which require Na+ to maintain normal cell metabolism most plant especially
Chapter 1 Literature review
3
glycophytes do not take Na+ as an essential molecule (Blumwald 2000) Thus
plants lack specialised Na+-pumps to extrude Na+ from root when exposed to
salinity stress (Garciadeblas et al 2001) It is believed that Na+ exclusion from
plant roots is mediated by the PM Na+H+ exchangers encoded by SOS1 gene (Zhu
2003 Ji et al 2013) This process is energised by the PM proton pump establishing
an H+ electrochemical potential gradient across the PM as driving force for Na+
exclusion (Palmgren and Nissen 2011) Salt tolerant wheat (Cuin et al 2011) and
the halophyte Thellungiella (Oh et al 2010) were observed with higher SOS1
andor SOS1-like Na+H+ exchanger activity Moreover overexpression of SOS1
or its homologues have been shown to result in enhanced salt tolerance in
Arabidopsis (Shi et al 2003 Yang et al 2009) and tobacco (Yue et al 2012)
123 Vacuolar Na+ sequestration
Plants are also capable of handling excessive cytosolic Na+ by moving it into
vacuole across the tonoplast to maintain cytosol sodium content at non-toxic levels
upon salinity stress (Blumwald et al 2000 Shabala and Shabala 2011) This
process is called ldquoNa+ sequestrationrdquo and is mediated by the tonoplast-localized
Na+H+ antiporters (Blumwald et al 2000) and energised by vacuolar H+-ATPase
(V-ATPase) and H+-PPase (V-PPase) (Zhang and Blumwald 2001 Fukuda et al
2004a) Na+H+ exchanger (NHX) genes are known to operate Na+ sequestration
and express in both roots and leaves Arabidopsis Na+H+ antiporter gene AtNHX1
was the first NHX homolog identified in plants (Rodriacuteguez-Rosales et al 2009)
and another five isoforms of AtNHX gene were then identified and characterised
(Yokoi et al 2002 Aharon et al 2003 Bassil et al 2011a Bassil et al 2011b
Qiu 2012 Barragan et al 2012) Overexpression of NHX1 in Arabidopsis (Apse
et al 1999) rice (Fukuda et al 2004b) maize (Yin et al 2004) wheat (Xue et al
2004) tomato (Zhang and Blumwald 2001) canola (Zhang et al 2001) and
tobacco (Lu et al 2014) result in enhanced salt tolerance in transformed plants
indicating the importance of Na+ transporting into vacuole in conferring plants
salinity stress tolerance (Ismail and Horie 2017) Besides the tonoplast NSCCs -
SV (slow vacuolar channel) and FV (fast vacuolar channel) - have been shown to
control Na+ leak back to the cytoplasm (Bonales-Alatorre et al 2013) which
further make Na+ sequestration more efficient
Chapter 1 Literature review
4
124 Control of xylem Na+ loading
Plant roots are responsible for absorption of nutrients and inorganic ions The
latter are generally loaded into xylem vessels from where they are transported to
shoot via the transpiration stream of the plant (Wegner and Raschke 1994 Munns
and Tester 2008) This makes toxic ion such as Na+ accumulate in shoot easily
under salinity stress Higher concentration of Na+ in mesophyll cells is always
harmful as it compromises plantrsquos leaf photochemistry and thus whole plant
performance One of the strategies to reduce Na+ accumulation in shoot is control
of xylem Na+ loading which can be achieved by either minimizing Na+ entry into
the xylem from the root or maximizing the retrieval of Na+ from the xylem before
it reaches sensitive tissues in the shoot (Tester and Davenport 2003 Katschnig et
al 2015)
The high-affinity K+ transporter (HKT) proteins (especially HKT1 subfamily)
which mainly express in the xylem parenchyma cells show their Na+-selective
transporting activity and play major role in Na+ unloading from xylem in several
plant species such as Arabidopsis rice and wheat (Munns and Tester 2008)
AtHKT11 (Sunarpi et al 2005 Davenport et al 2007 Moslashller et al 2009) and
OsHKT15 (Ren et al 2005) were reported to function in these processes
Moreover OsHKT14 (expressed in both rice leaf sheaths and stems Cotsaftis et
al 2012) and OsHKT11 (strongly expressed in the vicinity of the xylem in rice
leaves Wang et al 2015) were also suggested contributing to Na+ unloading from
the xylem of these tissues In durum wheat TmHKT14 and TmHKT15 were
identified as causal genes of NA+ EXCLUSION 1 ( Nax1 Huang et al 2006) and
NA+ EXCLUSION 2 (Nax2 Byrt et al 2007) respectively Both function by
removing Na+ from roots and the lower parts of leaves making Na+ concentration
low in leaf blade (James et al 2011) Recently introgression of TmHKT15-A into
a salt-sensitive durum wheat cultivar substantially decreased Na+ concentration in
leaves of transformed plants making their grain yield in saline soils increased by
up to 25 (Munns et al 2012) indicating the applicability of targeting this trait
for salinity stress tolerance breeding
Chapter 1 Literature review
5
125 Na+ retrieval from the shoot
Another strategy to prevent shoot Na+ over-accumulation is removal of Na+
from this tissue which was believed to be mediated by HKT1 in the recirculation
of Na+ back to the root by the phloem (Maathuis et al 2014) AtHKT11
(Berthomieu et al 2003) and OsHKT11 (Wang et al 2015) were suggested to
contribute to this process Moreover studies in salinity tolerant wild tomato
(Alfocea et al 2000) and the halophyte reed plants (Matsushita and Matoh 1991)
have revealed that they exhibited higher extent of Na+ recirculation than their
domestic tomato counterparts and the salt-sensitive rice plants respectively
Nevertheless it seems this notion does not hold in all the cases By using an hkt11
mutant Davenport et al (2007) demonstrated that AtHKT11 was not involved in
this process in the phloem which requires further investigation regarding this trait
126 K+ retention
The reason for Na+ being toxic molecule in plants lies in its inhibition of
enzymatic activity especially for those require K+ for functioning (Maathuis and
Amtmann 1999) Since over 70 metabolic enzymes are activated by K+ (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) it is likely that it is the cytosolic K+Na+ ratio
but not the absolute quantity of Na+ that determines plantrsquos ability to survive in
saline soils (Shabala and Cuin 2008) Therefore except for cytosolic Na+ exclusion
efficient cytosolic K+ retention may be another essential factor in the maintenance
of higher K+Na+ ratio to sustain cell metabolism under salinity stress Indeed a
strong positive correlation between K+ retention ability in root tissue and the overall
salinity stress tolerance has been reported in a wide range of plant species including
barley (Chen et al 2005 2007ac) wheat (Cuin et al 2008 2009) lucerne
(Smethurst et al 2008 Guo et al2016) Arabidopsis (Sun et al 2015) pepper
(Bojorquez-Quintal et al 2016) cotton (Wang et al 2016b) and cucumber
(Redwan et al 2016) Likewise a recent study in barley also emphasized the
importance of K+ retention in leaf mesophyll to confer plants salinity stress
tolerance (Wu et al 2015) K+ leakage through PM of both root and shoot tissues
is known to be mediated by two channels namely GORKs (guard cell outward-
rectifying K+ channels) and NSCCs (Shabala and Pottosin 2014) which play major
Chapter 1 Literature review
6
role in cytosolic K+ homeostasis maintenance However until now no salt tolerant
germplasm regarding this trait has been established
127 Reactive oxygen species (ROS) detoxification
The loading of toxic Na+ into plant cytosol not only interferes with various
physiological processes but also leads to the overproduction and accumulation of
reactive oxygen species (ROS) which cause oxidative stress and have major
damage effect to macromolecules (Vellosillo et al 2010 Karuppanapandian et al
2011) A large amount of antioxidant components (enzymes and low molecular
weight compounds) can be found in plants which constitute their defence system
to detoxify excessive ROS and protect cells from oxidative damage Therefore it
seems plausible that plants with higher antioxidant activity (in other words lower
ROS level) may be much more salt tolerant This is the case in many halophytes
and a range of glycophytes with higher salinity tolerance (reviewed in Bose et al
2014b) However ROS are also indispensable signalling molecules involved in a
broad range of physiological processes (Mittler 2017) detoxification of ROS may
interfere with these processes and cause pleiotropic effects (Bose et al 2014b)
making the link between antioxidant activity and salinity stress tolerance
complicated This can be reflected in a range of reports which failed to reveal or
showed negative correlation between the above traits (Bose et al 2014b)
13 Oxidative component of salinity stress
131 Major types of ROS
Reactive oxygen species (ROS) are inevitable by-products of various
metabolic pathways occurring in chloroplast mitochondria and peroxisomes (del
Riacuteo et al 2006 Navrot et al 2007) The major types of ROS are composed of
superoxide radicals (O2-) hydroxyl radical (bullOH) perhydroxy radical (HOObull)
alkoxy radicals (RObull) hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Mittler
2002 Gill and Tuteja 2010)
132 ROS friends and foes
ROS have long been considered as unwelcome by-products of aerobic
metabolism (Mittler 2002 Miller et al 2008) While numerous reports have
Chapter 1 Literature review
7
demonstrated that ROS are acting as signalling molecules to control a range of
physiological processes such as deference responses and cell death (Bethke and
Jones 2001 Mittler 2002) gravitropism (Joo et al 2001) stomatal closure (Pei et
al 2000 Yan et al 2007) cell expansion and polar growth (Coelho et al 2002
Foreman et al 2003) hormone signalling (Mori and Schroeder 2004 Foyer and
Noctor 2009) and leaf development (Yue et al 2000 Rodrıguez et al 2002 Lu
et al 2014)
Under optimal growth conditions ROS production in plants is programmed
and beneficial for plants at both physiological (Foreman et al 2003) and genetical
(Mittler et al 2004) levels However when exposed to stress conditions (eg
drought salinity extreme temperature heavy metals pathogens etc) ROS are
dramatically overproduced and accumulated which ultimately results in oxidative
stress (Apel and Hirt 2004) As highly reactive and toxic substances detrimental
effects of excessive ROS produced during adverse environmental conditions are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and the impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
133 ROS production in plants under saline conditions
Major sources of ROS in plants
ROS are formed as a result of a multistep reduction of oxygen (O2) in aerobic
metabolism pathway in living organisms (Asada 2006 Saed-Moucheshi et al 2014
Nita and Grzybowski 2016) In plants subcellular compartments such as
chloroplasts mitochondria and peroxisomes are the main sources that contribute
to ROS production (Mittler et al 2004 Asada 2006) O2- forms at the first step of
oxygen reduction and then quickly catalysed to H2O2 by superoxide dismutases
(SODs) (Ozgur et al 2013 Bose et al 2014b) In the presence of transition metals
such as Fe2+ and Cu+ H2O2 can be converted to highly toxic bullOH (Rodrigo-Moreno
et al 2013b) bullOH has a really short half-life (less than 1 μs) while H2O2 is the
most stable ROS with half-life in minutes (Pitzschke et al 2006 Bose et al 2014b)
Apart from the cellular compartments mentioned above ROS can also be produced
in the apoplastic spaces These sources include plasma membrane (PM) NADPH
oxidases cell-wall-bound peroxidases amine oxidases pH-dependent oxalate
Chapter 1 Literature review
8
peroxidases and germin-like oxidases (Bolwell and Wojtaszek 1997 Mittler 2002
Hu et al 2003 Walters 2003)
Changes in ROS production under saline conditions
In green tissue of plant cells ROS are mainly generated from chloroplasts and
peroxisomes especially under light condition (Navrot et al 2007) In non-green
tissue or dark condition mitochondria are the major source for ROS production
(Foyer and Noctor 2003 Rhoads et al 2006) Normally ROS homeostasis is able
to keep ROS in a lower non-toxic level (Mittler 2002 Miller et al 2008) However
elevated cytosolic ROS level is deleterious which can be observed when plants are
exposed to saline conditions (Hernandez et al 2001 Tanou et al 2009)
PSI (photosystem I) and PSII (photosystem II) reaction centres in thylakoids
are major sites involved in chloroplastic ROS production (Pfannschmidt 2003
Asada 2006 Gill and Tuteja 2010) Under normal circumstances the
photosynthetic product oxygen accepts electrons passing through the
photosystems and form superoxide radicals by Mehler reaction at the antenna
pigments in PSI (Asada 1993 Polle 1996 Asada 2006) After being reduced to
NADPH the electron flow then enters the Calvin cycle and fixes CO2 (Gill and
Tuteja 2010) Under saline conditions both osmotically-induced stomatal closure
and accumulation of high levels of cytosolic Na+ impair photosynthesis apparatus
and reduce plantrsquos capacity to assimilate CO2 in conjunction with fully utilise light
absorbed by photosynthetic pigments (Biswal et al 2011 Ozgur et al 2013) As
a result the excessive light captured allow overwhelming electrons passing through
electron transport chain (ETC) and lead to enhanced generation of superoxide
radicals (Asada 2006 Ozgur et al 2013) In mitochondria ETC the ROS
generation sites complexes I and complexes III overreduce ubiquinone (UQ) pool
upon salt stress and pass electron to O2 lead to increased production of O2minus (Noctor
2006) which readily catalysed into H2O2 by SODs (Raha and Robinson 2000
Moslashller 2001 Quan et al 2008) Peroxisomes are single membrane-bound
organelles which can generate H2O2 effectively during photorespiration by the
oxidation of glycolate to glyoxylate via glycolate oxidase reaction (Foyer and
Noctor 2009 Bauwe et al 2010) Salinity stress-induced stomatal closure reduces
CO2 content in leaf mesophyll cells leading to enhanced photorespiration and
increased glycolate accumulation and therefore elevated H2O2 production in these
Chapter 1 Literature review
9
organelles (Hernandez et al 2001 Karpinski et al 2003) Salinity-induced
apoplastic ROS generation is generally regulated by the plasma membrane NADPH
oxidases which is activated by elevated cytosolic free Ca2+ following NaCl-
induced activation of depolarization-activated Ca2+ channels (DACC) (Chen et al
2007a Demidchik and Maathuis 2007) This PM NADPH oxidase-mediated ROS
production plays a vital role in the regulation of acclimation to salinity stress
(Kurusu et al 2015) ROS production pattern is detailed in Figure11
Figure 11 ROS production pattern in plants From Bose et al (2014) J Exp Bot
65 1242-1257
Genetic variability in ROS production
Plantsrsquo ability to produce ROS under unfavourable environment varies which
may indicate their variability in salt stress tolerance Comparative analysis of two
rice genotypes contrasting in their salinity stress tolerance revealed higher level of
H2O2 in the salt sensitive cultivar in response to either short-term (Vaidyanathan et
al 2003) or long-term (Mishra et al 2013) salt stimuli A comparative study
Chapter 1 Literature review
10
between a cultivated tomato Solanum lycopersicum L and its salt tolerant
counterparts ndash wild tomato S pennellii - have demonstrated that the latter had less
oxidative damage by increasing the activities of related antioxidants indicating less
ROS were produced under salinity stress (Shalata et al 2001) Similar scenario
was also found between salt-sensitive Plantago media and salt-tolerant P
maritima (Hediye Sekmen et al 2007) The ROS production pattern between
Cakile maritime (halophyte) and Arabidopsis thaliana (glycophyte) also varies
with the latter had continuous increasing of H2O2 concentration during the 72 h
NaCl treatment while H2O2 level of the former declined after 4 h onset of salt
application (Ellouzi et al 2011)
134 Mechanisms for ROS detoxification
Two major types of antioxidants - enzymatic or non-enzymatic - constitute the
major defence mechanism that protect plant cells against oxidative damage by
quenching excessive ROS without converting themselves to deleterious radicals
(Scandalios 1993 Mittler et al 2004 Bose et al 2014b)
Enzymatic mechanisms
The enzymatic components of the antioxidative defence system comprise of
antioxidant enzymes such as superoxide dismutase (SOD) catalase (CAT)
ascorbate peroxidase (APX) peroxidase (POX) glutathione peroxidase (GPX)
monodehydroascorbate reductase (MDAR) dehydroascorbate reductase (DHAR)
and glutathione reductase (GR) (Saed-Moucheshi et al 2014) They are involved
in the process of converting O2- to H2O2 by SOD andor H2O2 to H2O by CAT
ascorbatendashglutathione cycle (Asc-GSH Figure 12) and glutathione peroxidase
cycle (GPX Figure 13) (Apel and Hirt 2004 Asada 2006)
Figure 12 Model of ROS detoxification by Asc-GSH cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Chapter 1 Literature review
11
Figure 13 Model of ROS detoxification by GPX cycle From Apel and Hirt
(2004) Annu Rev Plant Biol 55 373-399
Non-enzymatic mechanisms
Non-enzymic components of the antioxidative defense system comprise
of Asc GSH α-tocopherol carotenoids and phenolic compounds (Apel and Hirt
2004 Ahmad et al 2010 Das and Roychoudhury 2014) They are able to scavenge
the highly toxic ROS such as 1O2 and bullOH protect numerous cellular components
from oxidative damage and influence plant growth and development as well (de
Pinto and De Gara 2004)
14 ROS control over plant ionic homeostasis salinity
stress context
141 ROS impact on membrane integrity and cellular structures
The detrimental effects of excess ROS produced under salinity stress are a
result of their ability to cause lipid peroxidation DNA damage protein
denaturation carbohydrate oxidation pigment breakdown and an impairment of
enzymatic activity (Ozgur et al 2013 Choudhury et al 2017)
Lipid peroxidation occurs when ROS level reaches above the threshold
During this process ROS attack carbon-carbon double bond(s) and the ester linkage
between glycerol and the fatty acid making polyunsaturated fatty acids (PUFAs)
more prone to be attacked Oxidation of lipids is particularly dangerous once
initiated it will propagate free radicals through the ldquochain reactionsrdquo until
termination products are produced (Anjum et al 2015) during which a single bullOH
can result in peroxidation of many PUFAs in irreversible manner (Sharma et al
2012) The main products of lipid peroxidation are lipid hydroperoxides
(LOOH) Among the many different aldehydes terminal products
malondialdehyde (MDA) 4-hydroxy-2-nonenal 4-hydroxy-2-hexenal and acrolein
are taken as markers of oxidative stress (Del Rio et al 2005 Farmer and Mueller
Chapter 1 Literature review
12
2013) The excessively produced ROS especially bullOH can attack the sugar and
base moieties of DNA results in deoxyribose oxidation strand breakage
nucleotides removal DNA-protein crosslinks and nucleotide bases modifications
which may lead to malfunctioned or inactivated encoded proteins (Sharma et al
2012) They also attack and modify proteins directly through nitrosylation
carbonylation disulphide bond formation and glutathionylation (Yamauchi et al
2008) Indirectly the terminal products of lipid peroxidation MDA and 4-
hydroxynonenal are capable of reacting and oxidizing a range of amino acids such
as cysteine and methionine (Davies 2016) The role of carbohydrate oxidation in
stress signalling are obscure and much less studied However this process may be
harmful to plants as well as bullOH can react with xyloglucan and pectin breaking
them down and causing cell wall loosening (Fry et al 2002)
142 ROS control over plant ionic homeostasis
Salinity-induced plasma membrane depolarization (Jayakannan et al 2013)
and generation of ROS (Cuin and Shabala 2008) are the major reasons to cause
cytosolic ion imbalance ROS are capable of activating non-selective cation
channels (NSCCs) and guard cell outward-rectifying K+ channels (GORKs)
inducing ionic conductance and transmembrane fluxes of important ions such as K+
and Ca2+ (Demidchik et al 2003 20072010) Nowadays plant regulatory
networks such as stress perception action of signalling molecules and stimulation
of elongation growth have included ROS-activated channels as key components
The interest in these systems are mainly in linking ions transmembrane fluxes (such
as Ca2+ K+) to the production of ROS Both phenomena are ubiquitous and crucial
for plants as they together control a wide range of physiological and
pathophysiological reactions (Demidchik 2018)
Non-selective cation channels
Plant ROS-activated NSCCs were initially discovered in the charophyte
Nitella flexilis by Demidchik et al (1996 1997ab 2001) who showed that
exposure of intact cells to redox-active transition metals Cu+ and Fe2+ lead to the
production of hydroxyl radicals (bullOH) which induced instantaneous voltage-
independent and non-selective cationic conductance that allow passage of different
cations This idea was then examined in higher plants (Demidchik et al 2003
Chapter 1 Literature review
13
Foreman et al 2003 Inoue et al 2005) with the bullOH generating mixture-activated
cation-selective channels in permeability series of K+ (100) asymp NH4+ (091) asymp Na+
(071) asymp Cs+ (067) gt Ba2+ (032) asymp Ca2+ (024) in Arabidopsis root epidermal cells
The ROS activation of Ca2+-permeable NSCCs in a range of physiological
pathways will be discussed in detail below
K+ permeable channels
ROS are known to activate a certain class of K+ permeable NSCC channels
(Demidchik et al 2003 Shabala and Pottosin 2014) and GORK channels
(Demidchik et al 2010) resulting in massive K+ leak from cytosol and a rapid
decline of the cytosolic K+ pool (Shabala et al 2006) Since maintaining
intracellular K+ homeostasis is essential for turgor maintenance cytosolic pH
homeostasis maintenance enzyme activation protein synthesis stabilization
charge balance and membrane potential formation (Shabala 2003 Dreyer and
Uozumi 2011) the ROS-induced depletion of cytosolic K+ pool compromise these
functions Also it can activate caspase-like proteases and trigger programmed cell
death (PCD) (Shabala 2009) ROS-activated K+ leakage was first detected in the
green alga Chlorella vulgaris treated with copper ions (McBrien and Hassall 1965)
The idea was later extended to root tissue of higher plants Agrostis tenuis
(Wainwright and Woolhouse 1977) and Silene cucubalus (De Vos et al 1989) and
leaf tissue of Avena sativa (Luna et al 1994)
In Arabidopsis studies have shown that exogenous bullOH application to mature
roots can activate cation currents (Demidchik et al 2003) However H2O2-
activated cation currents can only be found when it was added to the cytosolic side
of the PM (Demidchik et al 2007) indicating the existence of a transition metal-
binding site in the cation channel mediating ROS-activated K+ efflux (Rodrigo-
Moreno et al 2013a) Using Metal Detector ver 20 software (Universities of
Florence and Trento Florence Italy) Demidchik et al(2014) identified the putative
CuFe binding sites in CNGC19 and CNGC20 with Cys 102 107 and 110 of
CNGC19 and Cys 133 138 and 141 of CNCG20 coordinating CuFe and
assembling them into the metal-binding sites in a probability close to 100 Given
that bullOH is extremely short-lived and unable to act at a distance gt 1 nm from the
generation site these identified sites may be crucial for the activation of bullOH
Chapter 1 Literature review
14
Guard cells are able to accumulate K+ for stomatal opening (Humble and
Raschke 1971) or release K+ for stomatal closing (MacRobbie 1981) The latter
was then observed with high GORK gene expression levels in Arabidopsis as
suggested by quantitative RT-PCR analyses (Ache et al 2000) and proved to be
mediated by GORK channels (Schroeder 2003 Hosy et al 2003) These
observations demonstrated that GORK channels play a key role in the control of
stomatal movements to allow plant to reduce transpirational water loss during stress
conditions
GORK channels are also highly expressed in root epidermis Using
electrophysiological means Demidchik et al (2003 2010) showed that exogenous
bullOH (generated by the mixture of Cu2+ and ascorbateH2O2) application to
Arabidopsis mature root results in massive K+ efflux which was inhibited in
Arabidopsis K+ channel knockout mutant Atgork1-1 indicating channels mediating
K+ efflux are encoded by the GORK GORK transcription was up-regulated upon
salt stress (Becker et al 2003) which may result from salt-induced ROS
production lead to an increased activity of this channel and massive K+ efflux (Tran
et al 2013) This efflux may operate as a ldquometabolic switchrdquo decreasing metabolic
activity under stress condition by releasing K+ and turn plant cells into a lsquohibernated
statersquo for stress acclimation (Shabala and Pottosin 2014)
SKOR (stellar K+ outward rectifier) channels found within the xylem
parenchyma of root tissue and mediated K+ loadingleaking from root stelar cells
into xylem (Gaymard et al 1998) can be activated by H2O2 through oxidation of
the Cys residue - Cys168 - within the S3 α-helix of the voltage sensor complex This
is very similar to the structure of GORK with its Cys residue exposed to the outside
when the GORK channel is in the open conformation Moreover substitution of
this cysteine moieties in SKOR channels abolished their sensitivity to H2O2
indicating that Cys168 is a critical target for H2O2 which may regulate ROS-
mediated control of the K+ channel in mineral nutrient partitioning in the plant
More recently Michard et al (2017) demonstrated that SKOR may also express in
pollen tube and showed its ROS sensitivity
Ca2+ permeable channels
ROS-induced Ca2+ influx from extracellular space to the cytosol was initially
found in the higher plants dayflower (Price 1990) and tobacco (Price et al 1994)
Chapter 1 Literature review
15
exogenously treated with H2O2 or paraquat (a ROS-generating chemical) The
similar observation was later reported by Demidchik et al (2003 2007) who treated
Arabidopsis mature root protoplast using bullOH-generating mixtures (Cu2+
H2O2ascorbate) or H2O2 and showed that ROS-induced Ca2+ uptake was mediated
by Ca2+-permeable NSCC with channel activation of bullOH is in a direct manner
from the extracellular spaces and H2O2 acts only at the cytosolic side of the mature
root epidermal PM The fact that H2O2 did induce inward Ca2+ currents in
protoplasts isolated from the Arabidopsis elongation root epidermis may indicate
that either Ca2+-permeable NSCCs have different structure andor regulatory
properties between root mature and elongation zones or cells in the latter zones
harbor a higher density of H2O2-permeable aquaporins in their PM allowing H2O2
diffuse into the cytosol (Demidchik and Maathuis 2007)
ROS-activated Ca2+-permeable NSCCs play a key role in mediating stomatal
closure in guard cells (Pei et al 2000) and elongationexpansion of plant cells
(Foreman et al 2003 Demidchik et al 2003 2007) Environmental stresses such
as drought and salt decrease water availability in plants leading to increased
production of ABA in guard cells (Cutler et al 2010 Kim et al 2010) ABA
however is able to stimulate NADPH oxidase-mediated production of H2O2
leading to the activation of Ca2+-permeable NSCCs in the guard cells PM for Ca2+
uptake and mediating stomatal closure (Pei et al 2000 Sah et al 2016) During
this process the PM localized NADPH oxidase can be activated by elevated Ca2+
with its subunit genes AtrbohD and AtrbohF responsible for the subsequent
production of H2O2 (Kwak et al 2003) Moreover the plasma membrane intrinsic
protein 21 (PIP21) aquaporin is likely mediating H2O2 enters into guard cell for
channel activation (Grondin et al 2015) In root tissues the growing root cells
from root hairs and root elongation zones show higher Ca2+-permeable NSCCs
activity than cells from mature zones (Demidchik and Maathuis 2007) This results
in enhanced Ca2+ influx into cytosol of elongating cells which stimulates
actinmyosin interaction to accelerate exocytosis polar vesicle embedment and
sustains cell expansion (Carol and Dolan 2006) In a study conducted by Foreman
et al (2003) the rhd2-1 mutants lacking NADPH oxidase was observed with far
less produced extracellular ROS exhibited stunted expansion in root elongation
zones and formed short root hairs indicating the importance of this process in
mediating cell elongation Similar to guard cell the PM NADPH oxidase in root
Chapter 1 Literature review
16
growing tissues is also responsible for the production of ROS required for the
activation of Ca2+-permeable NSCCs and can be stimulated by elevated cytosolic
Ca2+ (Figure 14) These processes form a self-amplifying lsquoROS- Ca2+ hubrdquo to
enhance and transduce Ca2+ and ROS signals (Demidchik and Shabala 2018) The
same ideas are also applicable for pollen tube growth (Malho et al 2006 McInnis
et al 2006 Potocky et al 2007) The H2O2-activated Ca2+ influx conductance has
been demonstrated in pollen tube protoplasts of pear (Wu et al 2010) and pollen
grain protoplasts of lily (Breygina et al 2016) mediating pollen tube growth and
pollen grain germination The cytosol-localized annexins were proposed to form
Ca2+-permeable channels based on the observation that exogenous application of
corn-derived purified annexin protein to Arabidopsis root epidermal protoplasts
results in elevation of cytosolic free Ca2+ in the latter (Laohavisit et al 2009 2012
Baucher et al 2012)
Figure 14 ROS-activated Ca2+-permeable NSCCs in mediating root elongation
From Demidchik and Maathuis (2007) New Phytol 175 387-404
143 ROS signalling under stress conditions
ROS have long been known as toxic by-products in aerobic metabolism
(Mittler et al 2017) However ROS produced in organelles or through PM
Chapter 1 Literature review
17
NADPH oxidase under stress conditions can act as beneficial signal transduction
molecules to activate acclimation and defence mechanisms in plants to counteract
stress-associated oxidative stress (Mittler et al 2004 Miller et al 2008) During
these processes ROS signals may either be limited within cells between different
organelles by (non-)enzymatic AO or auto-propagated to transfer rapidly between
cells for a long distance throughout the plant (Miller et al 2009) The latter signal
is mainly generated by H2O2 due to its long half-life (1 ms) thus can accumulate to
high concentrations (Cheeseman 2006 Moslashller et al 2007) or diffuse freely
through peroxiporin membrane channels to adjacent subcellular compartments and
cross neighbouring cells (Neill et al 2002) However plant cells contain different
cellular compartments with specific sets of stress proteins H2O2 generated in these
sites process identical properties which unable to distinguish the particular
stimulus to selectively regulate nuclear genes and trigger an appropriate
acclimation response (Moslashller and Sweetlove 2010 Mittler et al 2011) This may
attribute to the associated functioning of ROS signal with other signals such as
peptides hormones lipids cell wall fragments or the ROS signal itself carries a
decoded message to convey specificity (Mittler et al 2011)
Besides ROS signalling generated under salt stress condition can also trigger
acclimation responses in association with other well-established cellular signalling
components such as plant hormone (eg ABA - abscisic acid SA - salicylic acid
JA - jasmonate ET - ethylene BR - brassinosteroid GA - gibberellin and SL -
strigolactone) Ca2+ NO and H2 (Bari and Jones 2009 Jin et al 2013 Xu et al
2013 Nakashima and Yamaguchi-Shinozaki 2013 Xie 2014 Xia et al 2015
Mignolet-Spruyt et al 2016)
15 Linking salinity and oxidative stress tolerance
Salinity stress in plants reduces cell turgor and induces entry of large amount
of Na+ into cytosol Mechanisms such as osmotic adjustment and Na+ exclusion
were used by plants in maintaining cell turgor pressure and minimizing sodium
toxicity which has long been taken as the major components of salinity stress
tolerance However excessive ROS production always accompanies salinity stress
making oxidative stress tolerance the third component of salinity stress tolerance
Therefore revealing the mechanism of oxidative stress tolerance in plants and
Chapter 1 Literature review
18
linking it with salinity stress tolerance may open new avenue in breeding
germplasms with improved salinity stress tolerance
151 Genetic variability in oxidative stress tolerance
Plants exhibit various abilities to oxidative stress tolerance due to their genetic
variability in stress response It has been shown that the existence of genetic
variability in stress tolerance is due to the existence of differential expression of
stress‐responsive genes it is also an essential factor for the development of more
tolerant cultivars (Senthil‐Kumar et al 2003 Bita and Gerats 2013) Since
oxidative stress is one of the components of salinity stress the genetic variability
for tolerance to oxidative stress present in plants could be exploited to screen
germplasm and select cultivars that exhibit superior salinity stress tolerance This
promotes a need to establish a link between oxidative stress and salinity stress
tolerance
Plants biochemical markers such as antioxidants levelactivities (eg SOD
APX CAT ndash Maksimović et al 2013 total phenolic compounds flavonoids ndash
Dbira et al 2018) the extend of oxidative damage or lipid peroxidation (eg MDA
level Gόmez et al 1999 Hernandez et al 2001 Liu and Huang 2000 Suzuki and
Mittler 2006) and physiological markers such as chlorophyll content (Kasajima
2017) have been used for oxidative stress tolerance in lots of studies These markers
were also tested as a tool for salt tolerance screening in Kunth (Luna et al 2000)
the pasture grass Cenchrus ciliaris L (Castelli et al 2010) and barley (Maksimović
et al 2013) In this case targeting oxidative stress tolerance may help breeders
achieve salinity stress tolerance and genetic variation in oxidative stress tolerance
among a wide range of varieties is ideal for the identification of QTLs (quantitative
trait loci) which was often quantified by AO activity as a simple measure Indeed
enhanced AO (especially the enzymatic AO) activity has been frequently
mentioned as a major trait of oxidative stress tolerance in plants and a range of
publication have revealed positive correlation between AO activity and salinity
stress tolerance in major crop plants such as wheat (El-Bastawisy 2010 Bhutta
2011) rice (Vaidyanathan et al 2003) maize (Azooz et al 2009) tomato (Mittova
et al 2002) and canola (Ashraf and Ali 2008) However the above link is not as
straightforward as one may expect because ROS have dual role either as beneficial
Chapter 1 Literature review
19
second messengers or toxic by-products making them have pleiotropic effects in
plants (Bose et al 2014b) This may be the reason why no or negative correlation
between oxidative and salinity stress were revealed in a range of plant species such
as barley (Fan et al 2014) rice (Dionisio-Sese and Tobita 1998) radish (Noreen
and Ashraf 2009) and turnip (Noreen et al 2010) Moreover Frary et al (2010)
identified 125 AO QTLs associated with salinity stress tolerance in a tomato
introgression line indicating that the use of this trait is practically unfeasible This
prompts a need to find other physiological markers for oxidative stress tolerance
and link them with salinity stress tolerance in cereals Previous studies from our
laboratory reported that H2O2-induced K+ flux from root mature zone were
markedly different showed genetic variability between two barley varieties
contrasting in their salinity stress tolerance (Chen et al 2007a Maksimović et al
2013) with the salt tolerant variety leaking less K+ than its sensitive counterpart
indicating the possibility of using this trait as a novel physiological marker for
oxidative stress tolerance
152 Tissue specificity of ROS signalling and tolerance
The signalling role of ROS in regulating plant responses to abiotic and biotic
stress have been characterized mainly functioning in leaves andor roots (Maruta et
al 2012) Due to the cell type specificity in these tissues their ROS production
pathways vary with chloroplasts and peroxisomes the major generation site in
leaves and mitochondria being responsible for this process in roots (Foyer and
Noctor 2003 Rhoads et al 2006 Navrot et al 2007) Stress-induced ROS
generation in these organelles are capable of triggering a cascade of changes in the
nuclear transcriptome and influencing gene expression by modifying transcription
factors (Apel and Hirt 2004 Laloi et al 2004) However it is now believed that
the roles of ROS signalling are attributed to the differences of RBOHs (respiratory
burst oxidase homologues also known as NADPH oxidases) regulation in various
signal transduction pathways activated in assorted tissue and cell types under stress
conditions (Baxter et al 2014)
NADPH oxidases-derived ROS are known to activate a range of ion channels
to perform their signalling roles The most frequently mentioned example is H2O2-
induced stomatal closure in plant guard cells via the activation of Ca2+-permeable
NSCCs under stress conditions which has been detailed in the previous section
Chapter 1 Literature review
20
regarding Ca2+-permeable channel This indicates a link between ROS and Ca2+
signalling network as the flux kinetics of the latter ion (uptake into cytosol) is
known as the early signalling events in plants in response to salinity stress (Baxter
et al 2014) Similar mechanism can be found in growing tissues (ie root tips root
hairs pollen tubes) under normal growth condition where elevated cytosolic Ca2+
induced by ROS facilitates exocytosis to sustains cell expansion and elongation
(Demidchik and Maathuis 2007)
ROS activated K+ efflux from the cytosol is also of great significance In leaves
this phenomenon plays key role in mediating stress-associated stomatal closure
(MacRobbie 1981) In root tissues ROS-induced K+ efflux is several-fold higher
of magnitude in elongation root zone compared with the mature root zone
(Demidchik et al 2003 Adem et al 2014) which probably indicated that there
are major differences in ROS productiondetoxification pattern or ROS-sensitive
channelstransporters between the two root zones (Shabala et al 2016) Besides
ROS-induced K+ efflux from root epidermis was in a dose-dependent manner (Cuin
and Shabala 2007) and it was shown that salt-induced accumulation of ROS in
barley root was highly tissue specific and observed only in root elongation zone
indicating that the increased production of ROS in elongation zone may be able to
induce greater K+ loss (Shabala et al 2016) This phenomenon may be the reason
of elongation root zone with higher salt sensitivity However ROS-induced higher
K+ efflux in this tissue may be of some specific benefits As per Shabala and Potosin
(2014) the massive K+ leakage from the young active root apex results in a decline
of cytosolic K+ content which may enable cells transition from normal metabolism
to a ldquohibernated staterdquo during the first stage of salt stress onset This mechanism
may be essential for cells from this root zone to reallocate their ATP pool towards
stress defence responses (Shabala 2017)
16 Aims and objectives of this study
161 Aim of the project
As discussed in this chapter oxidative stress is one of the components of
salinity stress and the previous studies on the relationship between salinity and
oxidative stress were largely focused on the antioxidant system in conferring
salinity stress tolerance ignoring the fact that ROS are essential molecules for plant
Chapter 1 Literature review
21
development and play signalling role in plant biology Until now applying major
enzymatic AOs level as the biochemical markers of salinity stress tolerance have
been explored in cereals However the attempts to identify specific genes
controlling the above process have been not characterised Therefore our main aim
in this study was to establish a causal link between oxidative stress and salinity
stress tolerance in cereals by other means (such as MIFE microelectrode ion flux
estimation) develop a convenient inexpensive and quick method for crop
screening and pyramid major oxidative stress-related QTLs in association with
salinity stress tolerance
It has been commonly known that excessive ROS in plant tissues can be
destructive to key macro-molecules and cellular structures However ROS impact
on plant ionic homeostasis may occur well before such damage is observed
Electrophysiological methods have demonstrated that ROS are able to activate a
broad range of ion channels resulting in disequilibrium of the cytosolic ions pools
and leading to the occurrence of PCD The major ions involved in ROS activation
are K+ and Ca2+ as retention of the former and elevation of the latter ion in cytosol
under stress conditions has been widely reported in salinity stress studies Therefore
the ROS-induced K+ and Ca2+ fluxes ldquosignaturesrdquo may be used as prospective
physiological markers in breeding programs aimed at improving salinity stress
tolerance In order to validate this hypothesis and develop high throughput
phenotyping methods for oxidative stress tolerance in cereals this work employed
electrophysiological methods (specifically non-invasive microelectrode ion flux
estimation MIFE technique) to measure ROS-induced K+ and Ca2+ fluxes in a
range of barley and wheat varieties Our ultimate aim is to link kinetics of ion flux
responses with salinity stress tolerance and provide breeders with appropriate tools
and novel target traits to be used in genetic improvement of the salinity tolerance
in cereal crops
In the light of the above four main objectives of this project were as follows
1) To investigate a suitability of the non-invasive MIFE (microelectrodes
ion flux measurements) technique as a proxy for oxidative stress tolerance in
cereals
Chapter 1 Literature review
22
The main objective of this work was to establish a causal link between
oxidative stress and salinity stress tolerance and then determine the most suitable
parameter(s) to be used as a physiological marker in future studies
2) To validate developed MIFE protocols and reveal the identity of ions
transport system in cereals mediating ROS-induced ion fluxes
In this part a large number of contrasting barley bread wheat and durum
wheat accessions were used Their ROS-induced Ca2+ and K+ fluxes from specific
root zones were acquired and correlated with their overall salinity stress tolerance
The pharmacological experiments were conducted using different channel blockers
andor specific enzymatic inhibitors to investigate the role of specific transport
systems as downstream targets of salt-induced ROS signalling
3) To map QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
The main objective of this part was to identify major QTLs controlling ROS-
induced K+ and Ca2+ fluxes with the premise of revealing a causal correlation
between oxidative stress and salinity stress tolerance in barley Data for QTL
analysis were acquired from a double haploid barley population (eg derived from
CM72 and Gairdner) using the developed MIFE protocols
4) To develop a simple and reliable high-throughput phenotyping method to
replace the complicated MIFE technique for screening
Several simple alternative high-throughput assays were developed and
assessed for their suitability in screening germplasm for oxidative stress tolerance
as a proxy for the skill-demanding electrophysiological MIFE methods
162 Outline of chapters
Chapter 1 Literature review
Chapter 2 General materials and methods
Chapter 3 Hydrogen peroxide-induced root Ca2+ and K+ fluxes correlate with
salt tolerance in cereals towards the cell-based phenotyping
Chapter 4 Validating using MIFE technique-measured H2O2-induced ion
fluxes as physiological markers for salinity stress tolerance breeding in wheat and
barley
Chapter 1 Literature review
23
Chapter 5 QTLs for ROS-induced ions fluxes associated with salinity stress
tolerance in barley
Chapter 6 Developing a high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
Chapter 7 General discussion and future prospects
Chapter 2 General materials and methods
24
Chapter 2 General materials and methods
21 Plant materials
All the cereal genotypes used in this research were acquired from the
Australian Winter Cereal Collection and reproduced in our laboratory These
include a range of barley bread wheat and durum wheat varieties and a double
haploid (DH) population originated from the cross of two barley varieties CM72
and Gairdner
22 Growth conditions
221 Hydroponic system
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were grown in basic
salt medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in aerated hydroponic
system in darkness at 24 plusmn 1 for 4 days Seedlings with root length between 60
and 80 mm were used in all the electrophysiological experiments in this study
222 Paper rolls
Seeds were surface sterilized with ten-fold diluted commercial bleach for 10
min and then rinsed thoroughly with tap water Sterilized seeds were germinated in
Petri dishes on wet filter paper for 1 d Uniformly germinated seeds were then
chosen placed in paper rolls (Pandolfi et al 2010) and grown in a basic salt
medium (BSM 01 mM CaCl2 and 05 mM KCl pH 56) in darkness at 24 plusmn 1
for another 3 d
23 Microelectrode Ion Flux Estimation (MIFE)
231 Ion-selective microelectrodes preparation
Net ion fluxes were measured with ion-selective microelectrodes non-
invasively using MIFE technique (University of Tasmania Hobart Australia)
(Newman 2001) Blank microelectrodes were pulled out from borosilicate glass
capillaries (GC150-10 15 mm OD x 086 mm ID x 100 mm L Harvard Apparatus
Chapter 2 General materials and methods
25
UK) using a vertical puller then dried at 225 overnight in an oven and then
silanized with chlorotributylsilane (282707-25G Sigma-Aldrich Sydney NSW
Australia) Silanized electrode tips were flattened to a diameter of 2 - 3 microm and
backfilled with respective backfilling solutions (200 mM KCl for K+ and 500 mM
CaCl2 for Ca2+) Electrode tips were then front-filled with respective commercial
ionophore cocktails (Cat 99311 for K+ and 99310 for Ca2+ Sigma-Aldrich) Filled
microelectrodes were mounted in the electrode holders of the MIFE set-up and
calibrated in a set of respective calibration solutions (250 500 1000 microM KCl for
calibrating K+ electrode and 100 200 400 microM CaCl2 for calibrating Ca2+ electrode)
before and after measurements Electrodes with a slope of more than 50 mV per
decade for K+ and more than 25 mV per decade for Ca2+ and correlation
coefficients of more than 09990 have been used
232 Ion flux measurements
Net fluxes of Ca2+ and K+ were measured from mature (2 - 3 cm from root
apex) and elongation (1 - 2 mm from root apex) root zones To do this plant roots
were immobilized in a measuring chamber containing 30 ml of BSM solution and
left for 40 min adaptation prior to the measurement The calibrated electrodes were
co-focused and positioned 40ndash50 microm away from the measuring site on the root
before starting the experiment After commencing a computer-controlled stepper
motor (hydraulic micromanipulator) moved microelectrodes 100 microm away from the
site and back in a 12 s square-wave manner to measure electrochemical gradient
potential between two positions The CHART software was used to acquire data
(Shabala et al 1997 Newman 2001) and ion fluxes were then calculated using the
MIFEFLUX program (Newman 2001)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
26
Chapter 3 Hydrogen peroxide-induced root Ca2+
and K+ fluxes correlate with salt tolerance in
cereals towards the cell-based phenotyping
31 Introduction
Salinity stress is one of the major environmental constraints limiting crop
production worldwide that results in massive economic penalties especially in arid
and semi-arid regions (Schleiff 2008 Shabala et al 2014 Gorji et al 2015)
Because of this plant breeding for salt tolerance is considered to be a major avenue
to improve crop production in salt affected regions (Genc et al 2016) According
to the classical view two major components - osmotic stress and specific ion
toxicity - limit plant growth in saline soils (Deinlein et al 2014) Unsurprisingly
in the past decades many attempts have been made to target these two components
in plant breeding programs The major efforts were focused on either improving
plant capacity to exclude Na+ from uptake by targeting SOS1 (Martinez-Atienza et
al 2007 Xu et al 2008 Feki et al 2011) and HKT1 (Munns et al 2012 Byrt et
al 2014 Suzuki et al 2016) genes or increasing de novo synthesis of organic
osmolytes for osmotic adjustment (Sakamoto et al 1998 Sakamoto and Murata
2000 Wani et al 2013) However none of these approaches has resulted in truly
tolerant crops in the farmersrsquo fields and even the best performing genotypes created
showed a 50 of yield loss when grown under saline conditions (Munns et al
2012)
One of the reasons for the above detrimental effects of salinity on plant growth
is the overproduction and accumulation of reactive oxygen species (ROS) under
saline condition (Miller et al 2010 Bose et al 2014) The increasing level of ROS
in green tissues under saline condition results from the impairment of the
photosynthetic apparatus and a limited capability for CO2 assimilation in a
conjunction with plantrsquos inability to fully utilize light captured by photosynthetic
pigments (Biswal et al 2011 Ozgur et al 2013) However the leaf is not the only
site of ROS generation as they can also be produced in root tissues under saline
condition (Luna et al 2000 Mittler 2002 Miller et al 2008 2010 Turkan and
Demiral 2009) In Arabidopsis roots increasing hydroxyl radicals (OH)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
27
(Demidchik et al 2010) and H2O2 (Xie et al 2011) levels were observed under
salt stress Accumulation of NaCl-induced H2O2 was also observed in rice (Khan
and Panda 2008) and pea roots (Bose et al 2014c)
When ROS are accumulated in excessive quantities in plant tissues significant
damage to key macromolecules and cellular structures occurs (Vellosillo et al
2010 Karuppanapandian et al 2011) However the disturbance to cell metabolism
(and associated growth penalties) may occur well before this damage is observed
ROS generation in root tissues occurs rapidly in response to salt stimuli and leads
to the activation of a broad range of ion channels including Na+-permeable non-
selective cation channels (NSCCs) and outward rectifying efflux K+ channels
(GORK) This results in a disequilibrium of the cytosolic ions pools and a
perturbation of cell metabolic processes When the cytosolic K+Na+ ratio is shifted
down beyond some critical threshold the cell can undergo a programmed cell death
(PCD) (Demidchik et al 2014 Shabala and Pottosin 2014) Taken together these
findings have prompted an idea of improving salinity stress tolerance via enhancing
plant antioxidant activity (Kim et al 2005 Hasanuzzaman et al 2012) However
despite numerous attempts (Dionisio-Sese and Tobita 1998 Sairam et al 2005
Gill and Tuteja 2010) the practical outcomes of this approach are rather modest
(Allen 1995 Rizhsky et al 2002)
One of the reasons for the above failure to improve plant stress tolerance via
constitutive expression of enzymatic antioxidants is the fact that ROS also play an
important signaling role in plant adaptive and developmental responses (Mittler
2017) Therefore scavenging ROS by constitutive expression of enzymatic
antioxidants (AOs) may interfere with these processes and cause pleiotropic effects
As a result the reported association between activity of AO enzymes and salinity
stress tolerance is often controversial (Maksimović et al 2013) and the entire
concept ldquothe higher the AO activity the betterrdquo does not hold in many cases
(Mandhania et al 2006 Noreen and Ashraf 2009a Seckin et al 2009)
ROS are known to activate Ca2+ and K+-permeable plasma membrane channels
in root epidermis (Demidchik et al 2003) resulting in elevated Ca2+ and depleted
K+ pool in the cytosol with a consequent disturbance to intracellular ion homeostasis
A pivotal importance of K+ retention under salinity stress is well known and has been
widely reported to correlate positively with the overall salinity tolerance in roots of
both barley and wheat as well as many other species (reviewed by Shabala 2017)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
28
Elevation in the cytosolic free Ca2+ is also observed in response to a broad range of
abiotic and biotic stimuli and has long been considered an essential component of
cell stress signaling mechanism (Chen et al 2010 Bose et al 2011 Wang et al
2013) In the light of the above and given the dual role of ROS and their involvement
in multiple signaling transduction pathways (Mittler 2017) should salt tolerant
species and genotypes be more or less sensitive to ROS Is this sensitivity the same
for all tissues or does it show some specificity Can the magnitude of the ROS-
induced ion fluxes across the plasma membrane be used as a physiological marker in
breeding programs to improve plant salinity stress tolerance To the best of our
knowledge none of the previous studies has examined ROS-sensitivity of ion
transporters in the context of tissue-specificity or explored a causal link between two
types of ROS applied and stress-induced changes in plant ionic homeostasis in the
context of salinity stress tolerance This gap in our knowledge was addressed in this
work by employing the non-invasive microelectrode ion flux estimation (MIFE)
technique and investigating the correlation between oxidative stress-induced ion
responses and plantrsquos overall salinity stress tolerance
32 Materials and methods
321 Plant materials and growth conditions
Eight barley (seven Hordeum vulgare L and one H vulgare ssp Spontaneum)
and six wheat (bread wheat Triticum aestivum) varieties contrasting in salinity
tolerance were used in this study The list of cultivars is shown in Table 31
Seedlings for experiment were grown in hydroponic system (see section 221 for
details)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
29
Table 31 List of barley and wheat varieties used in this study Scores represent
quantified damage degree of cereals under salinity stress reported as damage
index score from 0 to 10
Barley Wheat
Tolerant Sensitive Tolerant Sensitive
Varieties Score Varieties Score Varieties Score Varieties Score
SYR01 025 Gairdner 400 Titmouse S 183 Seville20 383
TX9425 100 ZUG403 575 Cranbrook 250 Iran118 417
CM72 125 Naso Nijo 750 Westonia 300 340 550
ZUG293 175 Unicorn 950
0 - highest overall salinity tolerance 10 - lowest level of salt tolerance Data collected from
our previous study from Wu et al 2014 2015
322 K+ and Ca2+ fluxes measurements
All details for ion-selective microelectrodes preparation and ion flux
measurements protocols are available in the section 23
323 Experimental protocols for microelectrode ion flux estimation
(MIFE) measurements
Two types of ROS were tested - hydrogen peroxide (H2O2) and hydroxyl
radicals (OH) A final working concentration of H2O2 in BSM was achieved by
adding H2O2 stock to the measuring chamber As the half-life of H2O2 in the
absence of transition metals is of an order of magnitude of several (up to 10) hours
(Yazici and Deveci 2010) and the entire duration of our experiments did not exceed
30 min one can assume that bath H2O2 concentration remained stable during
measurements A mixture of coppersodium ascorbate (CuA 0310 mM) was
used to generate OH (Demidchik et al 2003) The measuring solution containing
05 mM KCl and 01 mM CaCl2 was buffered with 4mM MESTris to achieve pH
56 Net Ca2+ and K+ fluxes were measured from mature and elongation zones of a
root for 4 to 5 min to ensure the stability of initial ion fluxes Then a stressor (either
H2O2 or OH) was added to the bath and Ca2+ and K+ fluxes were acquired for
another 20 min The first 30 ndash 60 s after adding the treatment solution (H2O2 or
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
30
CuA mixture) were discarded during data analyses in agreement with the MIFE
theory that requires non-stirred conditions (Newman 2001)
324 Quantifying plant damage index
The extent of plant salinity tolerance was quantified by allocating so-called
ldquodamage index scorerdquo to each plant The use of such damage index is a widely
accepted practice by plant breeders (Zhu et al 2015 Wu et al 2014 2015) This
index is based on evaluation of the extent of leaf chlorosis and plant survival rate
and relies on the visual assessment of plant performance after about 30 days of
exposure to high salinity The score ranges between 0 (no stress symptoms) and 10
(completely dead plant) and it was shown before that the damage index score
correlated strongly with the grain yield under stress conditions (Zhu et al 2015)
325 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at p lt 005 level
33 Results
331 H2O2-induced ion fluxes are dose-dependent
Two parameters were identified and analyzed from transient response curves
(Figure 31) The first one was peak value defined as the maximum flux value
measured after the treatment and the second was the end value defined as a
baseline flux 20 min after the treatment application
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
31
Figure 31 Descriptions (see inserts in each panel) of cereal root ion fluxes in
response to H2O2 and hydroxyl radicals (OH) in a single experiment (AB) Ion
flux kinetics in root elongation zone (A) and mature zone (B) in response to
H2O2 (CD) Ion flux kinetics in root elongation zone (C) and mature zone (D)
in response to OH Two distinctive flux points were identified in kinetics of
responses peak value-identified as a maximum flux value measured after a
treatment end value-identified 20 min after the treatment application An arrow
in each panel represents when oxidative stress was imposed
Two barley varieties (TX9425 salinity tolerant Naso Nijo salinity sensitive)
were used for optimizing the dosage of H2O2 treatment Accordingly TX9425 and
Naso Nijo roots were treated with 01 03 10 30 and 10 mM H2O2 and ion fluxes
data were acquired from both root mature and elongation zones for 15 min after
application of H2O2 We found that except for 01 mM all the H2O2 concentrations
triggered significant ion flux responses in both root zones (Figures 32A 32B and
33A 33B) In the elongation root zone an initial K+ efflux (negative flux values
Figure 32A) and Ca2+ uptake (positive flux values Figure 33A) were observed
Application of H2O2 to the root led to a more intensive K+ efflux and a reduced Ca2+
influx (the latter turned to efflux when concentration of H2O2 was ge 1 mM) (Figures
32A and 33A) In the mature root zone the initial K+ uptake (Figure 32B) and Ca2+
efflux (Figure 33B) were observed Application of H2O2 to the bath led to a dramatic
K+ efflux and Ca2+ uptake (Figures 32B and 33B) Ca2+ flux has returned to pre-
stress level after reaching a peak (Figures 33A 33B) Fluxes of K+ however
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
32
remained negative after reaching the respective peak (Figure 32A 32B) The time
required to reach a peak increased with an increase in H2O2 concentration (Figures
32A 32B and 33A 33B)
The peak values for both Ca2+ and K+ fluxes showed a clear dose-dependency
for H2O2 concentrations used (Figures 32C 32D and 33C 33D) The biggest
significant difference (p ˂ 005) in ion flux responses of contrasting varieties was
observed at 10 mM H2O2 for both K+ (Figure 32C 32D) and Ca2+ fluxes (Figure
33C 33D) Accordingly 10 mM H2O2 was chosen as the most suitable
concentration for further experiments
Figure 32 (AB) Net K+ fluxes measured from barley variety TX9425 root
elongation zone (A) - about 1 mm from the root tip and mature zone (B) - about
30mm from the root tip with respective H2O2 concentrations (CD) Dose-
dependency of H2O2-induced K+ fluxes from root elongation zone (C) and
mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks indicate
statistically significant differences between two varieties ( p lt 005 Studentrsquos
t-test) Responses from Naso Nijo were qualitatively similar to those shown for
TX9425
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
33
Figure 33 (AB) Net Ca2+ fluxes measured from barley variety TX9425 root
elongation zone (A) and mature zone (B) with respective H2O2 concentrations
(CD) Dose-dependency of H2O2-induced Ca2+ fluxes from root elongation zone
(C) and mature zone (D) Error bars are means plusmn SE (n = 6 minus 8) Asterisks
indicate statistically significant differences between two varieties ( p lt 005
Studentrsquos t-test) Responses from Naso Nijo were qualitatively similar to those
shown for TX9425
332 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
barley
Once the optimal H2O2 concentration was chosen eight barley varieties
contrasting in their salt tolerance (see Table 31) were tested for their ability to
maintain K+ and Ca2+ homeostasis under 10 mM H2O2 treatment (Figures 34 and
35) The kinetics of K+ flux responses were qualitatively similar and the
magnitudes were dramatically different between mature and elongation zones as
well as between the varieties tested (Figure 34A 34B) Highest and smallest peak
and end fluxes of K+ were observed in Naso Nijo and CM72 respectively in the
elongation root zone (Figure 34C 34D) The same trend was found in the mature
root zone for K+ peak fluxes with a small difference in K+ end fluxes where the
highest flux was observed in another cultivar Unicorn (Figure 34E 34F) Ca2+
peak flux responses varied among cultivars (Figure 35A 35B) with the highest
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
34
and smallest Ca2+ fluxes observed in SYR01 and Gairdner in elongation zone
(Figure 35C) and Naso Nijo and ZUG403 in mature zone (Figure 35D)
We then used a quantitative scoring system (Wu et al 2015) to correlate the
magnitude of measured flux responses with the salinity tolerance of each genotype
The overall salinity tolerance of barley was quantified as a damage index score
ranging between 0 and 10 with 0 representing most tolerant and 10 representing
most sensitive variety (Table 31) Peak and end flux values of K+ and Ca2+ were
then plotted against respective tolerance scores A significant (p lt 005) positive
correlation was found between H2O2-induced K+ efflux (Figure 34I 34J) the Ca2+
uptake (Figure 35F) and the salinity damage index score in the mature root zone
At the same time no correlation was found in the elongation zone for either K+
(Figure 34G 34H) or Ca2+ flux (Figure 35E)
Figure 34 Kinetics of K+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6 minus 8) (CDGH) Peak (C)
and end (D) K+ fluxes of eight barley varieties in response to 10 mM H2O2 and
their correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of eight barley varieties in response to
10 mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
35
Figure 35 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of eight barley varieties in response to 10 mM H2O2 and their correlation
with damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of
eight barley varieties in response to 10 mM H2O2 and their correlation with
damage index (F) in root mature zone
333 Genotypic variation in H2O2-induced Ca2+ and K+ fluxes in
wheat
Six wheat varieties contrasting in their salt tolerance were used to check
whether the above trends observed in barley are also applicable to wheat species
Transient K+ and Ca2+ flux responses to 10 mM H2O2 in wheat were qualitatively
identical to those measured from barley roots in both zones (Figures 36A 36B
and 37A 37B) When peak and end flux values were plotted against the salinity
damage index (Table 31 Wu et al 2014) a strong positive correlation was found
between H2O2-induced K+ (Figure 36E 36F) and Ca2+ (Figure 37D) fluxes and
the overall salinity tolerance (Table 31) in wheat root mature zone (p lt 001 for
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
36
Figure 36I 36J p lt 005 for Figure 37F) Similar to barley no correlation was
found between salt damage index (Table 31) and the magnitude of ion flux
responses (Figures 36C 36D and 37C) in the root elongation zone of wheat
(Figures 36G 36H and 37E)
Figure 36 Kinetics of K+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and
end (D) K+ fluxes of six wheat varieties in response to 10 mM H2O2 and their
correlation with damage index (GH respectively) in root elongation zone
(EFIJ) Peak (E) and end (F) K+ fluxes of six wheat varieties in response to 10
mM H2O2 and their correlation with damage index (IJ respectively) in root
mature zone
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
37
Figure 37 Kinetics of Ca2+ fluxes from three representative wheat varieties in
response to 10 mM H2O2 treatment from both root elongation zone (A) and
mature zone (B) Error bars are means plusmn SE (n = 6minus8) (CE) Peak Ca2+ fluxes
(C) of six wheat varieties in response to 10 mM H2O2 and their correlation with
damage index (E) in root elongation zone (DF) Peak Ca2+ fluxes (D) of six
wheat varieties in response to 10 mM H2O2 and their correlation with damage
index (F) in root mature zone
Taken together the above results suggest that the H2O2-induced fluxes of Ca2+
and K+ in mature root zone correlate well with the damage index but no such
correlation exists in the elongation zone
334 Genotypic variation of hydroxyl radical-induced Ca2+ and
K+ fluxes in barley
Using eight barley varieties listed in Table 31 we then repeated the above
experiments using a hydroxyl radical the most aggressive ROS species of which
can be produced during Fenton reaction between transition metal and ascorbate
(Halliwell and Gutteridge 2015) Hydroxyl radicals (OH) were generated by
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
38
applying 0310 mM Cu2+ascorbate mixture (Demidchik et al 2003) This
treatment caused a dramatic K+ efflux (6ndash8 fold greater than the treatment with
H2O2 data not shown) with fluxes reaching their peak efflux magnitude after 3 to
4 min of stress application in elongation zone and 7 to 13 min in the mature zone
(Figure 38A 38B) The mean peak values ranged from minus3686 plusmn 600 to minus8018 plusmn
536 nmol mminus2middotsminus1 and from minus7669 plusmn 27 to minus11930 plusmn 619 nmolmiddotmminus2middotsminus1 respectively
for the two zones (data not shown)
Figure 38 Kinetics of K+ fluxes from three representative barley varieties in
response to 031 OH treatment from both root elongation zone (A) and mature
zone (B) Error bars are means plusmn SE (n = 6minus8) (CDGH) Peak (C) and end (D)
K+ fluxes of eight barley varieties in response to OH and their correlation with
damage index (GH respectively) in root elongation zone (EFIJ) Peak (E)
and end (F) K+ fluxes of eight barley varieties in response to OH and their
correlation with damage index (IJ respectively) in root mature zone
Contrary to H2O2 treatment a dramatic and instantaneous net Ca2+ efflux was
observed in both zones immediately after application of OH-generation mixture to
the bath (Figure 39A 39B) This Ca2+ efflux was short lived and net Ca2+ influx
was measured after about 2 min from elongation and after 8 min from mature root
zones respectively (Figure 39A 39B) No significant correlation between overall
salinity tolerance (damage index see Table 31) and either Ca2+ or K+ fluxes in
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
39
response to OH treatment was found in either zone (Figures 38G - 38J and 39E
39F)
Figure 39 Kinetics of Ca2+ fluxes from three representative barley varieties in
response to 031 mM Cu2+ascorbate (OH) treatment from both root
elongation zone (A) and mature zone (B) Error bars are means plusmn SE (n = 6minus8)
(CE) Peak Ca2+ fluxes (C) of eight barley varieties in response to OH and their
correlation with damage index (E) in root elongation zone (DF) Peak Ca2+
fluxes (D) of eight barley varieties in response OH and their correlation with
damage index (F) in root mature zone
34 Discussion
ROS are the ldquodual edge swordsrdquo that are essential for plant growth and
signaling when they are maintained at the non-toxic level but that can be
detrimental to plant cells when ROS production exceeds a certain threshold (Mittler
2017) This is particularly true for the role of ROS in plant responses to salinity
Salt-stress induced ROS production is considered to be an essential step in
triggering a cascade of adaptive responses including early stomatal closure (Pei et
al 2000) control of xylem Na+ loading (Jiang et al 2012 Zhu et al 2017) and
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
40
sodium compartmentalization (de la Garma et al 2015) At the same time
excessive ROS accumulation may have negative impact on intracellular ionic
homeostasis under saline conditions Of specific importance is ROS-induced
cytosolic K+ loss that stimulates protease and endonuclease activity promoting
program cell death (Demidchik et al 2010 2014 Shabala and Pottosin 2014
Hanin et al 2016) Here in this study we show that ROS regulation of ion fluxes
is highly plant tissue-specific and differs between various ROS species
341 The magnitude of the hydroxyl radical-induced K+ and Ca2+
fluxes does not correlate with salinity stress tolerance in barley
Hydroxyl radicals (OH) are considered to be very short-lived (half-life of 1
ns) and highly aggressive agents that are a prime cause of oxidative damage to
proteins and nucleic acids as well as lipid peroxidation during oxidative stress
(Demidchik 2014) At physiologically relevant concentrations they have the
greatest potency to induce activation of Ca2+ and K+ channels leading to massive
fluxes of these ions across cellular membranes (Demidchik et al 2003 2010) with
detrimental effects on cell metabolism This is clearly demonstrated by our data
showing that OH-induced K+ efflux was an order of magnitude stronger compared
with that induced by H2O2 for the appropriate variety and a root zone (eg Figures
34 and 38) Due to their short life they can diffuse over very short distances (lt 1
nm) (Sies 1993) and thus are less suitable for the role of the signaling molecules
Importantly OH cannot be scavenged by traditional enzymatic antioxidants and
the control of OH level in cells is achieved via an elaborate network of non-
enzymatic antioxidants (eg polyols tocopherols polyamines ascorbate
glutathione proline glycine betaine polyphenols carotenoids reviewed by Bose
et al 2014b) It was shown that exogenous application of some of these non-
enzymatic antioxidants prevented OH-induced K+ efflux from plant cells (Cuin
and Shabala 2007) and resulted in improved salinity stress tolerance (Ashraf and
Foolad 2007 Chen and Murata 2008 Pandolfi et al 2010) Thus an ability of
keeping OH levels under control appears to be essential for plant survival under
salt stress conditions and all barley genotypes studied in our work appeared to
possess this ability (although most likely by different means)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
41
A recent study from our laboratory (Shabala et al 2016) has shown that higher
sensitivity of the root apex to salinity stress (as compared to mature root zone) was
partially explained by the higher population of OH-inducible K+-permeable efflux
channels in this tissue At the same time root apical cells responses to salinity stress
by a massive increase in the level of allantoin a substance with a known ability to
mitigate oxidative damage symptoms (Watanabe et al 2014) and alleviate OH-
induced K+ efflux from root cells (Shabala et al 2016) This suggests an existence
of a feedback mechanism that compensates hypersensitivity of some specific tissue
and protects it against the detrimental action of OH From our data reported here
we speculate that the same mechanism may exist amongst diverse barley
germplasm (eg those salt sensitive varieties but with less OH-induced K+ efflux)
Thus from the practical point of view the lack of significant correlation between
OH-induced ion fluxes and salinity stress tolerance (Figures 38 and 39) makes
this trait not suitable for salinity breeding programs
342 H2O2-induced K+ and Ca2+ fluxes in cereals correlate with
their overall salinity stress tolerance but only in mature zone
Earlier observations showed that salt sensitive barley varieties (with higher
damage index) have higher K+ efflux in response to H2O2 compared to salt tolerant
varieties (Chen et al 2007a Maksimović et al 2013) In this study we extrapolated
these initial observations made on a few selected varieties to a larger number of
genotypes We have also shown that (1) the same trend is also applicable to wheat
species (2) larger K+ efflux is mirrored by the higher Ca2+ uptake in H2O2-treated
roots and (3) the correlation between salinity tolerance and H2O2-induced ion flux
responses exist only in mature but not elongation root zone
Over the last decade an ability of various plant tissues to retain potassium
under stress conditions has evolved as a novel and essential mechanism of salinity
stress tolerance in plants (reviewed by Shabala and Pottosin 2014 and Shabala et
al 2014 2016) Reported initially for barley roots (Chen et al 2005 2007ac) a
positive correlation between the overall salinity stress tolerance and the ability of a
root tissue to retain K+ was later expanded to many other species (reviewed by
Shabala 2017) and also extrapolated to explain the inter-specific variability in
salinity stress tolerance (Sun et al 2009 Lu et al 2012 Chakraborty et al 2016)
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
42
In roots this NaCl-induced K+ efflux is mediated predominantly by outward-
rectifying K+ channels GORK that are activated by both membrane depolarization
(Very et al 2014) and ROS (Demidchik et al 2010) as shown in direct patch-
clamp experiments Thus the reduced H2O2 sensitivity of roots of tolerant wheat
and barley genotypes may be potentially explained by either smaller population of
ROS-sensitive GORK channels or by higher endogenous level of enzymatic
antioxidants in the mature root zone It is not clear at this stage if H2O2 is less prone
to induce K+ efflux (eg root cells are less sensitive to this ROS) in salt tolerant
plants or the ldquoeffectiverdquo H2O2 concentration in root cells is lower in salt-tolerant
plants due to a higher scavenging or detoxificating capacity However given the
fact that the activity of major antioxidant enzymes has been shown to be higher in
salt sensitive barley cultivars in both control and H2O2 treated roots (Maksimović
et al 2013) the latter hypothesis is less likely to be valid
The molecular identity of ROS-sensitive transporters should be revealed in the
future pharmacological and (forward) genetic experiments Previously we have
shown that H2O2-induced Ca2+ and K+ fluxes were significantly attenuated in
Arabidopsis Atann1 mutants and enhanced in overexpressing lines (Richards et al
2014) making annexin a likely candidate to this role Further H2O2-induced Ca2+
uptake in Arabidopsis roots was strongly suppressed by application of 30 microM Gd3+
a known blocker of non-selective cation channels (Demidchik et al 2007 ) and
roots pre-treatment with either cAMP or cGMP significantly reduced H2O2-induced
K+-leakage and Ca2+-influx (Ordontildeez et al 2014) implicating the involvement of
cyclic nucleotide-gated channels (one type of NSCC) (Demidchik and Maathuis
2007)
The lack of the above correlation between H2O2-induced K+ efflux and salinity
tolerance in the elongation root zone is very interesting and requires some further
discussion In recent years a ldquometabolic switchrdquo concept has emerged (Demidchik
2014 Shabala 2017) which implies that K+ efflux from metabolically active cells
may be a part of the mechanism inhibiting energy-consuming anabolic reactions
and saving energy for adaptation and reparation needs This mechanism is
implemented via transient decrease in cytosolic K+ concentration and accompanied
reduction in the activity of a large number of K+-dependent enzymes allowing a
redistribution of ATP pool towards defense responses (Shabala 2017) Thus high
K+ efflux from the elongation zone in salt-tolerant varieties may be an important
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
43
part of this adaptive strategy This suggestion is also consistent with the observation
that plants often respond to salinity stress by the increase in the GORK transcript
level (Adem et al 2014 Chakraborty et al 2016)
It should be also commented that salt tolerant varieties used in this study
usually have lower grain yield under control condition (Chen et al 2007c Cuin et
al 2009) showing a classical trade-off between tolerance and productivity (Weis
et al 2000) most likely as a result of allocation of a larger metabolic pool towards
constitutive defense traits such as maintenance of more negative membrane
potential in plant roots (Shabala et al 2016) or more reliance on the synthesis of
organic osmolytes for osmotic adjustment
343 Reactive oxygen species (ROS)-induced K+ efflux is
accompanied by an increased Ca2+ uptake
Elevation in the cytosolic free calcium is crucial for plant growth
development and adaptation Calcium influx into plant cells may be mediated by a
broad range of Ca2+-permeable channels Of specific interest are ROS-activated
Ca2+-permeable channels that form so-called ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) This mechanism implies that Ca2+-activated NADPH oxidases work
in concert with ROS-activated Ca2+-permeable cation channels to generate and
amplify stress-induced Ca2+ and ROS signals (Demidchik et al 2003 2007
Demidchik and Maathuis 2007 Shabala et al 2015) This self-amplification
mechanism may be essential for early stress signaling events as proposed by
Shabala et al 2015 and may operate in the root apex where the salt stress sensing
most likely takes place (Wu et al 2015) In the mature zone however continues
influx of Ca2+ may cause excessive apoplastic O2 production where it is rapidly
reduced to H2O2 By interacting with transition metals (Cu+ and Fe2+) in the cell
wall the hydroxyl radicals are formed (Demidchik 2014) activating K+ efflux
channels This may explain the observed correlation between the magnitude of
H2O2-induced Ca2+ influx and K+ efflux measured in this tissue (Figures 34I 34J
35F 36I 36J and 37F) This notion is further supported by the previous reports
that in Arabidopsis mature root cell protoplasts hydroxyl radicals were proved to
activate and mediate inward Ca2+ and outward K+ currents (Demidchik et al 2003
2007) while exogenous H2O2 failed to activate inward Ca2+ currents (Demidchik
Chapter 3 ROS-induced ions fluxes in root of several cereal varieties
44
et al 2003) The conductance resumed when H2O2 was applied to intact mature
roots (Demidchik et al 2007) This indicated that channel activation by H2O2 may
be indirect and mediated by its interaction with cell wall transition (Fry 1998
Halliwell and Gutteridge 2015)
344 Implications for breeders
Despite great efforts made in plant breeding for salt tolerance in the past
decades only limited success was achieved (Gregorio et al 2002 Munns et al
2006 Shahbaz and Ashraf 2013) It becomes increasingly evident that the range of
the targeted traits needs to be extended shifting a focus from those related to Na+
exclusion from uptake (Shi et al 2003 Byrt et al 2007 James et al 2011 Suzuki
et al 2016) to those dealing with tissue tolerance The latter traits have become the
center of attention of many researchers in the last years (Roy et al 2014 Munns et
al 2016) However to the best of our knowledge none of the previous works
provided an unequivocal causal link between salinity-stress tolerance and ROS
activation of root ion transporters mediating ionic homeostasis in plant cells We
took our first footstep to fill this gap in our knowledge by the current study
Taken together our results indicate high tissue specificity of root ion flux
response to ROS and suggest that measuring the magnitude of H2O2-induced net
K+ and Ca2+ fluxes from mature root zone may potentially be used as a tool for
cell-based phenotyping in breeding programs aimed to improve salinity stress
tolerance in cereals The next step in this process will be a full-scale validation of
the proposed method and finding QTLs associated with ROS-induced ion fluxes in
plant roots
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
45
Chapter 4 Validating using MIFE technique-
measured H2O2-induced ion fluxes as physiological
markers for salinity stress tolerance breeding in
wheat and barley
41 Introduction
Wheat and barley are known as important staple food worldwide (Baik and
Ullrich 2008 Shewry 2009) According to FAO
(httpwwwfaoorgworldfoodsituationcsdben) data the world annual wheat and
barley production in 2017 is forecasted at 755 and 148 million tonnes respectively
making them the second and fourth most-produced cereals However the
production rates are increasing rather slow and hardly sufficient to meet the demand
of feeding the estimated 93 billion populations by 2050 (Tester and Langridge
2010) To the large extent this mismatch between potential supply and demand is
determined by the impact of agricultural food production from abiotic stresses
among which soil salinity is one of such factors
The salinity stress tolerance mechanisms of cereals in the context of oxidative
stress tolerance specifically ROS-induced ion fluxes has been investigated and
correlated with the former in our previous study (Chapter 3) By using the MIFE
technique we measured transient ion fluxes from the root epidermis of several
contrasting barley and wheat varieties in response to different types of ROS Being
confined to mature root zone and H2O2 treatment we reported a strong correlation
between H2O2-induced K+ efflux and Ca2+ uptake and their overall salinity stress
tolerance in this root zone with salinity tolerant varieties leaking less K+ and
acquiring less Ca2+ under this stress condition While these finding opened a new
and previously unexplored opportunity to use these novel traits (H2O2-induced K+
and Ca2+ fluxes) as potential physiological markers in breeding programs the
number of genotypes screened was not large enough to convince breeders in the
robustness of this new approach This calls for the validation of the above approach
using a broader range of genotypes In order to validate the applicability of the
above developed MIFE protocol for breeding and examine how robust the above
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
46
correlation is we extend our work to 44 barley 20 bread wheat and 20 durum wheat
genotypes contrasting in their salinity stress tolerance
Another aim of this study is to reveal the physiological andor molecular
identity of the downstream targets mediating above ion flux responses to ROS
Pharmacological experiments were further conducted using different channel
blockers andor specific enzymatic inhibitors to address this issue and explore the
molecular identity of H2O2-responsive ion transport systems in cereal roots
42 Materials and methods
421 Plant materials and growth conditions and Ca2+ and K+ flux
measurements
Forty-four barley (43 Hordeum vulgare L 1 H vulgare ssp Spontaneum
SYR01) twenty bread wheat (Triticum aestivum) and twenty durum wheat
(Triticum turgidum spp durum) varieties were employed in this study Seedlings
were grown hydroponically as described in the section 221 All details for ion-
selective microelectrodes preparation and ion flux measurements protocols are
available in the section 23 Based on our findings in chapter 3 ions fluxes were
measured from the mature root zone in response to 10 mM H2O2
422 Pharmacological experiments
Mechanisms mediating H2O2-induced Ca2+ and K+ fluxes in root mature zone
in cereals were investigated by the introduction of pharmacological experiments
using one barley (Naso Nijo) and wheat (durum wheat Citr 7805) variety Prior to
the application of H2O2 stress for MIFE measurements roots pre-treated for 1 h
with one of the following chemicals 20 mM tetraethylammonium chloride (TEA+
a known blocker of K+-selective plasma membrane channels) 01 mM gadolinium
chloride (Gd3+ a known blocker of NSCCs) or 20 microM diphenylene iodonium (DPI
a known inhibitor of NADPH oxidase) All chemicals were from Sigma-Aldrich
423 Statistical analysis
Statistical significance of mean plusmn SE values was determined by the standard
Studentrsquos t -test at P lt 005 level
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
47
43 Results
431 H2O2-induced ions kinetics in mature root zone of cereals
Consistent with our previous study in chapter 3 net K+ uptake was measured
in the mature root zone of cereals in resting state (Figure 41A) along with slight
efflux for Ca2+ (Figure 41B) Acute (10 mM) H2O2 treatment caused an immediate
and massive K+ efflux (Figure 41A) and Ca2+ uptake (Figure 41B) with a
gradually recovery of Ca2+ after 20 min of H2O2 application (Figure 41B) The K+
flux never recovered in full and remained negative (Figure 41A)
Figure 41 Descriptions (see inserts in each panel) of net K+ (A) and Ca2+ (B)
flux from cereals root mature zone in response to 10 mM H2O2 in a
representative experiment Two distinctive flux points were marked on the
curves a peak value ndash identified as maximum flux value measured after
treatment and an end value ndash values measured 20 min after the H2O2 treatment
application The arrow in each panel represents the moment when H2O2 was
applied Figures derived from chapter 3
432 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity tolerance in barley
After imposition of 10 mM H2O2 K+ flux changed from net uptake to efflux
The smallest peak and end net flux (leaking less K+) was found in salt-tolerant
CM72 cultivar (-377 + 48 nmol m-2 s-1 and -269 + 39 nmol m-2 s-1 respectively)
The highest peak and end K+ efflux was observed in varieties Naso Nijo (-185 + 35
nmol m-2 s-1) and Dash (-113 + 11 nmol m-2 s-1) (Figures 42A and 42C) At the
same time this treatment resulted in various degree of Ca2+ influx among all the
forty-four barley varieties with the mean peak Ca2+ flux ranging from 155 plusmn 25
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
48
nmol m-2 s-1 in SYR01 (salinity tolerant) to 652 plusmn 43 nmol m-2 s-1 in Naso Nijo
(salinity sensitive) (Figure 42E) A linear correlation between the overall salinity
stress tolerance (quantified as the salt damage index see Wu et al 2015 and Table
41 for details) and the H2O2-induced ions fluxes were plotted Pronounced and
negative correlations (at P ˂ 0001 level) were found in H2O2-induced of K+ efflux
(Figures 42B and 42D) and Ca2+ uptake (Figure 42F) In our previous study on
chapter 3 conducted on eight contrasting barley genotypes we showed the same
significant correlation between oxidative stress and salinity stress tolerance Here
we validated the finding and provided a positive conclusion about the casual
relationship between salinity stress and oxidative stress tolerance in barley H2O2-
induced Ca2+ uptake and K+ deprivation in barley root mature zone correlates with
their overall salinity tolerance
Table 41 List of barley varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected from our previous study by Wu et
al 2015
Damage Index Score of Barley
SYR01 025 RGZLL 200 AC Burman 267 Yan89110 450
TX9425 100 Xiaojiang 200 Clipper 275 Yiwu Erleng 500
CM72 125 YU6472 200 93-3143 C60 300 YPSLDM 500
Honen 150 Barque73 225 Lixi143 300 ZUG403 575
YWHKSL 150 CXHKSL 225 Schooner 300 Dash 600
YYXT 150 Mundah 225 YSM3 300 Macquarie 700
Flagship 175 Dayton 250 Franklin 325 Naso Nijo 750
Gebeina 175 Skiff 250 Hu93-045 325 Haruna Nijo 775
Numar 175 Yan90260 250 Aizao3 350 YF374 800
ZUG293 175 Yerong 250 Gairdner 400 Kinu Nijo 850
DYSYH 200 Zhepi2 250 Sahara 400 Unicorn 950
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
49
Figure 42 Genetic variability of oxidative stress tolerance in barley Peak K+
flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of forty-four barley
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the root mature zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
433 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in bread
wheat
H2O2-induced ions fluxes in bread wheat were similar with those in barley By
comparing K+ and Ca2+ fluxes of the twenty bread wheat varieties we found salt
tolerant cultivar Titmouse S and sensitive Iran 118 exhibited smallest and biggest
K+ and Ca2+ peak fluxes respectively (Figures 43A and 43E) Similar
observations were found for K+ end flux values for contrasting Berkut and Seville
20 varieties respectively (Figure 43C) A significant (P ˂ 005) correlation
between salinity damage index (Wu et al 2014 Table 42) and H2O2-induced Ca2+
and K+ fluxes were found for bread wheat (Figures 43B 43D and 43F) which
was consistent with our previous results conducted on six contrasting bread wheat
genotypes
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
50
Table 42 List of wheat varieties used in this study Scores represent quantified
extent of damage imposed by salinity to barley plants on a 0 to 10 scale (0 ndash
highest overall salinity tolerance no visual stress symptoms 10 ndash lowest level
of salt tolerance dead plants) Data collected based on our previous study by Wu
et al 2014
Damage Index Score of Bread Wheat Damage Index Score of Durum Wheat
Berkut 183 Gladius 350 Alex 400 Timilia 633
Titmouse S 183 Kukri 350 Zulu 533 Jori 650
Cranbrook 250 Seville20 383 AUS12746 583 Hyperno 650
Excalibur 250 Halberd 383 Covelle 583 Tamaroi 650
Drysdale 283 Iraq43 417 Jandaroi 600 Odin 683
Persia6 317 Iraq50 417 Kalka 600 AUS19762 733
H7747 317 Iran118 417 Tehuacan60 617 Caparoi 750
Opata 317 Krichauff 450 AUS16469 633 C250 783
India38 333 Sokoll 500 Biskiri ac2 633 Towner 783
Persia21 333 Janz 517 Purple Grain 633 Citr7805 817
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
51
Figure 43 Genetic variability of oxidative stress tolerance in bread wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty bread wheat
varieties in response to 10 mM H2O2 and their correlation with the damage index
(B D and F respectively) Fluxes were measured from the mature root zone of
4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point (in B D
and F) represents a single variety
434 H2O2-induced K+ efflux and Ca2+ uptake from the mature root
zone correlates with the overall salinity stress tolerance in durum
wheat
Similar to barley and bread wheat H2O2-induced K+ efflux and Ca2+ influx
also correlated with their overall salinity tolerance (Figure 44)
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
52
Figure 44 Genetic variability of oxidative stress tolerance in durum wheat Peak
K+ flux (A) end K+ flux value (C) and peak Ca2+ flux (E) of twenty durum
wheat varieties in response to 10 mM H2O2 and their correlation with the damage
index (B D and F respectively) Fluxes were measured from the mature root
zone of 4-d-old seedlings Means plusmn SE (n = 6-8 individual plants) Each point
(in B D and F) represents a single variety
435 Barley tends to leak less K+ and acquire less Ca2+ than wheat
in mature root zone upon oxidative stress
A general comparison of K+ and Ca2+ fluxes in response to H2O2 among barley
bread wheat and durum wheat is given in Figure 45 Net flux was calculated as
mean value in each species group (eg 44 barley 20 bread wheat and 20 durum
wheat respectively Figures 45A and 45B) At resting state both bread wheat and
durum wheat showed stronger K+ uptake ability than barley (180 plusmn 12 and 225 plusmn
18 vs 130 plusmn 7 nmol m-2 middot s-1 respectively P ˂ 001 Figure 45C) but no significant
difference was found in their Ca2+ kinetics (Figure 45D) After being treated with
10 mM H2O2 the peak K+ flux did not exhibit obvious significance among the three
species (Figure 45C) while Ca2+ loading from wheat was twice as high as the
loading in barley (52 vs 26 nmol m-2 middot s-1 respectively P ˂ 0001 Figure 45D)
The net mean leakage of K+ and acquisition of Ca2+ showed clear difference among
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
53
these species with K+ loss and Ca2+ acquisition from barley mature root zone
generally less than bread wheat and durum wheat (Figures 45E and 45F) The
overall trend in H2O2-induced K+ efflux and Ca2+ uptake followed the pattern
durum wheat gt bread wheat gt barley reflecting differences in salinity stress
tolerance between species (Munns and Tester 2008)
Figure 45 General comparison of H2O2-induced net K+ (A) and Ca2+ (B) fluxes
initialpeak K+ flux (C) and Ca2+ flux (D) values net mean K+ efflux (E) and
Ca2+ (F) uptake values from mature root zone in barley bread wheat and durum
wheat Mean plusmn SE (n = 44 20 and 20 genotypes respectively)
436 H2O2-induced ion flux in root mature zone can be prevented
by TEA+ Gd3+ and DPI in both barley and wheat
Pharmacological experiments using two K+-permeable channel blockers (Gd3+
blocks NSCCs TEA+ blocks K+-selective plasma membrane channels) and one
plasma membrane (PM) NADPH oxidase inhibitor (DPI) were conducted to
identify the likely candidate ion transporting systems mediating the above
responses in barley and wheat H2O2-induced K+ efflux and Ca2+ uptake in the
mature root zone was significantly inhibited by Gd3+ TEA+ and DPI (Figure 46)
Both Gd3+ and TEA+ caused a similar (around 60) block to H2O2-induced K+
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
54
efflux in both species the blocking effect in DPI pre-treated roots was 66 and
49 respectively (Figures 46A and 46B) At the same time the NSCCs blocker
Gd3+ results in more than 90 inhibition of H2O2-induced Ca2+ uptake in both
barley and wheat the K+ channel blocker TEA+ also affected the acquisition of Ca2+
to higher extent (88 and 71 inhibition respectively Figures 46C and 46D)
The inactivation of PM NADPH oxidase caused significant inhibition (up to 96)
of Ca2+ uptake in barley while 51 inhibition was observed in wheat samples
(Figures 46C and 46D)
Figure 46 Effect of DPI (20 microm) Gd3+ (01 mM) and TEA+ (20 mM) pre-
treatment (1 h) on H2O2-induced net mean K+ and Ca2+ fluxes from the mature
root zone of barley (A and C respectively) and wheat (B and D respectively)
Mean plusmn SE (n = 5 ndash 6 plants)
44 Discussion
441 H2O2-induced ions fluxes from root mature zone as a novel
physiological trait to explore mechanisms of salinity stress
tolerance
H2O2 is known for its signalling role and has been implicated in a broad range
of physiological processes in plants (Choudhury et al 2017 Mittler 2017) such as
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
55
plant growth development and differentiation (Schmidt and Schippers 2015)
pathogen defense and programmed cell death (Dangl and Jones 2001 Gechev and
Hille 2005 Torres et al 2006) stress sensing signalling and acclimation (Slesak
et al 2007 Baxter et al 2014 Dietz et al 2016) hormone biosynthesis and
signalling (Bartoli et al 2013) root gravitropism (Joo et al 2001) and stomatal
closure (Pei et al 2000) This role is largely explained by the fact that H2O2 has a
long half-life (minutes) and thus can diffuse some distance from the production site
(Pitzschke et al 2006) However excessive production and accumulation of ROS
can be toxic leading to oxidative stress Salinity is one of the abiotic factors causing
such oxidative damage (Hernandez et al 2000) Therefore numerous efforts aimed
at increasing major antioxidants (AO) activity had been taken in breeding for
oxidative stress tolerance associated with salinity tolerance while the outcome
appears unsatisfactory because of the failure in either revealing a correlation
between AO activity and salinity tolerance in a range of species (Dionisio-Sese and
Tobita 1998 Noreen and Ashraf 2009b Noreen et al 2010 Fan et al 2014) or
pyramiding major AO QTLs (Frary et al 2010) Here in this work by using the
seminal MIFE technique we established a causal link between the oxidative and
salinity stress tolerance We showed that H2O2-induced K+ efflux and Ca2+ uptake
in the mature root zone in cereals correlates with their overall salinity tolerance
(Figures 42 43 and 44) with salinity tolerant varieties leak less K+ and acquire
less Ca2+ and vice versa The reported findings here provide additional evidence
about the importance of K+ retention in plant salinity stress tolerance and new
(previously unexplored) thoughts in the ldquoCa2+ signaturerdquo (known as the elevation
in the cytosolic free Ca2+ at the bases of the PM Ca2+-permeable channels
activation during this process (Richards et al 2014) The K+ efflux and the
accompanying Ca2+ uptake upon H2O2 may indicate a similar mechanism
controlling these processes
The existence of a causal association between oxidative and salinity stress
tolerance allows H2O2-induced K+ and Ca2+ fluxes being used as physiological
markers in breeding programs The next step would be creation of the double
haploid population to be used for QTL mapping of the above traits This can be
achieved using varieties with weaker (eg CM72 for barley Titmouse S for bread
wheat AUS 12748 for durum wheat) and stronger (eg Naso Nijo for barley Iran
118 for bread wheat C250 for durum wheat) K+ efflux and Ca2+ flux responses to
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
56
H2O2 treatment as potential parental lines to construct DH lines The above traits
which are completely new and previously unexplored may be then used to create
salt tolerant genotypes alongside with other mechanisms through the ldquopyramidingrdquo
approach (Flowers and Yeo 1995 Tester and Langridge 2010 Shabala 2013)
442 Barley tends to retain more K+ and acquire less Ca2+ into
cytosol in root mature zone than wheat when subjected to oxidative
stress
All the barley and wheat varieties screened in this study varied largely in their
initial root K+ uptake status (data not shown) and H2O2-induced K+ and Ca2+ flux
(Figures 42 43 and 44 left panels) while their general tendency is comparable
(Figures 45A and 45B) Barley is considered to be the most salt tolerant cereal
followed by the moderate tolerant bread wheat and sensitive durum wheat (Munns
and Tester 2008) In this study the highest K+ uptake ability in root mature zone at
resting state was observed in the salt sensitive durum wheat (Figure 45C) followed
by bread wheat and barley which is consistent with previous reports that leaf K+
content (mmolmiddotg-1 DW) was found highest in durum wheat (146) compared with
bread wheat and barley (126 and 112 respectively) (Wu et al 2014 2015)
According to the concept of ldquometabolic hypothesisrdquo put forward by Demidchik
(2014) K+ a known activator of more than 70 metabolic enzymes (Dreyer and
Uozumi 2011 Anschuumltz et al 2014) and with high concentration in cytosol may
activate the activity of metabolic enzymes and draw the major bulk of available
energy towards the metabolic processes driven by these conditions When plants
encountered stress stimuli a large pool of ATP will be redirected to defence
reactions and energy balance between metabolism and defence determines plantrsquos
stress tolerance (Shabala 2017) Therefore in this study the salt sensitive durum
wheat may utilise the majority bulk of K+ pool for cell metabolism thus the amount
of available energy is limited to fight with salt stress Taken together these findings
further revealed that either higher initial K+ content (Wu et al 2014) or higher
initial K+ uptake value has no obvious beneficial effect to the overall salinity
tolerance in cereals
Unlike the case of steady K+ under control conditions K+ retention ability
under stress conditions has been intensively reported and widely accepted as an
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
57
essential mechanism of salinity stress tolerance in a range of species (Shabala 2017)
In this study we also revealed a higher K+ retention ability in response to oxidative
stress in the salt tolerant barley variety compared with salt sensitive wheat variety
(Figure 45E) which was accompanied with the same trend in their Ca2+ restriction
ability upon H2O2 exposure (Figure 45F) This may be attributed to the existence
of more ROS sensitive K+ and Ca2+ channels in the latter species While Ca2+
kinetics between the two wheat clusters seems to be another situation Although
H2O2-induced Ca2+ uptake in bread was as higher as that of durum wheat (Figures
45B 45D and 45F) the former cluster was not equally salt sensitive as the latter
(damage index score 355 vs 638 respectively Plt0001 Wu et al 2014) The
physiological rationale behind this observation may be that bread wheat possesses
other (additional) mechanisms to deal with salinity such as a higher K+ retention
(Figure 45E) or Na+ exclusion abilities (Shah et al 1987 Tester and Davenport
2003 Sunarpi et al 2005 Cuin et al 2008 2011 Horie et al 2009) to
compensate for the damage effect of higher Ca2+ in cytosol
443 Different identity of ions transport systems in root mature
zone upon oxidative stress between barley and wheat
Earlier studies reported that ROS is able to activate GORK channel
(Demidchik et al 2010) and NSCCs (Demidchik et al 2003 Shabala and Pottosin
2014) in the root epidermis mediating K+ efflux and Ca2+ influx respectively The
specific oxidant that directly activates these channels is known as bullOH which can
be converted by interaction between H2O2 and cell wall transition metals (Shabala
and Pottosin 2014) We believe that the similar ions transport system is also
applicable to cereals in response to H2O2 At the same time the so-called ldquoROS-
Ca2+ hubrdquo mechanism (Demidchik and Shabala 2018) with the involvement of PM
NADPH oxidase should not be neglected However whether the underlying
mechanisms between barley and wheat are different or not remains elusive As
expected Gd3+ (the NSCCs blocker) and TEA+ (the K+-selective channel blocker)
inhibited H2O2-induced K+ efflux from both cereals (Figures 46A and 46B) The
fact that the extent of inhibition of both blockers was equal in both cereals may be
indicative of an equivalent importance of both NSCC and GORK involved in this
process At the same time Gd3+ caused gt 90 inhibition of Ca2+ uptake in both
Chapter 4 H2O2-induced ions fluxes in root of various cereal varieties
58
barley and wheat roots (Figures 46C and 46D) This suggests that H2O2-induced
Ca2+ uptake from the root mature zone of cereals is predominantly mediated by
ROS-activated Ca2+-permeable NSCCs (Demidchik and Maathuis 2007) These
findings suggested that barley and wheat are likely showing similar identities in
ROS sensitive channels
In the case of 1 h pre-treatment with DPI an inhibitor of NADPH oxidase H2O2-
induced Ca2+ uptake was suppressed in both barley and wheat (Figures 46C and
46D) This is fully consistent with the idea that PM NADPH oxidase acts as the
major ROS generating source which lead to enhanced H2O2 production in
apoplastic area under stress conditions (Demidchik and Maathuis 2007) The
apoplastic H2O2 therefore activates Ca2+-permeable NSCC and leads to elevated
cytosolic Ca2+ content which in turn activates PM NADPH oxidase to form a so
called self-amplifying ldquoROS-Ca2+ hubrdquo thus enhancing and transducing Ca2+ and
redox signals (Demidchik and Shabala 2018) Given the fact that K+-permeable
channels (such as GORK and NSCCs) are also activated by ROS the inhibition of
H2O2-induced Ca2+ uptake may lead to major alterations in intracellular ionic
homeostasis which reflected and supported by the observation that DPI pre-
treatment lead to reduced H2O2-induced K+ efflux (Figures 46A and 46B)
However the observation that DPI pre-treatment results in much higher inhibition
effect of H2O2-induced Ca2+ uptake in barley (as high as the Gd3+ pre-treatment
for direct inhibition Figure 46C) compared with wheat (96 vs 51 Figures
46C and 46D) in this study may be indicative of the existence of other Ca2+-
independent Ca2+-permeable channels in the latter cereal The Ca2+-permeable
CNGCs (cyclic nucleotide-gated channels one type of NSCC) therefore may
possibly be involved in this process in wheat mature root cells (Gobert et al
2006 Ordontildeez et al 2014)
Chapter 5 QTLs identification in DH barley population
59
Chapter 5 QTLs for ROS-induced ions fluxes
associated with salinity stress tolerance in barley
51 Introduction
Soil salinity is one of the most major environmental constraints reducing crop
yield and threatening global food security (Munns and Tester 2008 Shahbaz and
Ashraf 2013 Butcher et al 2016) Given the fact that salt-free land is dwindling
and world population is exploding creating salt tolerant crops becomes an
imperative (Shabala 2013 Gupta and Huang 2014)
Salinity stress is complex trait that affects plant growth by imposing osmotic
ionic and oxidative stresses on plant tissues (Adem et al 2014) In this term the
tolerance to each of above components is conferred by numerous contributing
mechanisms and traits Because of this using genetic modification means to
improve crop salt tolerance is not as straightforward as one may expect It has a
widespread consensus that altering the activity of merely one or two genes is
unlikely to make a pronounced change to whole plant performance against salinity
stress Instead the ldquopyramiding approachrdquo was brought forward (Flowers 2004
Yamaguchi and Blumwald 2005 Munns and Tester 2008 Tester and Langridge
2010 Shabala 2013) which can be achieved by the use of marker assisted selection
(MAS) MAS is an indirect selection process of a specific trait based on the
marker(s) linked to the trait instead of selecting and phenotyping the trait itself
(Ribaut and Hoisington 1998 Collard and Mackill 2008) which has been
extensively explored and proposed for plant breeding However not much progress
was achieved in breeding programs based on DNA markers for improving
quantitative whole-plant phenotyping traits (Ben-Ari and Lavi 2012) Taking
salinity stress tolerance as an example although considerable efforts has been made
by prompting Na+ exclusion and organic osmolytes production of plants in
responses to this stress breeding of salt-tolerant germplasm remains unsatisfying
which propel researchers to take oxidative stress (one of the components of salinity
stress tolerance) into consideration
One of the most frequently mentioned traits of oxidative stress tolerance is an
enhanced antioxidants (AOs) activity in plants While a positive correlation
Chapter 5 QTLs identification in DH barley population
60
between salinity stress tolerance and the level of enzymatic antioxidants has been
reported from a wide range of plant species such as wheat (Bhutta 2011 El-
Bastawisy 2010) rice (Vaidyanathan et al 2003) tomato (Mittova et al 2002)
canola (Ashraf and Ali 2008) and maize (Azooz et al 2009) equally large number
of papers failed to do so (barley - Fan et al 2014 rice - Dionisio-Sese and Tobita
1998 radish - Noreen and Ashraf 2009 turnip - Noreen et al 2010) Also by
evaluating a tomato introgression line (IL) population of S lycopersicum M82
and S pennellii LA716 Frary (Frary et al 2010) identified 125 AO QTLs
(quantitative trait loci) associated with salinity stress tolerance Obviously the
number is too big to make QTL mapping of this trait practically feasible (Bose et
al 2014b)
Previously in Chapter 3 and 4 we have revealed a causal relationship between
oxidative stress and salinity stress tolerance in barley and wheat and explored the
oxidative stress-related trait H2O2-induced Ca2+ and K+ fluxes as potential
selection criteria for crop salinity stress tolerance Here in this chapter we have
applied developed MIFE protocols to a double haploid (DH) population of barley
to identify QTLs associated with ROS-induced root ion fluxes (and overall salinity
tolerance) Three major QTLs regarding to oxidative stress-induced ions fluxes in
barley were identified on 2H 5H and 7H respectively This finding suggested the
potential of using oxidative stress-induced ions fluxes as a powerful trait to select
salt tolerant germplasm which also provide new thoughts in QTL mapping for
salinity stress tolerance based on different physiological traits
52 Materials and methods
521 Plant material growth conditions and Ca2+ and K+ flux
measurements
A total of 101 double haploid (DH) lines from a cross between CM72 (salt
tolerant) and Gairdner (salt sensitive) were used in this study Seedlings were
grown hydroponically as described in the section 221 All details for ion-selective
microelectrodes preparation and ion flux measurements protocols are available in
the section 23 Based on our previous findings ions fluxes were measured from
the mature root zone in response to 10 mM H2O2
Chapter 5 QTLs identification in DH barley population
61
522 QTL analysis
Two physiological markers namely H2O2-induced peak K+ and Ca2+ fluxes
were used for QTL analysis The genetic linkage map was constructed using 886
markers including 18 Simple Sequence Repeat (SSR) and 868 Diversity Array
Technology (DArT) markers The software package MapQTL 60 (Ooijen 2009)
was used to detect QTL QTL analysis was first conducted by interval mapping
(IM) For this the closest marker at each putative QTL identified using interval
mapping was selected as a cofactor and the selected markers were used as genetic
background controls in the approximate multiple QTL model (MQM) A logarithm
of the odds (LOD) threshold values ge 30 was applied to declare the presence of a
QTL at 95 significance level To determine the effects of another trait on the
QTLs for salinity tolerance the QTLs for salinity tolerance were re-analysed using
another trait as a covariate Two LOD support intervals around each QTL were
established by taking the two positions left and right of the peak that had LOD
values of two less than the maximum (Ooijen 2009) after performing restricted
MQM mapping The percentage of variance explained by each QTL (R2) was
obtained using restricted MQM mapping implemented with MapQTL60
523 Genomic analysis of potential genes for salinity tolerance
The sequences of markers bpb-8484 (on 2H) bpb-5506 (on 5H) and bpb-3145
(on 7H) associated with different QTL for oxidative stress tolerance were used to
identify candidate genes for salinity tolerance The sequences of these markers were
downloaded from the website httpwwwdiversityarrayscom followed by a blast
search on the website httpwebblastipkgaterslebendebarley to identify the
corresponding morex_contig of these markers The morex_contig_48280
morex_contig_136756 and morex_contig_190772 were found to be homologous
with bpb-8484 (Identities = 684703 97) bpb-5506 (Identities = 726736 98)
and bpb-3145 (Identities = 247261 94) respectively The genome position of
these contigs were located at 7691 cM on 2H 4413 cM on 5H and 12468 cM on
7H Barley genomic data and gene annotations were downloaded from
httpwebblastipk-gaterslebendebarley_ibscdownloads Annotated high
confidence genes between 6445 and 8095 cM on 2H 4299 and 4838 cM on 5H
Chapter 5 QTLs identification in DH barley population
62
11983 and 14086 cM on 7H were deemed to be potential genes for salinity
tolerance
53 Results
531 Peak K+ and Ca2+ fluxes of the DH lines under H2O2 treatment
As shown in Table 51 two parental lines showed significant difference in
H2O2-induced peak K+ and Ca2+ flux with the salt tolerant cultivar CM72 leaking
less K+ (less negative) and acquiring less Ca2+ (less positive) than the salt sensitive
cultivar Gairdner DH lines from the cross between CM72 and Gairdner also
showed significantly different Ca2+ (from 15 to 60 nmolmiddotm-2middots-1) and K+ (from -43
to -190 nmolmiddotm-2middots-1) fluxes in response to 10 mM H2O2 Figure 51 shows the
frequency distribution of peak K+ flux and peak Ca2+ flux upon H2O2 treatment in
101 DH lines
Table 51 H2O2-induced peak K+ and Ca2+ flux from parental and DH lines
Cultivars Peak K+ flux (nmolmiddotm-2middots-1) Peak Ca2+ flux (nmolmiddotm-2middots-1)
CM72 -47 plusmn 33 264 plusmn 35
Gairdner -122 plusmn 134 404 plusmn12
DH lines average -97 plusmn 174 335 plusmn 39
DH lines range -43 to -190 15 to 60
Data are Mean plusmn SE (n = 6)
Figure 51 Frequency distribution for Peak K+ flux (A) and Peak Ca2+ flux (B)
of DH lines derived from a cross of CM72 and Gairdner exposed to 10 mM H2O2
treatment
Chapter 5 QTLs identification in DH barley population
63
532 QTLs for H2O2-induced peak K+ flux and peak Ca2+ flux
Three QTLs for H2O2-induced peak K+ flux were identified on chromosomes
2H 5H and 7H which were designated as QKFCG2H QKFCG5H and
QKFCG7H respectively (Table 52 Figure 52) The nearest marker for
QKFCG2H is bPb-4482 which explained 92 of phenotypic variation The bPb-
5506 is the nearest marker for QKFCG5H and explained 103 of phenotypic
variation The third one QKFCG7H accounts for 117 of phenotypic variation
with bPb-0773 being the closest marker
Two QTLs for H2O2-induced Peak Ca2+ flux were identified on chromosomes
2H (QCaFCG2H) and 7H (QCaFCG7H) (Table 52 Figure 52) with the nearest
marker is bPb-0827 and bPb-8823 respectively The former explained 113 of
phenotypic variation while the latter explained 148
Table 52 QTLs for H2O2-induced peak K+ and Ca2+ flux in the DH line of CM72
and Gairdner QTL for H2O2-induced peak K+ flux when using H2O2-induced
peak Ca2+ flux as a covariate
Traits QTL
Linkage
group
Nearest
marker
Position
(cM) LOD
R2
() Covariate
KF
QKFCG2H 2H bPb-4482 126 312 92
QKFCG5H 5H bPb-5506 507 348 103 NA
QKFCG7H 7H bPb-0773 166 391 117
CaF QCaFCG2H 2H bPb-0827 1128 369 113
NA QCaFCG7H 7H bPb-8823 156 425 148
KF
QKFCG2H 2H
NS NS
CaF QKFCG5H 5H bPb-0616 47 514 145
QKFCG7H 7H
NS NS
KFCaF H2O2-induced peak K+ Ca2+ flux NS not significant NA not applicable
Chapter 5 QTLs identification in DH barley population
64
Figure 52 QTLs associated with H2O2-induced peak K+ flux (in red) and H2O2-
induced peak Ca2+ flux (in blue) For better clarity only parts of the chromosome
regions next to the QTLs are shown
533 QTL for KF when using CaF as a covariate
As shown in Table 52 QTLs related to oxidative stress induced peak K+ flux
and Ca2+ flux were observed on 2H 5H and 7H By compare the physical position
of the linkage map QTLs on 2H for peak K+ and Ca2+ flux and on 7H were located
at similar positions indicating a possible relationship between these two traits
(Table 52 Figures 53A and 53B) To further confirm this a QTL analysis for KF
was conducted by using CaF as a covariate Of the three QTLs for H2O2-induced
peak K+ flux only QKFCG5H was not affected (LOD = 347 R2 = 101) when
CaF was used as a covariate The other two QTLs QKFCG2H and QKFCG7H
which located at similar positions to those for H2O2-induced peak Ca2+ flux
became insignificant (LOD ˂ 2) (Figure 53C)
Chapter 5 QTLs identification in DH barley population
65
Figure 53 Chart view of QTLs for H2O2-induced peak K+ (A) and Ca2+ (B) flux
in the DH line (C) Chart view of QTLs for H2O2-induced peak K+ flux when
using H2O2-induced peak Ca2+ flux as covariate Arrows (peaks of LOD value)
in panels indicate the position of associated markers
534 Potential genes for H2O2-induced K+ and Ca2+ flux on 2H 5H
and 7H
Three QTLs were identified for H2O2-induced K+ and Ca2+ flux with QTLs
from 2H and 7H being involved in both H2O2-induced K+ and Ca2+ fluxes and QTL
from 5H being associated with H2O2-induced K+ flux only By blast searching of
the three closely linked markers bpb-8484 on 2H bpb-5506 on 5H and bpb-3145
on 7H high confidence genes were extracted near these markers Among all
annotated genes a total of eight genes in these marker regions were chosen as the
candidate genes for these traits (Table 53) which can be used for in-depth study in
the near future
Chapter 5 QTLs identification in DH barley population
66
Table 53 Candidate genes for H2O2-induced K+ and Ca2+ flux
Chromosome Candidate genes
2H Calcium-dependent lipid-binding (CaLB domain) family
protein 1
Annexin 8 1
5H NAC transcription factor 2
AP2-like ethylene-responsive transcription factor 2
7H
Calcium-binding EF-hand family protein 1
Calmodulin like 37 (CML37) 1
Protein phosphatase 2C family protein (PP2C) 3
WRKY family transcription factor 2
1 Calcium-dependent proteins 2 transcription factors 3 other proteins
54 Discussion
541 QTL on 2H and 7H for oxidative stress control both K+ and
Ca2+ flux
Salinity stress is one of the major yield-limiting factors and plantrsquos tolerance
mechanisms to this stress is highly complex both physiologically and genetically
(Negratildeo et al 2017) Three major components are involved in salinity stress in
crops osmotic stress specific ion toxicity and oxidative stress Among them
improving plant ability to synthesize organic osmotica for osmotic adjustment and
exclude Na+ from uptake have been targeted to create salt tolerant crop germplasm
(Sakamoto and Murata 2000 Martinez-Atienza et al 2007 Munns et al 2012
Wani et al 2013 Byrt et al 2014) However these efforts have been met with a
rather limited success (Shabala et al 2016)
Until now no QTL associated with oxidative stress-induced control of plant
ion homeostasis have been reported yet for any crop species Here we identified
two QTLs on 2H and 7H controlling H2O2-induced K+ flux (QKFCG2H and
Chapter 5 QTLs identification in DH barley population
67
QKFCG7H respectively) and Ca2+ flux (QCaFCG2H and QCaFCG7H
respectively) and one QTL on 5H related to H2O2-induced K+ flux (QKFCG5H)
in the seedling stage from a DH population originated from the cross of two barley
cultivars CM72 and Gairdner Further analysis on the QTL for KF using CaF as a
covariate confirmed that same genes control KF and CaF on both 2H and 7H
(Figure 53C) QKFCG5H was less affected (Figure 53C) when CaF was used as
a covariate indicating the exclusive involvement of this QTL in H2O2-induced K+
efflux Therefore all these three major QTL (one on each 2H 5H and 7H) identified
in this work could be candidate loci for further oxidative stress tolerance study The
genetic evidence for oxidative stress tolerance revealed in this study may also be of
great importance for salinity stress tolerance Plantsrsquo K+ retention ability under
unfavorable conditions has been largely studied in a range of species in recent years
indicating the important role of this trait played in conferring salinity stress
tolerance (Shabala 2017) This can be reflected by the fact that K+ content in plant
cell is more than 100-fold than in the soil (Dreyer and Uozumi 2011) It is also
involved in various key physiological pathways including enzyme activation
membrane potential formation osmoregulation cytosolic pH homeostasis and
protein synthesis (Veacutery and Sentenac 2003 Gierth and Maumlser 2007 Dreyer and
Uozumi 2011 Wang et al 2013 Anschuumltz et al 2014 Cheacuterel et al 2013) making
the maintenance of high cytosolic K+ content highly required (Wu et al 2014) On
the other hand plants normally maintain a constant and low (sub-micromolar) level
of free calcium in cytosol to use it as a second messenger in many developmental
and signaling cascades Upon sensing salinity cytosolic free Ca2+ levels are rapidly
elevated (Bose et al 2011) prompting a cascade of downstream events One of
them is an activation of the NADPH oxidase This plasma membrane-based protein
is encoded by RBOH (respiratory burst oxidase homolog) genes and has two EF-
hand motifs in the hydrophilic N-terminal region and is synergistically activated by
Ca2+-binding to the EF-hand motifs along with phosphorylation (Marino et al
2012) Ca2+ binding then triggers a conformational change that results in the
activation of electron transfer originating from the interaction between the N-
terminal Ca2+-binding domain and the C-terminal superdomain (Baacutenfi et al 2004)
Plant plasma membranes also harbor various non-selective cation channels
(NSCCs) which are permeable to Ca2+ and may be activated by both membrane
depolarisation and ROS (Demidchik and Maathuis 2007) Together RBOH and
Chapter 5 QTLs identification in DH barley population
68
NSCC forms a positive feedback loop termed ldquoROS-Ca2+ hubrdquo (Demidchik and
Shabala 2018) that amplifies stress-induced Ca2+ and ROS transients While this
process is critical for plant adaptation the inability to terminate it may be
detrimental to the organism Thus lower ROS-induced Ca2+ uptake seems to give
plant a competitive advantage
By using the same DH population as in this study a QTL associated with leaf
temperature (one of the traits for drought tolerance) was reported at the similar
position with our QTLs for oxidative stress tolerance on 2H (Liu et al 2017)
Moreover meta-analysis of major QTL for abiotic stress tolerance in barley also
indicated a high density of QTL for drought salinity and waterlogging stress at this
location on 2H (Zhang et al 2017) The same publication also summarized a range
of major QTLs for salinity stress tolerance at the position of 5H as in this study
(Zhang et al 2017) Another study using TX9425Naso Nijo DH population
reported a QTL associated with waterlogging stress tolerance at the similar position
of 7H with this study (Xu et al 2012) While both drought and water logging stress
are able to induce transient Ca2+ uptake to cytosol (Bose et al 2011) and K+ efflux
to extracellular spaces (Wang et al 2016) then ROS produced due to drought
stress-induced stomatal closure and water logging stress-induced oxygen
deprivation may be one of the factors facilitate these processes Therefore as ROS
production under stress conditions is a common denominator (Shabala and Pottosin
2014) the QTLs for oxidative stress identified in this study which associated with
salinity stress tolerance may at least in part possess similar mechanisms with the
mentioned stresses above
542 Potential genes contribute to oxidative stress tolerance
ROS (especially bullOH) are known to activate a number of K+- and Ca2+-
permeable channels (Demidchik et al 2003 2007 2010 Demidchik and Maathuis
2007 Zepeda-Jazo et al 2011) prompting Ca2+ influx into and K+ efflux from
cytosol especially in cells from the mature root zone Therefore the identified
QTLs for H2O2-induced ions fluxes might be probably closely related to these ions
transporting systems or act as subunit of these channels In our previous chapter
(Chapter 4) we explored the molecular identity of ion transport system upon H2O2
treatment in root mature zone of both barley and wheat and revealed an
involvement of NSCCs GORK channels and PM NADPH oxidase in this process
Chapter 5 QTLs identification in DH barley population
69
The ROS-activated K+-permeable NSCCs and GORK channels mediated H2O2-
induced K+ efflux At the same time ROS-activated Ca2+-permeable NSCCs
mediated H2O2-induced Ca2+ uptake with the activation of PM NADPH oxidase
by elevated cytosolic Ca2+ It is not clear at this stage which specific genes
contribute to these processes Plants utilise transmembrane osmoreceptors to
perceive and transduce external oxidative stress signal inducing expression of
functional response genes associated with these ion channels or other processes
(Liu et al 2017) Therefore genes in these pathways have higher possibility to be
taken as candidate genes In this study the nearest markers of the QTL detected
were located around 7691 cM on 2H 4413 cM on 5H and 12468 cM on 7H
Several candidate genes in the vicinity of the reported markers appear to be present
associated with ions fluxes These include calcium-dependent proteins
transcription factors and other stress related proteins (Table 53)
Since H2O2-induced Ca2+ acquisition was spotted therefore proteins binding
Ca2+ or contributing to Ca2+ signalling can be deemed as candidates It is claimed
that many signals raise cytosolic Ca2+ concentration via Ca2+-binding proteins
among which three quarters contain Ca2+-binding EF-hand motif(s) (Day et al
2002) making calcium-binding EF-hand family protein as one of the potential
genes One example is PM-based NADPH oxidase mentioned above Other
candidates that possess Ca2+-binding property is calmodulin like proteins (CML
such as CML 37) and Ca2+-dependent lipid-binding (CaLB) domains The former
are putative Ca2+ sensors with 50 family and varying number of EF hands reported
in Arabidopsis (Vanderbeld and Snedden 2007 Zeng et al 2015) the latter also
known as C2 domains are a universal Ca2+-binding domains (Rizo and Sudhof
1998 de Silva et al 2011) Both were shown to be involved in plant response to
various abiotic stresses (Zhang et al 2013 Zeng et al 2015) Annexins are a group
of Ca2+-regulated phospholipid and membrane-binding proteins which have been
frequently mentioned to catalyse transmembrane Ca2+ fluxes (Clark and Roux 1995
Davies 2014) and contributes to plant cell adaptation to various stress conditions
(Laohavisit and Davies 2009 2011 Clark et al 2012) In Arabidopsis AtANN1 is
the most abundant annexin and a PM protein that regulates H2O2-induced Ca2+
signature by forming Ca2+-permeable channels in planar lipid bilayers (Lee et al
2004 Richards et al 2014) Its role in other species such as cotton (GhAnn1 -
Zhang et al 2015) potato (STANN1 - Szalonek et al 2015) rice (OsANN1 - Qiao
Chapter 5 QTLs identification in DH barley population
70
et al 2015) brassica (AnnBj1 - Jami et al 2008) and lotus (NnAnn1 - Chu et al
2012) was also reported While reports about Annexin 8 are rare a study by
overexpressing AnnAt8 in Arabidopsis and tobacco showed enhanced abiotic stress
tolerance in the transgenic lines (Yadav et al 2016) Therefore the identified
candidate gene Annexin 8 could be taken into consideration for the QTL found in
2H in this study
Transcription factors (TFs) are DNA-binding domains containing proteins that
initiate the process of converting DNA to RNA (Latchman 1997) which regulate
downstream activities including stress responsive genes expression (Agarwal and
Jha 2010) In Arabidopsis thaliana 1500 TFs were described to be involved in this
process (Riechmann et al 2000) According to our genomic analysis in this study
three transcription factors in the vicinity of nearest markers were observed
including NAC transcription factor and AP2-like ethylene-responsive transcription
factor on 5H and WRKY family transcription factor on 7H (Table 53) Indeed
previous studies about these transcription factors have been well-documented
(Nakashima et al 2012 Licausi et al 2013 Nuruzzaman et al 2013 Rinerson et
al 2015 Guo et al 2016 Jiang et al 2017) indicating their role in plant stress
responses
Protein phosphatases type 2C (PP2Cs) may also be potential target genes
They constitute one of the classes of protein serinethreonine phosphatases sub-
family which form a structurally and functionally unique class of enzymes
(Rodriguez 1998 Meskiene et al 2003) They are also known as evolutionary
conserved from prokaryotes to eukaryotes and playing vital role in stress signalling
pathways (Fuchs et al 2013) Recent studies have demonstrated that
overexpression of PP2C in rice (Singh et al 2015) and tobacco (Hu et al 2015)
resulted in enhanced salt tolerance in the related transgenic lines Its function in
barley deserves further verification
Chapter 6 High-throughput assay
71
Chapter 6 Developing a high-throughput
phenotyping method for oxidative stress tolerance
in cereal roots
61 Introduction
Both global climate change and unsustainable agricultural practices resulted
in significant soil salinization thus reducing crop yields (Horie et al 2012 Ismail
and Horie 2017) Until now more than 20 of the worldrsquos agricultural land (which
accounts for 6 of the worldrsquos total land) has been affected by excessive salts this
number is increasing daily ( Ismail and Horie 2017 Gupta and Huang 2014) Given
the fact that more food need to be acquired from the limited arable land to feed the
expanding world population in the next few decades (Brown and Funk 2008 Ruan
et al 2010 Millar and Roots 2012) generating crop germplasm which can grow
in high-salt-content soil is considering a major avenue to fully utilise salt-affected
land (Shabala 2013)
One of constraints imposed by salinity stress on plants is an excessive
production and accumulation of reactive oxygen species (ROS) causing oxidative
stress This results in a major perturbation to cellular ionic homeostasis (Demidchik
2015) and in extreme cases has severe damage to plant lipids DNA proteins
pigments and enzymes (Ozgur et al 2013 Choudhury et al 2017) Plants deal
with excessive ROS production by increased activity of antioxidants (AO)
However given the fact that AO profiles show strong time- and tissue- (and even
organelle-specific) dependence and in 50 cases do not correlate with salinity
stress tolerance (Bose et al 2014b) the use of AO activity as a biochemical marker
for salt tolerance is highly questionable (Tanveer and Shabala 2018)
In chapter 3 and 4 we have shown that roots of salt-tolerant barley and wheat
varieties possessed greater K+ retention and lower Ca2+ uptake when challenged
with H2O2 These ionic traits were measured by using the MIFE (microelectrode
ion flux estimation) technique We have then applied MIFE to DH (double haploid)
barley lines revealing a major QTL for the above flux traits in chapter 5 These
findings open exciting prospects for plant breeders to screen germplasm for
oxidative stress tolerance targeting root-based genes regulating ion homeostasis
Chapter 6 High-throughput assay
72
and thus conferring salinity stress tolerance The bottleneck in application of this
technique in breeding programs is a currently low throughput capacity and
technical complications for the use of the MIFE method
The MIFE technique works as a non-invasive mean to monitor kinetics of ion
transport (uptake or release) across cellular membranes by using ion-selective
microelectrodes (Shabala et al 1997) This is based on the measurement of
electrochemical gradients near the root surface The microelectrodes are made on a
daily basis by the user by filling prefabricated pulled microcapillary with a sharp
tip (several microns diameter) with specific backfilling solution and appropriate
liquid ionophore specific to the measured ion Plant roots are mounted in a
horizontal position in a measuring chamber and electrodes are positioned in a
proximity of the root surface using hand-controlled micromanipulators Electrodes
are then moved in a slow square-wave 12 sec cycle measuring ion diffusion
profiles (Shabala et al 2006) Net ion fluxes are then calculated based on measured
voltage gradients between two positions close to the root surface and some
distance (eg 50 microm) away The method is skill-demanding and requires
appropriate training of the personnel The initial setup cost is relatively high
(between $60000 and $100000 depending on a configuration and availability of
axillary equipment) and the measurement of one specimen requires 20 to 25 min
Accounting for the additional time required for electrodes manufacturing and
calibration one operator can process between 15 and 20 specimens per business
day using developed MIFE protocols in chapter 3 As breeders are usually
interested in screening hundreds of genotypes the MIFE method in its current form
is hardly applicable for such a work
In this work we attempted to seek much simpler alternative phenotyping
methods that can be used to screen cereal plants for oxidative stress tolerance In
order to do so we developed and compared two high-throughput assays (a viability
assay and a root growth assay) for oxidative stress screening of a representative
cereal crop barley (Hordeum vulgare) The biological rationale behind these
approaches lies in a fact that ROS-induced cytosolic K+ depletion triggers
programmed cell death (Shabala 2007 Shabala 2009 Demidchik at al 2010) and
results in the loss of cell viability This effect is strongest in the root apex (Shabala
et al 2016) and is associated with an arrest of the root growth Reliability and
Chapter 6 High-throughput assay
73
feasibility of these high-throughput assays for plant breeding for oxidative stress
tolerance are discussed in this paper
62 Materials and methods
621 Plant materials and growth conditions
Eleven barley (ten Hordeum vulgare L and one H vulgare ssp Spontaneum)
varieties contrasting in salinity tolerance were used in this study All seeds were
obtained from the Australian Winter Cereal Collection The list of varieties is
shown in Table 61 Seedlings for experiment were grown in paper roll (see 222
for details)
Treatment with H2O2 was started at two different age points 1 d and 3 d and
lasted until plant seedlings reached 4 d of growth at which point assessments were
conducted so that in both cases 4-d old plants were assayed Concentrations of H2O2
ranged from 0 to 10 mM Fresh solutions were made on a daily basis to compensate
for a possible decrease of H2O2 activity
Table 61 Barley varieties used in the study The damage index scores represent
quantified damage degree of barley under salinity stress with scores from 0 to
10 indicating barley overall salinity tolerance from the best (0) to the worst (10)
(see Wu et al 2015 for details)
Varieties Damage Index Score
SYR01 025
TX9425 100
CM72 120
YYXT 145
Numar 170
ZUG293 170
Hu93-045 325
ZUG403 570
Naso Nijo 750
Kinu Nijo 6 845
Unicorn 945
Chapter 6 High-throughput assay
74
622 Viability assay
Viability assessment of barley root cells was performed using a double staining
method that included fluorescein diacetate (FDA Cat No F7378 Sigma-Aldrich)
and propidium iodide (PI Cat No P4864 Sigma-Aldrich) (Koyama et al 1995)
Briefly control and H2O2-treated root segments (about 5 mm long) were isolated
from both a root tip and a root mature zone (20 to 30 mm from the root tip) stained
with freshly prepared 5 microgml FDA for 5 min followed by 3 microgml PI for 10 min
and washed thoroughly with distilled water Stained root segment was placed on a
microscope slide covered with a cover slip and assessed immediately using a
fluorescent microscope Staining and slide preparation were done in darkness A
fluorescent microscope (Leica MZ12 Leica Microsystems Wetzlar Germany)
with I3-wavelength filter (Leica Microsystems) and illuminated by an ultra-high-
pressure mercury lamp (Leica HBO Hg 100 W Leica Microsystems) was used to
examine stained root segments The excitation and emission wavelengths for FDA
and PI were 450 ndash 495 nm and 495 ndash 570 nm respectively Photographs were taken
by a digital camera (Leica DFC295 Leica Microsystems) Images were acquired
and processed by LAS V38 software (Leica Microsystems) The exposure features
of the camera were set to constant values (gain 10 x saturation 10 gamma 10) in
each experiment allowing direct comparison of various genotypes For untreated
roots the exposure time was 591 ms for H2O2-treated roots it was increased to 19
s The overview of the experimental protocol for viability assay by the FDA - PI
double staining method is shown in Figure 61 The ImageJ software was used to
quantify red fluorescence intensity that is indicative of the proportion of dead cells
Images of H2O2-treated roots were normalised using control (untreated) roots as a
background
Chapter 6 High-throughput assay
75
Figure 61 Viability staining and fluorescence image acquisition (A) Isolated
root segments from control (C) and treatment (T) seedlings placed in a Petri dish
(35 mm diameter) separated with a cut yellow pipette tip for convenience
stained with FDA followed by PI (B) Stained and washed root segments
positioned on a glass slide and covered with a cover slip The prepared slide was
then placed on a fluorescent microscope mechanical stage (C) Sample area
observed under the fluorescent light (D) A typical root fluorescent image
acquired by the LAS V38 software from mature root zone of a control plant
623 Root growth assay
Root lengths of 4-d old barley seedlings were measured after 3 d of treatments
with various concentrations of H2O2 ranging between 0 and 10 mM (0 01 03 1
Chapter 6 High-throughput assay
76
3 10 mM) The relative root lengths (RRL) were estimated as percentage of root
lengths to controls of the respective genotypes
624 Statistical analysis
Statistical significance of mean values was determined by the standard
Studentrsquos t -test at P lt 005 level
63 Results
631 H2O2 causes loss of the cell viability in a dose-dependent
manner
Barley variety Naso Nijo was used to study dose-dependent effects of H2O2 on
cell viability The concentrations of H2O2 used were from 03 to 10 mM Both 1 d-
(Figure 62A) and 3 d- (Figure 62B) exposure to oxidative stress caused dose-
dependent loss of the root cell viability One-day H2O2 treatment was less severe
and was observed only at the highest H2O2 concentration used (Figure 62A) When
roots were treated with H2O2 for 3 days the red fluorescence signal can be readily
observed from H2O2 treatments above 3 mM (Figure 62B)
Figure 62 Viability staining of Naso Nijo roots (elongation and mature zones)
exposed to 0 03 1 3 10 mM H2O2 for 1 day (A) and 3 days (B) One (of five)
typical images is shown from each concentration and root zone Bar = 1 mm
Chapter 6 High-throughput assay
77
Quantitative analyses of the red fluorescence intensity were implemented in
order to translate images into numerical values (Figure 63) Mild root damage was
observed upon 1 d H2O2 treatment and there was no significant difference between
elongation zone and mature zone for any concentration used (Figure 63A) Similar
findings (eg no difference between two zones) were observed in 3 d H2O2
treatment when the concentration was low (le 3 mM) (Figure 63B) Application of
10 mM H2O2 resulted in severe damage to root cells and clearly differentiated the
insensitivity difference between the two root zones with elongation zone showing
more severe root damage compared to the mature zone (Figure 63B significant at
P ˂ 005) Accordingly 10 mM H2O2 with 3 d treatment was chosen as the optimum
experimental treatment for viability staining assays on contrasting barley varieties
Figure 63 Red fluorescence intensity (in arbitrary units) measured from roots
of Naso Nijo upon exposure to various H2O2 concentrations for either one day
(A) or three days (B) Mean plusmn SE (n = 5 individual plants)
632 Genetic variability of root cell viability in response to 10 mM
H2O2
Five contrasting barley varieties (salt tolerant CM72 and YYXT salt sensitive
ZUG403 Naso Nijo and Unicorn) were employed to explore the extent of root
damage upon oxidative stress by the means of viability staining of both elongation
and mature root zones A visual assessment showed clear root damage upon 3 d-
exposure to 10 mM H2O2 in all barley varieties and both root zones and damage in
the elongation zone was more severe than in the mature zone (Figures 62B and
64)
Chapter 6 High-throughput assay
78
Figure 64 Viability staining of root elongation (A) and mature (B) zones of four
barley varieties (CM72 YYXT ZUG403 Unicorn) exposed to 10 mM H2O2 for
3 days One (of five) typical images is shown for each zone Bar = 1 mm
The quantitative analyses of the fluorescence intensity revealed that salt
sensitive varieties showed stronger red fluorescence signal in the root elongation
zone than tolerant ones (Figure 65A) indicating much severe root damage of the
sensitive genotypes By pooling sensitive and tolerant varieties into separate
clusters a significant (P ˂ 001) difference between two contrasting groups was
observed (Figure 65B) In mature root zone however no significant difference
was observed amongst the root cell viability of five contrasting varieties studied
(Figure 65C)
Chapter 6 High-throughput assay
79
Figure 65 Quantitative red fluorescence intensity from root elongation (A) and
mature zones (C) of five barley varieties exposed to 10 mM H2O2 for 3 d (B)
Average red fluorescence intensity measured from root elongation zone of salt
tolerant and salt sensitive barley groups Mean plusmn SE (n = 6) Asterisks indicate
statistically significant differences between salt tolerant and sensitive varieties
at P lt 001 (Studentrsquos t-test)
The results in this section were consistent with our findings in chapter 3 and 4
using MIFE technique which elucidated that not only oxidative stress-induced
transient ions fluxes but also long-term root damage correlates with the overall
salinity tolerance in barley
Based on these findings we can conclude that plant oxidative and salinity
stress tolerance can be quantified by the viability staining of roots treated with 10
mM H2O2 for 3 days that would include staining the root tips with FDA and PI and
then quantifying intensity of the red fluorescence signal (dead cells) from root
elongation zone This protocol is simpler and quicker than MIFE assessment and
requires only a few minutes of measurements per sample making this assay
compliant with the requirements for high throughput assays
Chapter 6 High-throughput assay
80
633 Methodological experiments for cereal screening in root
growth upon oxidative stress
Being a high throughput in nature the above imaging assay still requires
sophisticated and costly equipment (eg high-quality fluorescence camera
microscope etc) and thus may be not easily applicable by all the breeders This
has prompted us to go along another avenue by testing root growth assays Two
contrasting barley varieties TX9425 (salt tolerant) and Naso Nijo (salt sensitive)
were used for standardizing concentration of ROS (H2O2) treatment in preliminary
experiments After 3 d of H2O2 treatment root length declined in both the varieties
for any given concentration tested (01 03 1 3 10 mM) and salt tolerant variety
TX9425 grew better (had higher relative root length RRL) than salt sensitive
variety Naso Nijo at each the treatment used (Figure 66A) The decreased RRL
showed the dose-dependency upon increasing H2O2 concentration with a strong
difference (P ˂ 0001) occurring from 1 to 10 mM H2O2 treatments between the
contrasting varieties (Figure 66A) The biggest difference in RRL between the
varieties was observed under 1 mM H2O2 treatment (Figure 66A) which was
chosen for screening assays
Chapter 6 High-throughput assay
81
Figure 66 (A) Relative root length of TX9425 and Naso Nijo seedlings treated
with 0 01 03 1 3 10 mM H2O2 for 3 d Mean plusmn SE (n =14) Asterisks indicate
statistically significant differences between two varieties at P lt 0001 (Studentrsquos
t-test) (B) Genetic variability in the relative root length in 11 barley varieties
treated with 1 mM H2O2 for 3 d Mean plusmn SE (n =14) (C) Correlation between
H2O2ndashtreated relative root length and the overall salinity tolerance (damage
index see Table 61) of 11 barley varieties
634 H2O2ndashinduced changes of root length correlate with the
overall salinity tolerance
Eleven barley varieties were selected to test the relationship between the root
growth under oxidative stress and their overall salinity tolerance under 1 mM H2O2
treatment After 3 d exposure to 1 mM H2O2 the relative root length (RRL) of all
the barley varieties reduced rapidly ranging from the lowest 227 plusmn 03 (in the
variety Unicorn) to the highest 632 plusmn 2 (in SYR01) (Figure 66B) The RRL
values were then correlated with the ldquodamage index scoresrdquo (Table 61) a
quantitative measure of the extent of salt damage to plants provided by the visual
assessment on a 0 to 10 score (0 = no symptoms of damage 10 = completely dead
Chapter 6 High-throughput assay
82
plants see section 324 for more details) A significant correlation (r2 = 094 P ˂
0001) between RRL and the overall salinity tolerance was observed (Figure 66C)
indicating a strong suitability of the RRL assay method as a proxy for
oxidativesalinity stress tolerance Given the ldquono cost no skillrdquo nature of this
method it can be easily taken on board by plant breeders for screening the
germplasm and mapping QTLs for oxidative stress tolerance (one of components
of the salt tolerance mechanism)
64 Discussion
641 H2O2 causes a loss of the cell viability and decline of growth
in barley roots
H2O2 is one of the major ROS produced in plant tissues under stress conditions
that leads to oxidative damage The effect of this stable oxidant on plant cell
viability and root growth was investigated in this study Both parameters decreased
in a dose- andor time-dependent manner upon H2O2 exposure (Figures 62 and
66A 66B) The physiological rationale behind these observations may lay in a
fact that exogenous application of H2O2 causes instantaneous [K+]cyt and [Ca2+]cyt
changes in different root zones
Stress-induced enhanced K+ leakage from root epidermis results in depletion
of cytosolic K+ pool (Shabala et al 2006) thus activating caspase-like proteases
and endonucleases and triggering PCD (Shabala 2009 Demidchik et al 2014)
leading to deleterious effect on plant viability (Shabala 2017) This is reflected in
our findings that roots lost their viability after being treated with H2O2 especially
upon higher dosage and long-term exposure (Figure 63) Furthermore K+ is
required for root cell expansion (Walker et al 1998) and plays a key role in
stimulating growth (Nieves-Cordones et al 2014 Demidchik 2014) Therefore
the loss of a large quantity of cytosolic K+ might be the primary reason for the
inhibition of the root elongation in our experiments (Figure 66A 66B) This is
consistent with root growth retardation observed in plants grown in low-K+ media
(Kellermeier et al 2013)
High concentration of cytosolic K+ is essential for optimizing plant growth
and development Also essential is maintenance of stable (and relatively low)
Chapter 6 High-throughput assay
83
levels of cytosolic free Ca2+ (Hepler 2005 Wang et al 2013) Therefore H2O2-
induced cytosolic Ca2+ disequilibrium may be another contributing factor to the
observed loss of cell viability and reported decrease in the relative root length in
this study (Figures 64 and 66A 66B) In our previous chapters we showed that
plants responded to H2O2 by increased Ca2+ uptake in mature root epidermis This
is expected to result in [Ca2+]cyt elevation that may be deleterious to plants as it
causes protein and nucleic acids aggregation initiates phosphates precipitation and
affects the integrity of the lipid membranes (Case et al 2007) It may also make
cell walls less plastic through rigidification thus inhibiting cell growth (Hepler
2005) In root tips however increased Ca2+ loading is required for the stimulation
of actinmyosin interaction to accelerate exocytosis that sustains cell expansion and
elongation (Carol and Dolan 2006) The rhd2 Arabidopsis mutant lacking
functional NADPH oxidase exhibited stunted roots as plants were unable to
produce sufficient ROS to activate Ca2+-permeable NSCCs to enable Ca2+ loading
into the cytosol (Foreman et al 2003)
642 Salt tolerant barley roots possess higher root viability in
elongation zone after long-term ROS exposure
It was argued that the ROS-induced self-amplification mechanism between
Ca2+-activated NADPH oxidases and ROS-activated Ca2+-permeable cation
channels in the plasma membrane and transient K+ leakage from cytosol may be
both essential for the early stress signalling (Shabala et al 2015 Shabala 2017
Demidchik and Shabala 2018) As salt sensing mechansim is most likely located in
the root meristem (Wu et al 2015) this may explain why the correlation between
the overall salinity tolerance and H2O2-induced transient ions fluxes was not found
in this zone in short-term experiments (see Chapter 3 for detailed finding) Under
long-term H2O2 exposures however (as in this study) we observed less severe root
damage in the elongation zone in salt tolerant varieties (Figure 65A 65B) This
suggested a possible recovery of these genotypes from the ldquohibernated staterdquo
(transferred from normal metabolism by reducing cytosolic K+ and Ca2+ content for
salt stress acclimation) to stress defence mechanisms (Shabala and Pottosin 2014)
which may include a superior capability in maintaining more negative membrane
potential and increasing the production of metabolites in this zone (Shabala et al
Chapter 6 High-throughput assay
84
2016) This is consistent with a notion of salt tolerant genotypes being capable of
maintaining more negative membrane potential values resulting from higher H+-
ATPases activity in many species (Chen et al 2007b Bose et al 2014a Lei et al
2014) and the fact that a QTL for the membrane potential in root epidermal cells
was colocated with a major QTL for the overall salinity stress tolerance (Gill et al
2017)
In the mature root zone the salt-sensitive varieties possessed a higher transient
K+ efflux in response to H2O2 yet no major difference in viability staining was
observed amongst the genotypes in this root zone after a long-term (3 d) H2O2
exposure (Figure 64B and 65C) This is counterintuitive and suggests an
involvement of some additional mechanisms One of these mechanisms may be a
replenishing of the cytosolic K+ pool on the expense of the vacuole As a major
ionic osmoticum in both the cytosolic and vacuolar pools potassium has a
significant role in maintaining cell turgor especially in the latter compartment
(Walker et al 1996) Increasing cytosolic Ca2+ was first shown to activate voltage-
independent vacuolar K+-selective (VK) channels in Vicia Faba guard cells (Ward
and Schroeder 1994) mediating K+ back leak into cytosol from the vacuole pool
This observation was later extended to cell types isolated from Arabidopsis shoot
and root tissues (Gobert et al 2007) as well as other species such as barley rice
and tobacco (Isayenkov et al 2010) Thus the higher Ca2+ influx in sensitive
varieties upon H2O2 treatment is expected to increase their cytosolic free Ca2+
concentration thus inducing a strong K+ leak from the vacuole to compensate for
the cytosolic K+ loss from ROS-activated GORK channel This process will be
attenuated in the salt tolerant varieties which have lower H2O2-induced Ca2+ uptake
As a result 3 days after the stress onset the amount of K+ in the cytosol in mature
root zone may be not different between contrasting varieties explaining the lack of
difference in viability staining
643 Evaluating root growth assay screening for oxidative stress
tolerance
A rapid and revolutionary progress in plant molecular breeding has been
witnessed since the development of molecular markers in the 1980s (Nadeem et al
2018) At the same time the progress in plant phenotyping has been much slower
Chapter 6 High-throughput assay
85
and in most cases lack direct causal relationship with the traits targeted However
future breeding programmes are in a need of sensitive low cost and efficient high-
throughput phenotyping methods The novel approach developed in chapter 3
allowed us to use the MIFE technique for the cell-based phenotyping for root
sensitivity to ROS one of the key components of mechanism of salinity stress
tolerance Being extremely sensitive and allowing directly target operation of
specific transport proteins this method is highly sophisticated and is not expected
to be easily embraced by breeders In this study we provided an alternative
approach namely root growth assay which can be used as the high-throughput
phenotyping method to replace the sophisticated MIFE technique This screening
method has minimal space requirements (only a small growth room) and no
measuring equipment except a simple ruler Assuming one can acquire 5 length
measurements per minute and 15 biological replicates are sufficient for one
genotype the time needed for one genotype is just three minutes which means one
can finish the screening of 100 varieties in 5 h This is a blazing fast avenue
compared to most other methods This offers plant breeders a convenient assay to
screen germplasm for oxidative stress tolerance and identify root-based QTLs
regulating ion homeostasis and conferring salinity stress tolerance
Chapter 7 General conclusion and future prospects
86
Chapter 7 General discussion and future prospects
71 General discussion
Soil salinity is a major global issue threatening cereal production worldwide
(Shrivastava and Kumar 2015) The majority of cereals are glycophytes and thus
perform poorly in saline soils (Hernandez et al 2000) Therefore developing salt
tolerant crops is important to ensure adequate food supply in the coming decades
to meet the demands of the increasing population Generally the major avenues
used to produce salt tolerant crops have been conventional breeding and modern
biotechnology (Flowers and Flowers 2005 Roy et al 2014) However due to
some obvious practical drawbacks (Miah et al 2013) the former has gradually
given way to the latter Marker assisted selection (MAS) and genetic engineering
are the two known modern biotechnologies (Roy et al 2014) MAS is an indirect
selection process of a specific trait based on the marker(s) linked to the trait instead
of selecting and phenotyping the trait itself (Ribaut and Hoisington 1998 Collard
and Mackill 2008) While genetic engineering can be achieved by either
introducing salt-tolerance genes or altering the expression levels of the existing salt
tolerance-associated genes to create transgenic plants (Yamaguchi and Blumwald
2005) Given the fact that the application of transgenic crop plants is rather
controversial and the MAS technique can facilitate the process of pyramiding traits
of interest to improve crop salt tolerance substantially (Yamaguchi and Blumwald
2005 Collard and Mackill 2008) the latter may be more acceptable in plant
breeding pipeline However exploring the detailed characteristics of QTLs needs
the combination of both biotechnologies
Oxidative stress tolerance is one of the components of salinity stress tolerance
This trait has been usually considered in the context of ROS detoxification
However being both toxic agents and essential signalling molecules ROS may
have pleiotropic effects in plants (Bose et al 2014b) making the attempts in
pyramiding major antioxidants-associated QTLs for salinity stress tolerance
unsuccessful Besides ROS are also able to activate a range of ion channels to cause
ion disequilibrium (Demidichik et al 2003 2007 2014 Demidchik and Maathuis
2007) Indeed several studies have revealed that both H2O2 and bullOH-induced ion
Chapter 7 General conclusion and future prospects
87
fluxes showed their distinct difference between several barley varieties contrasting
in their salt stress tolerance (Chen et al 2007a Maksimović et al 2013 Adem et
al 2014) and different cell type showed different sensitivity to ROS (Demidichik
et al 2003) Since wheat and barley are two major grain crops cultivated all over
the world with sufficient natural genetic variations for exploitation the attempts of
producing salt tolerant cereals using proper selection processes (such as MAS) with
proper ROS-related physiological markers (such as ROS on cell ionic relations)
would deserve a trial Funded by Grain Research amp Development Corporation and
aimed at understanding ROS sensitivity in a range of cereal (wheat and barley)
varieties in various cell types and validating the applicability of using ROS-induced
ion fluxes as a physiological marker in breeding programs to improve plant salinity
stress tolerance we established a causal association between ROS-induced ion
fluxes and plants overall salinity stress tolerance validated the applicability of the
above marker identified major QTLs associated with salinity stress tolerance in
barley and found an alternative high-throughput phenotyping method for oxidative
stress tolerance in cereal roots
The major findings in this project were (i) the magnitude of H2O2-induced K+
and Ca2+ fluxes from root mature zone of both wheat and barley correlated with
their overall salinity stress tolerance (ii) H2O2-induced K+ and Ca2+ fluxes from
mature root zone of cereals can be used as a novel physiological trait of salinity
stress tolerance in plant breeding programs (iii) major QTLs for ROS-induced K+
and Ca2+ flux associated with salinity stress tolerance in barley were identified on
chromosome 2 5 and 7 (iv) root growth assay was suggested as an alternative
high-throughput phenotyping method for oxidative stress tolerance in cereal roots
H2O2 and bullOH are two frequently mentioned ROS in plants with the former
has a half-life in minutes and the latter less than 1 μs (Pitzschke et al 2006 Bose
et al 2014b) This determines the property of H2O2 to diffuse freely for long
distance making it suitable for the role of signalling molecule Therefore it is not
surprising that the correlation between cereals overall salinity stress tolerance and
ROS-induced K+ efflux and Ca2+ uptake were found under H2O2 treatment but not
bullOH At the same time we also found that H2O2-induced K+ and Ca2+ fluxes showed
some cell-type specificity with the above correlation only observed in root mature
zone The recently emerged ldquometabolic switchrdquo concept indicated that high K+
efflux from the elongation zone in salt-tolerant varieties can inactivate the K+-
Chapter 7 General conclusion and future prospects
88
dependent enzymes and redistribute ATP pool towards defence responses for stress
adaptation (Shabala 2007) which may explain the reason of the lack of the above
correlation in root elongation zone It should be also commented that different cell
types show diverse sensitivity to specific stimuli and are adapted for specific andor
various functions due to the different expression level of genes in that tissue so it
is important to pyramid trait in a specific cell type in breeding program
In order to validate the above correlations a range of barley bread wheat and
durum wheat varieties were screened using the developed protocol above We
showed that H2O2-induced K+ and Ca2+ fluxes in root mature zone correlated with
the overall salinity stress tolerance in barley bread wheat and durum wheat with
salt sensitive varieties leaking more K+ and acquiring more Ca2+ These findings
also indicate the applicability of using the MIFE technique as a reliable screening
tool and H2O2-induced K+ and Ca2+ fluxes as a new physiological marker in cereal
breeding programs Due to the fact that previous studies on oxidative stress mainly
focused on AO activity our newly developed oxidative stress-related trait in this
study may provide novel avenue in exploring the mechanism of salinity stress
Previous efforts in pyramiding AO QTLs associated with salinity stress
tolerance in tomato was unsuccessful because more than 100 major QTLs has been
identified (Frary et al 2010) making QTL mapping of this trait practically
unfeasible Besides no major QTL associated with oxidative stress-induced control
of plant ion homeostasis has been reported yet in any crop species Here in this
study by using the aforementioned physiological marker of salinity stress tolerance
and genetic linkage map with DNA markers we identified three QTLs associated
with H2O2-induced Ca2+ and K+ fluxes for salinity stress tolerance in barley based
on the correlation found between these two traits These QTLs were located on
chromosome 2 5 and 7 respectively with the QTLs on 2H and 7H controlling both
K+ flux and Ca2+ flux and the QTL on 5H only involved in K+ flux H2O2-induced
K+ efflux is known to be mediated by GROK and K+-permeable NSCC
(Demidichik et al 2003 2014) while H2O2-induced Ca2+ uptake is mediated by
Ca2+-permeable NSCCs (Demidichik et al 2007 Demidchik and Maathuis 2007)
Taken together these two types of NSCC may exhibit some similarity since the
same QTLs from 2H and 7H were observed to control both ion flux While the one
on 5H controlling K+ efflux may be related to GORK channel Given the fact that
this is the very first time the major oxidative stress-associated QTLs being
Chapter 7 General conclusion and future prospects
89
identified it warrants in-depth study in this direction Accordingly several
potential genes comprise of calcium-dependent proteins protein phosphatase and
stress-related transcription factors were chosen for further investigation
The above findings open previously unexplored prospects of improving
salinity tolerance by pyramiding H2O2-induced Ca2+ and K+ fluxes However the
bottleneck of many breeding programs for salinity stress tolerance is a lack of
accurate plant phenotyping method In this study although we have proved that
H2O2-induced Ca2+ and K+ fluxes measured by using MIFE technique is reliable
for screening for salinity stress tolerance this method is too complicated with rather
low throughput capacity This poses a need to find a simple phenotyping method
for large scale screening Field screening for grain yield for example might be the
most reliable indicator Besides Plant above-ground performance such as plant
height and width plant senescence chlorosis and necrosis etc (Gaudet and Paul
1998) also reflect the overall plant performance as plant growth is an integral
parameter (Hunt et al 2002) However given the fact that these methods are time-
space- and labour-consuming and it is also affected by many other uncontrollable
factors such as temperature nutrition water content and wind screening in the
field becomes extremely unreliable and difficult Biochemical tests (measurements
of AO activity) are simple and plausible for screening But this method does not
work all the time because the properties of AO profiles are highly dynamic and
change spatially and temporally making it not reliable for screening Here we have
tested and compared two high-throughput phenotyping methods ndash root viability
assay and root growth assay ndash under H2O2 stress condition We then observed the
similar results with that of MIFE method and deemed root growth assay as a proxy
due to the fact that it does not need any specific skills and training and has the
minimal space and simple tool (a ruler) requirements which can be easily handled
by anyone
72 Future prospects
The establishment of a causal relationship between oxidative stress and
salinity stress tolerance in cereals using MIFE technique the identification of novel
QTLs for salinity tolerance under oxidative stress condition in barley and the
finding of using root growth assay as a simple high-throughput phenotyping
Chapter 7 General conclusion and future prospects
90
method for oxidative stress tolerance screening are valuable to salt stress tolerance
studies in cereals These findings improved our understanding on effects of stress-
induced ROS accumulation on cell ionic relations in different cell types and
opened previously unexplored prospects for improving salinity tolerance The
further progress in the field may be achieved addressing the following issues
i) Investigating the causal relationship between oxidative stress and other
stress factors in crops using MIFE technique
ROS production is a common denominator of literally all biotic and abiotic
stress (Shabala and Pottosin 2014) However studies in ROS has been largely
emphasised on their detoxification by a range of antioxidants ignoring the fact that
basal level of ROS are also indispensable and playing signalling role in plant
biology Although the generated ROS signal upon different stresses to trigger
appropriate acclimation responses may show some specificity (Mittler et al 2011)
our success in revealing a causal link between oxidative and salinity stress tolerance
by applying ROS exogenously and measuring ROS-induced ions flux may worth a
decent trial in correlation with other stresses such as drought flooding heavy metal
toxicity or temperature extremes
ii) Verifying chosen candidate genes and picking out the most likely genes
for further functional analysis
Using a DH population derived from CM72 and Gairdner three major QTLs
have been identified in this study and eight potential genes were chosen including
four calcium-dependent proteins three transcription factors and PP2C protein
through our genetic analysis A differential expression analysis of the potential
genes can be conducted to pick out the most likely genes for further functional
analysis Typically gene function can be investigated by changing its expression
level (overexpression andor inactivation) in plants (Sitnicka et al 2010) In this
study the identified QTLs were controlling K+ efflux andor Ca2+ uptake upon the
onset of ROS therefore any inactivation of the genes may have a positive effect
(eg plants leaking less K+ andor acquire less Ca2+) Conventionally the basic
principle of gene knockout was to introduce a DNA fragment into the site of the
target gene by homological recombination to block its expression This DNA
fragment can be either a non-coding fragment or deletion cassette (Sitnicka et al
2010) However this technique is less efficient with high expenses In recent years
Chapter 7 General conclusion and future prospects
91
researcher have developed alternative gene-editing techniques to achieve the above
goal such as ZNFs (Zinc finger nucleases) (Petolino 2015) TALENs
(Transcription activator-like effector nucleases) (Joung and Sander 2015) and
CRISPR (clustered regularly interspaced short palindromic repeats)Cas
(CRISPR-associated) system (Ran et al 2013 Ledford 2015) among which
CRISPRCas system has become revolutionized and the most widespread technique
in a range of research fields due to its high-efficiency target design simplicity and
generation of multiplexed mutations (Paul and Qi 2016)
CRISPRCas9 is a frequently mentioned version of the CRISPRCas system
which contains the Cas9 protein and a short non-coding gRNA (guide RNA) that
is composed of two components a target-specific crRNA (CRISPR RNA) and a
tracrRNA (trans-activating crRNA) The target sequence can be specified by
crRNA via base pairing between them and cleaved by Cas9 protein to induce a
DSB (double-stranded break) DNA damage repair machinery then occurs upon
cleavage which would then result in error-prone indel (insertiondeletion)
mutations to achieve gene knockout purpose (Ran et al 2013) This genetic
engineering technique has been widely used for genome editing in plants such as
Arabidopsis barley wheat rice soybean Brassica oleracea tomato cotton
tobacco etc (Malzahn et al 2017) Therefore after picking out the most likely
genes in this study it would be a good choice to perform the subsequent gene
functional analysis study using CRISPRCas9 gene editing technique
Functions of candidate genes in this study can also be investigated by
overexpression This can be achieved by vector construction for gene
overexpression (Lloyd 2003) and a subsequent Agrobacterium-mediated
transformation of the constructed vector into plant cell (Karimi et al 2002)
iii) Pyramiding the new developed trait (H2O2-induced Ca2+ and K+ fluxes)
alongside with other mechanisms of salinity stress tolerance
Salinity tolerance is a complex and multi-genic trait which is attributed to a
range of biological mechanisms (Shabala et al 2010 Wu et al 2015) Therefore
it is highly unlikely that modification of one gene would result in great
improvements Oxidative stress can occur in any biotic and abiotic stress conditions
When plants are under salinity stress the knockout of gene(s) controlling ROS-
induced Ca2+ andor K+ fluxes may partly relief the adverse effect caused by the
associated oxidative stress and confer plants salinity stress tolerance At the same
Chapter 7 General conclusion and future prospects
92
time if pyramiding the above process with other traditional mechanisms of salinity
stress tolerance such as Na+ exclusion and osmotic adjustment it may provide
double or several fold cumulative effect in improving plants salinity stress tolerance
This may include a knockout of the candidate gene in this study alongside with an
overexpression of the SOS1 or HKT1 gene or introduction of the glycine betaine
biosynthesis gene such as codA betA and betB into plants
References
93
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Ache P Becker D Ivashikina N Dietrich P Roelfsema MRG Hedrich R (2000)
GORK a delayed outward rectifier expressed in guard cells of Arabidopsis
thaliana is a K+‐selective K+‐sensing ion channel FEBS Lett 486 93ndash98
Adem GD Roy SJ Zhou M Bowman JP Shabala S (2014) Evaluating contribution
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Aharon GS Apse MP Duan SL Hua XJ Blumwald E (2003) Characterization of
a family of vacuolar Na+H+ antiporters in Arabidopsis thaliana Plant Soil
253 245ndash256
Ahmad P Jaleel CA Salem MA Nabi G Sharma S (2010) Roles of enzymatic and
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Alfocea FP Balibrea ME Alarcon JJ Bolarin MC (2000) Composition of xylem
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Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants Plant
Physiol 107 1049ndash1054
Agarwal PK Jha B (2010) Transcription factors in plants and ABA dependent and
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Amtmann A Fischer M Marsh EL Stefanovic A Sanders D Schachtman DP
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Amtmann A Sanders D (1998) Mechanisms of Na+ uptake by plant cells Adv Bot
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Anjum NA Sofo A Scopa A Roychoudhury A Gill SS Iqbal M Lukatkin AS
Pereira E Duarte AC Ahmad I (2015) Lipids and proteins ndash major targets of
oxidative modifications in abiotic stressed plants Environ Sci Pollut R 22
4099ndash4121
References
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Anschuumltz U Becker D Shabala S (2014) Going beyond nutrition regulation of
potassium homoeostasis as a common denominator of plant adaptive
responses to environment J Plant Physiol 171 670-687
Apel K Hirt H (2004) Reactive oxygen species metabolism oxidative stress and
signal transduction Annu Rev Plant Biol 55 373ndash399
Apse MP Aharon GS Snedden WA Blumwald E (1999) Salt tolerance conferred
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1256-1258
Asada K (1993) Molecular mechanism of production and scavenging of active
oxygen species in chloroplasts Nippon Nogeik Kaishi 67 1255-1263
Asada K (2006) Production and scavenging of reactive oxygen species in
chloroplasts and their functions Plant Physiol 141 391-396
Ashraf M Ali Q (2008) Relative membrane permeability and activities of some
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Azooz MM Ismail AM Elhamd MA (2009) Growth lipid peroxidation and
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Baik BK Ullrich SE (2008) Barley for food characteristics improvement and
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Baacutenfi B Tirone F Durussel I Knisz J Moskwa P Molnaacuter GZ Krause KH Cox
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Bari R Jones JD (2009) Role of plant hormones in plant defence responses Plant
Mol Biol 69 473ndash488
Barragan V Leidi EO Andres Z Rubio L De Luca A Fernandez JA Cubero B
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Bassil E Tajima H Liang YC Ohto M Ushijima K Nakano R Esumi T Coku A
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Chen Z Pottosin II Cuin TA Fuglsang AT Tester M Jha D Zepeda-Jazo I Zhou
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Chen Z Zhou M Newman IA Mendham NJ Zhang G Shabala S (2007c)
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Cuin TA Betts SA Chalmandrier R Shabala S (2008) A roots ability to retain K+
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Cuin TA Bose J Stefano G Jha D Tester M Mancuso S Shabala S (2011)
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Cuin TA Shabala S (2007) Compatible solutes reduce ROS-induced potassium
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Cuin TA Shabala S (2008) Compatible solutes mitigate damaging effects of salt
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Cuin TA Tian Y Betts SA Chalmandrier R Shabala S (2009) Ionic relations and
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Das K Roychoudhury A (2014) Reactive oxygen species (ROS) and response of
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Day IS Reddy VS Ali GS Reddy AS (2002) Analysis of EF-hand-containing
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Dbira S Al Hassan M Gramazio P Ferchichi A Vicente O Prohens J Boscaiu M
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Deinlein U Stephan AB Horie T Luo W Xu G Schroeder JI (2014) Plant salt-
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De la Garma JG Fernandez-Garcia N Bardisi E Pallol B Rubio-Asensio JS Bru
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Demidchik V Shabala S (2018) Mechanisms of cytosolic calcium elevation in
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Dietz KJ Mittler R Noctor G (2016) Recent progress in understanding the role of
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Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-
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Garciadeblas B Benito B Rodriguez-Navarro A (2001) Plant cells express several
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Gill SS Tuteja N (2010) Reactive oxygen species and antioxidant machinery in
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Gόmez JM Hernaacutendez JA Jimeacutenez A del Rίo LA Sevilla F (1999) Differential
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Gorji T Tanik A Sertel E (2015) Soil salinity prediction monitoring and mapping
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Gupta B Huang BR (2014) Mechanism of salinity tolerance in plants
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Halliwell B Gutteridge JMC (2015) In Free Radicals in Biology and Medicine 5th
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Hasanuzzaman M Hossain MA da Silva JAT Fujita M (2012) Plant response and
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Horie T Karahara I Katsuhara M (2012) Salinity tolerance mechanisms in
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Hosy E Vavasseur A Mouline K Dreyer I Gaymard F Poreacutee F Boucherez J
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Huang S Spielmeyer W Lagudah ES James RA Platten JD Dennis ES Munns
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Hu W Yan Y Hou X He Y Wei Y Yang G He G Peng M (2015) TaPP2C1 a
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Hu X Bidney DL Yalpani N Duvick JP Crasta O Folkerts O Lu G (2003)
Overexpression of a gene encoding hydrogen peroxide-generating oxalate
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Inoue H Kudo T Kamada H Kimura M Yamaguchi I Hamamoto H (2005)
Copper elicits an increase in cytosolic free calcium in cultured tobacco cells
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Ismail AM Horie T (2017) Genomics physiology and molecular breeding
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2939ndash2947
Jami SK Clark GB Turlapati SA Handley C Roux SJ Kirti PB (2008) Ectopic
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1019-1030
Jayakannan M Bose J Babourina O Rengel Z Shabala S (2013) Salicylic acid
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2268
Jiang CF Belfield EJ Mithani A Visscher A Ragoussis J Mott R Smith JAC
Harberd NP (2012) ROS-mediated vascular homeostatic control of root-to-
shoot soil Na delivery in Arabidopsis EMBO J 31 4359ndash4370
Jiang J Ma S Ye N Jiang M Cao J Zhang J (2017) WRKY transcription factors
in plant responses to stresses J Integr Plant Biol 59 86-101
Ji H Pardo JM Batelli G Van Oosten MJ Bressan RA Li X (2013) The Salt
Overly Sensitive (SOS) pathway established and emerging roles Mol Plant
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Jin Q Zhu K Cui W Xie Y Han BI Shen W (2013) Hydrogen gas acts as a novel
bioactive molecule in enhancing plant tolerance to paraquat‐induced
oxidative stress via the modulation of heme oxygenase‐1 signalling system
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Joo JH Bae YS Lee JS (2001) Role of auxin-induced reactive oxygen species in
root gravitropism Plant Physiol 126 1055ndash1060
Joung JK Sander JD (2013) TALENs a widely applicable technology for targeted
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Karimi M Inzeacute D Depicker A (2002) GATEWAYtrade vectors for Agrobacterium-
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Karuppanapandian T Moon JC Kim C Manoharan K Kim W (2011) Reactive
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Katschnig D Bliek T Rozema J Schat H (2015) Constitutive high-level SOS1
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Kellermeier F Chardon F Amtmann A (2013) Natural variation of Arabidopsis
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Kim SY Lim JH Park MR Kim YJ Park TI Se YW Choi KG Yun SJ (2005)
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Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde
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Laohavisit A Mortimer JC Demidchik V Coxon KM Stancombe MA
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Laohavisit A Shang Z Rubio L Cuin TA Veacutery AA Wang A Mortimer JC
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Laurie S Feeney KA Maathuis FJ Heard PJ Brown SJ Leigh RA (2002) A role
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Liu X Fan Y Mak M Babla M Holford P Wang F Chen G Scott G Wang G
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Luna C Seffino LG Arias C Taleisnik E (2000) Oxidative stress indicators as
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Lu W Guo C Li X Duan W Ma C Zhao M Gu J Du X Liu Z Xiao K (2014)
Overexpression of TaNHX3 a vacuolar Na+H+ antiporter gene in wheat
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Lu Y Li N Sun J Hou P Jing X Zhu H Deng S Han Y Huang X Ma X Zhao
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Maksimović JD Zhang J Zeng F Živanović BD Shabala L Zhou M Shabala S
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Malzahn A Lowder L Qi Y (2017) Plant genome editing with TALEN and
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Mandhania S Madan S Sawhney V (2006) Antioxidant defense mechanism under
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Marino D Dunand C Puppo A Pauly N (2012) A burst of plant NADPH oxidases
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Martinez-Atienza J Jiang X Garciadeblas B Mendoza I Zhu JK Pardo JM
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Maruta T Noshi M Tanouchi A Tamoi M Yabuta Y Yoshimura K Ishikawa T
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McInnis SM Desikan R Hancock JT Hiscock SJ (2006) Production of reactive
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Plant Physiol 173 91ndash111
Mignolet-Spruyt L Xu E Idanheimo N Hoeberichts FA Muhlenbock P Brosche
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Millar J Roots J (2012) Changes in Australian agriculture and land use
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Miller G Shulaev V Mittler R (2008) Reactive oxygen signaling and abiotic stress
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Mittler R (2017) ROS are good Trends Plant Sci 22 11ndash19
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Munns R James RA Lauchli A (2006) Approaches to increasing the salt tolerance
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Munns R Tester M (2008) Mechanisms of salinity tolerance Annu Rev Plant Biol
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Navrot N Rouhier N Gelhaye E Jacquot JP (2007) Reactive oxygen species
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Nieves-Cordones M Aleman F Martinez V Rubio F (2014) K+ uptake in plant
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Oh DH Dassanayake M Haas JS Kropornika A Wright C drsquoUrzo MP Hong H
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Qadir M Quillerou E Nangia V Murtaza G Singh M Thomas RJ Drechsel P
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Ren ZH Gao JP Li LG Cai XL Huang W Chao DY Zhu MZ Wang ZY Luan
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Richards SL Laohavisit A Mortimer JC Shabala L Swarbreck SM Shabala S
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Rizhsky L Hallak-Herr E Van Breusegem F Rachmilevitch S Barr JE Rodermel S
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Rodrıguez AA Grunberg KA Taleisnik EL (2002) Reactive oxygen species in the
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Roy SJ Negratildeo S Tester M (2014) Salt resistant crop plants Curr Opin Biotechnol
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Schmidt R Schippers JHM (2015) ROS-mediated redox signaling during cell
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Senthil‐Kumar M Srikanthbabu V Mohan Raju B Shivaprakash N Udayakumar
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Shabala S Cuin TA (2008) Potassium transport and plant salt tolerance Physiol
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Sitnicka D Figurska K Orzechowski S (2010) Functional analysis of genes Adv
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Slama I Abdelly C Bouchereau A Flowers T Savoure A (2015) Diversity
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Sunarpi Horie T Motoda J Kubo M Yang H Yoda K Horie R Chan WY Leung
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Suzuki N Mittler R (2006) Reactive oxygen species and temperature stresses a
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