Sugar-modulated gene expression and cell division in cell culture and seedlings of A. thaliana

Sabine Kunz

Umeå Plant Science Centre

Fysiologisk Botanik

Umeå 2014

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7601-174-4

Front cover by Sabine Kunz

Elektronisk version tillgänglig på http://umu.diva-portal.org/

Printed by: Servicecenter KBC, Umeå Universitet

Umeå, Sweden 2014

i

“Life is not easy for any of us. But what of that? We must have perseverance

and above all confidence in ourselves. We must believe that we are gifted

for something, and that this thing, at whatever cost, must be attained.”

Marie Curie, Nobel laureate (Chemistry, 1911; 1867-1934)

ii

Table of Contents

Table of Contents .................................................................................................. ii

Abstract ............................................................................................................... iv

Sammanfattning ................................................................................................... v

List of used Abbreviations .................................................................................... vi

List of Papers ........................................................................................................ ix

Contribution as an Author ....................................................................................... x

Introduction .......................................................................................................... 1

Plant Growth and Development ............................................................................. 1 Meristems .......................................................................................................... 1 Developmental phases ....................................................................................... 2

Carbohydrates (CH) as energy source .................................................................... 2 Sucrose production and transport ..................................................................... 2 Source and sink tissues ...................................................................................... 3 Sucrose degradation .......................................................................................... 3 Starch production and degradation ................................................................... 4 Maintenance of carbon homeostasis ................................................................. 4 Starvation ........................................................................................................... 5

Physiological responses to altered carbohydrate homeo-stasis ............................ 5 Development and growth in mutants impaired in carbohydrate metabolism .. 5 Development and growth in response to exogenous sugars ............................. 6 Carbohydrate regulation of photosynthesis and stress response ..................... 7

Sugar perception and signal integration ................................................................ 7 Sugar perception ................................................................................................ 7

The signalling molecule .................................................................................. 7 Signal generation: intracellular pools of signalling sugar species .................. 8

Signal transduction........................................................................................... 10 Hexose-signalling ............................................................................................. 11

HXK1, the Glc sensor .................................................................................... 11 HXK1-independent Glc-signalling ................................................................. 12 Convergent Glc- and Fru-signalling .............................................................. 12

Disaccharide-signalling ..................................................................................... 13 Suc, the transported signalling molecule ..................................................... 13 T6P, an essential signalling molecule ........................................................... 14

iii

Energy-signalling .............................................................................................. 14 TOR, an essential regulator of growth promotion ....................................... 15 SnRK1-dependent energy signalling ............................................................. 15

Sugars and gene expression ................................................................................. 16 Studying sugar-dependent gene expression – approaches and challenges ..... 17

Exogenous sugar application induces transcriptional changes .................... 17 Correlation between transcriptome and metabolome ................................ 18 Plant and organ cell composition affects the sugar-response ..................... 19

Sugars and cell division ........................................................................................ 20 Cell cycle ........................................................................................................... 20 Sugar control of phase transition ..................................................................... 21 Cell division (M-phase) ..................................................................................... 21 Sugar-control of cell division ............................................................................ 22

Aim of Study ....................................................................................................... 24

Results and Discussion ........................................................................................ 25

Xyl-grown Arabidopsis thaliana cell culture - A novel biological system ......... 25 Xylose – an alternative CH-source in A. thaliana cell suspension culture ........ 26 Growth on Xyl permits sugar-responses .......................................................... 28 Complementary experimental systems – solving a conceptual problem ........ 29 Sugar-modulated gene expression in A. thaliana cell culture and seedlings ... 30 Distinct sugar types differentially modulate gene expression ......................... 31 Xylose – an additional sugar signal in A. thaliana?........................................... 32 Dissection of sugar signal(s) – nature, perception and generation ................. 33 Generation of the signal requires sugar-uptake and interconversion ............. 35 Transcriptional regulation of bZIP63, At5g22920, TPS9 and BT2 through the

energy-signalling network ................................................................................ 35 Perturbation of carbohydrate homeostasis is required for the observation of

differential expression in planta ...................................................................... 36 bZIP63, At5g22920, TPS9 and BT2 expression potentially determines growth 37 Long-term real-time live cell imaging – an upgrade of the toolbox ................. 39 Sugar-type specific progression of the cell cycle is determined by the length of

the interphase .................................................................................................. 41 2dog-effects indicate a perturbation or collapse of the cellular

carbohydrate/energy homeostasis .................................................................. 42

Conclusion and Future Perspective ..................................................................... 44

Acknowledgements ............................................................................................. 46

References .......................................................................................................... 48

iv

Abstract

Throughout their life cycle, plants adjust growth in response to their

developmental and environmental situation within the limits of their

energetic capacities. This capacity is defined by the local sugar availability,

which is constantly modulated through synthesis, transport and

consumption of sugar. The monitoring of sugar presence is carried out by a

complex signalling network in which simple sugars (e.g. glucose, fructose

and sucrose) act as metabolic signals for the modulation of physiological

processes. However, often it remains unclear whether the regulation is

induced by the simple sugars themselves or by their derivatives generated

during sugar metabolism. This thesis focuses on the dissection of distinct

sugar signals, their generation, perception and impact on the modulation of

gene expression and cell division both in cell culture and young seedlings.

Based on a stem-cell-like A. thaliana cell culture, which could be sustained

in a hormone-free media, a new biological system, supplied with Xyl as the

only carbon source was developed. The performance of a variety of sugar and

sugar analogue treatments in this novel system allowed for the identification

of sugar-responsive candidate genes, which were specifically regulated by

glucose, fructose and sucrose. For several genes (e.g. bZIP63, AT5g22920,

TPS9, MGD2 and BT2), this regulation required both sugar transport into

the cytosol and metabolisation for the generation of the signal. Furthermore,

gene expression analyses in young A. thaliana seedlings indicated the

requirement for the catalytic activity of hexokinase 1 in the regulation of

bZIP63, Atg22920 and BT2 under conditions of a perturbed carbohydrate

balance. These findings have been combined in a proposed model for the

transcriptional regulation of bZIP63, AT5g22920, TPS9, MGD2 and BT2,

which further proposes a function of those genes in the regulation of cell

division.

The optimisation of a protocol for long-term real-time live-cell imaging

provided a valuable tool to show that, similar to gene expression, the

progression of cell division depended on a sugar-type-specific regulation at

the single-cell level; this regulation was most likely caused by prolongation of

the interphase. Together with the observation of cell death and growth arrest

of the primary root in intact seedlings in response to the glucose analogue

2dog, this led to the conclusion that sugar signals themselves were sufficient

to induce cell division. However, the continuation of cell cycle progression

and consequently organ growth over long-time required the availability of

the energy contained in the sugar.

v

Sammanfattning

Under hela sin livs-cykel måste växter anpassa sin tillväxt-hastighet utifrån

sitt utvecklings-stadie och den omgivande miljön, begränsat av sin energi-

kapacitet. Denna energi-kapacitet, definieras av den lokala tillgången på

socker, som konstant moduleras av syntes, transport och konsumtion.

Övervakningen av tillgången på socker styrs av ett komplext signal-nätverk, i

vilket enkla socker (t.ex. glukos, fruktos och sukros) fungerar som

metaboliska signaler för att reglera fysiologiska processer. Emellertid är det

ofta oklart huruvida regleringen induceras av dessa enkla socker eller av dess

derivat som produceras av socker-metabolismen. Denna avhandling

fokuserar på dessa distinkta socker-signaler, hur de genereras, uppfattas och

påverkar gen-expression och cell division, i både cellkultur och unga plantor.

Baserat på en stamcells-lik A. thaliana cellkultur, som kunde upprätthållas i

ett hormon-fritt media, kunde ett nytt biologiskt system utvecklas, till vilket

endast xylos tillfördes som kol-källa. Effekten från ett flertal socker och

socker-analog behandlingar på detta nya system ledde till att flera socker-

reglerade kandidat-gener kunde identifieras, dessa är specifikt reglerade av

de distinkta sockerarterna glukos, fruktos och sukros. För flera av generna

(t.ex. bZIP63, AT5g22920, TPS9, MGD2 and BT2) krävdes både att sockret

transporterades till cytosolen och att sockret kunde metaboliseras för att

signalen skulle genereras. Gen-expressions analys av unga A. thaliana

plantor, indikerade ett behov av katalytisk aktivitet från HXK1 för

regleringen av bZIP63, Atg22920 och BT2 vid rubbad kolhydrat-balans.

Dessa iakttagelser kombinerades till en modell för transkriptions reglering

av bZIP63, AT5g22920, TPS9, MGD2 and BT2, som tyder på en roll för

dessa gener i kontrollen av cell division.

Optimeringen av ett långsiktigt real-time live-cell imaging protokoll gav ett

verktyg för att visa hur en socker-specifik reglering, liknande den gen-

expressions baserade kontrollen, deltar i kontrollen av cell divisionens

framåtskridande på cellnivå. Regleringen sker förmodligen genom en

förlängning av interfasen. Detta tillsammans med observationer av celldöd

och stoppad tillväxt i primära rötter som svar på glukos-analogen 2dog,

ledde till antagandet att socker-signalen ensam kan inducera cell division,

emellertid kräver kontinuerlig cell divisionen och organ-utveckling över en

längre tid tillgång på lagrad energi.

vi

List of used Abbreviations

2dog 2-deoxyglucose

3OMG 3-O-methyl-glucose

A. thaliana Arabidopsis thaliana

ABA abscisic acid

ADP adenosine diphosphate

ADP-Glc ADP-glucose

AGPase ADP-Glc pyrophosphorylase

AMPK Adenosine monophosphate dependent kinase

AMY1 alpha-amylase 1

ANAC089 Arabidopsis transcription activation factor 1, 2 and Cup-shaped cotyledon 2 domain containing protein 89

Ara arabinose

ATG8 Autophagy 8

ATP adenosine triphosphate

BE branching enzyme

BT2 BTB and TAZ domain protein 2

bZIP basic leucin zipper transcription factor

CAB Chlorophyll a binding protein

CASP CDKA;1

CCAAT-displacement protein alternatively spliced product cyclin dependent kinase A;1

CDKB;1 cyclin dependent kinase B;1

CDS cell division site

CDZ cell division zone

cFBP cytosolic fructose 1,6 bisphosphatase

CH carbohydrate(s)

cINV cytosolic invertase

CLASP CLIP-associated protein

CO2 carbon dioxide

DBE debranching enzyme

Din6 dark inducable 6

DNA deoxyribonucleic acid

DPE disproportionating enzyme

e.g. for example (lat. "exempli gratia")

E2F E2 promoter binding factor

Elo3 Elongata protein 3

et al. and others ("et alii")

Fig. figure

FK fructokinase

FRET fluorescence energy transfer

Fru fructose

Fru-6-P fructose-6-phosphate

G1/2-phase gap 1/2-phase

G1P glucose-1-phosphate

G6P glucose-6-phosphate

GA gibberellic acid

vii

gin2.1 glucose insensitive 2.1

Glc glucose

Gso GASSHO; putative leucine-rich repeat transmembrane-type receptor kinase

GWD glucan water dikinase

HEC hecate

HKL hexokinase like

HXK hexokinase

INV invertase

ISA isoamylase

LST8 lethal with sec thirteen 8

Mal maltose

MAP65 microtubule associated protein 65

MEX1 maltose exporter 1

MGD2 monogalactosyldiacylglycerol synthase 2

mM millimolar

M-phase mitosis-phase

mRNA messenger ribonucleic acid

MS mitotic spindle

MYB75/PAP1 myeloblastosis protein 75/ product of anthocyanin accumulation 1

NAF non-aqueous fractionation

Nana encodes an aspartic protease

NEB nuclear envelope breakdown

NMR nuclear magnetic resonance

ORF open reading frame

P phosphate

Pal palatinose

pANT promoter of Aintegumenta

pATML1 promoter of Arabidopsis thaliana mildew resistance locus

PGI phosphoglucoisomerase

PGM phosphoglucomutase

pLFY promoter of Leafy

PMT1/2 polyol/monosaccharide transporter 1/2

PPB pre-prophase-band

PPP pentose-phosphate-pathway

ProDH2 proline-dehydrogenase 2

qRT-PCR quantitative reverse transcription polymerase chain reaction

R receptor

RAM root apical meristem

Rbr retinoblastoma related protein

RGF1/2/3 root meristem growth factor 1/2/3

RGS1 regulator of G-protein signalling 1

ROS reactive oxygen species

RPT5B regulatory particle 5B

SAM shoot apical meristem

SCR scarecrow

SEX starch excess

SnF1 sucrose-non-fermenting 1

SnRK1 sucrose-non-fermenting related kinase 1

viii

S-phase synthesis (DNA) - phase

SPP sucrose-phosphate-phosphatase

SPS sucrose-phosphate-synthase

SS starch synthase

Stimpy encodes a homeodomain protein

STP1 sugar-transporter 1

Suc sucrose

SUS sucrose synthase

SUT sucrose-transporter

T transporter

T6P trehalose-6-phosphate

Tab. table

TAN encodes a WD repeat protein

TF transcription factor

Tic time for coffee

TON1 Tonneau 1

TOR target of rapamycin

TP triose-phosphate

TPS trehalose-6-phosphate synthase

TPST tyrosylprotein sulfotransferase

TPT triose-phosphate transporter

Tre trehalose

TUA:GFP tubulin-alpha green fluorescent fusion protein

Tur turanose

UDP-Glc uridine-diphosphate-glucose

UGPase UDP-glc-pyrophosphorylase

uORF upstream open reading frame

UTR untranslated region

VHA-B1 V-ATPase B subunit 1

WOX Wuschel-related homeobox protein

WUS Wuschel

Xyl xylose

ix

List of Papers

I

Kunz S, Pesquet E and Kleczkowski LA (2014). Functional dissection of

sugar signals affecting gene expression in Arabdopsis thaliana. PLoS One

9(6):e100312

II

Kunz S, Gardeström P, Pesquet E and Kleczkowski LA. The metabolic

activity of HEXOKINASE 1 is required for glucose-induced repression of

bZIP63, At5g22920 and BT2 in Arabidopsis. Manuscript.

III

Kunz S, Ménard D, Kleczkowski LAK and Pesquet E. Monitoring the role of

distinct sugars on cell division in Arabidopsis plants cells and seedlings.

Manuscript.

x

Contribution as an Author

For Paper I, I designed and performed the experiments. I performed all

data analysis, except for the statistical analysis of the microarray data. I

wrote the manuscript.

For Paper II, I conceived, designed and performed all experiments, except

for the measurement of the adenylates (Fig. 7). I performed all data analysis

and visualisation. I wrote the manuscript.

For Paper III, I designed and performed the experiments involving the live

cell imaging. I conceived, designed and performed the experiments involving

whole plants. I performed all data analysis and visualisation. I wrote the

manuscript.

1

Introduction

Plant Growth and Development

Land plants are sessile multicellular organisms. This feature demands the

ability to adapt to an ever-changing environment. Adaptation is established

through the modulation of physiological processes and the subsequent

adjustment of growth and development.

Growth, an energy demanding process defined by an increase in biomass,

occurs throughout the whole day-night cycle (Smith and Stitt, 2007). Organ

growth may occur into all dimensions (width, length, thickness) and is

determined at the cellular level. Single cell growth, which is highly

coordinated depending on the environmental and nutritional state of the

plant, influences thereby organ shape and plant anatomy (Braidwood et al.,

2014). Plant growth, morphology and development are determined by

temporally and spatially coordinated cell division, cell elongation/expansion

and specific differentiation processes. Cell elongation and cell division are

two biological programs, which may similarly contribute to an increase in

biomass. As they determine the total number of cells and the average cell size

of a plant, these two variables are decisive for the final organ and plant size

(Mizukami, 2001).

Plants benefit from growth in the constant competition for resources like

light and nutrients; still it does not occur at all costs. In fact, it is tightly

regulated depending on the energy reserves available (Smith and Stitt,

2007), thus preventing famine and growth inhibition due to energy

deficiency. Carbohydrates (CH) represent the major energy source in plants

and act as potent signals within sugar and energy signalling networks which

establish molecular links between the sugar metabolism and the regulation

of plant growth and development. The progression of cell division and

elongation underlies strict regulation through these networks, which

integrates information on the metabolic state and environment of a plant

(Smith and Stitt, 2007; Ljung, 2013; Paparelli et al., 2013).

Meristems

In contrast to animals, cell division in plants is primarily localised to

specialised tissues, the meristems. There are four meristematic tissues in

plants: the shoot apical meristem (SAM), the root apical meristem (RAM),

the cambium of the vasculature and the cork cambium. While the two latter

2

tissues are essential for thickening of stems (and therefore are most

prominent in trees), the SAM and RAM are essential for longitudinal growth

and development of the shoot and the root, respectively (Aichinger et al.,

2012). Meristems consist mainly of undifferentiated cells and pluripotent

stem cells, which as a result of cell division provide new cells that might

undergo differentiation and thereby initiate the development of new organs

(Aichinger et al., 2012).

Developmental phases

The development of a plant from an embryo, contained in the seed, to a

reproductive plant producing new seeds ensures the survival of its species or

population. Subsequent to germination, plants undergo two developmental

phase-transitions: the embryonic-to-vegetative and the vegetative-to-

reproductive phase transition. Both transitions are accompanied by

substantial changes in morphology of the developing organs and an

adaptation of the metabolism to meet the demands of the new tissues and

structures (Poethig, 2010). The metabolic adaptation is realised through a

tight regulation of phase transitions in the meristems by an extensive

crosstalk between phytohormones and metabolic signals such as CH

(Eveland and Jackson, 2012; Matsoukas et al., 2013; Wahl et al., 2013; Yang

et al., 2013; Yu et al., 2013).

Carbohydrates (CH) as energy source

The ability to photosynthesize enables plants to fix carbon from CO2 into

reduced molecules, CH or sugars, which can be transported, stored and

metabolised to meet the demands for building blocks and energy throughout

the whole plant. Carbon-fixation and reduction is realised by light-

dependent enzyme reactions of the Calvin-Benson-Cycle in the chloroplast

stroma. The first net product of photosynthesis, triose-phosphate (TP), is the

precursor of all carbon-containing compounds, e.g. serving as the substrate

for glycolysis and being involved in the formation of other soluble sugars

[e.g. glucose (Glc), fructose (Fru), sucrose (Suc)], starch, amino acids, lipids,

nucleic acids, etc.

Sucrose production

The formation of Suc, which takes place in the cytosol, requires transport of

TP from the chloroplast stroma to the cytosol, a process which is guided by

the triose-phosphate/phosphate translocator (TPT). The exported TP is

converted to Suc via different hexose- and Suc-phosphate intermediates,

3

involving the action of cytosolic enzymes, such as Fru-1,6-bisphosphatase

(cFBP), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), UDP-

Glc-pyrophosphorylase (UGPase), Suc-phosphate-synthase (SPS) and Suc-

phosphate-phosphatase (SPP).

Source and sink tissues – Suc transport

Photosynthetically active (autotrophic) tissues or organs represent the so-

called metabolic source tissues. They supply carbon to the so-called

metabolic sinks, which are non-photosynthetically active (heterotrophic),

such as roots, seeds and flowers. The Suc, generated from TP, is either stored

in the vacuole or transported through the phloem from source to sink

tissues. Long-distance Suc-transport requires the activity of proteins with

Suc-transporter ability, such as SUT- or certain SWEET-proteins (Srivastava

et al., 2008; Weise et al., 2008; Chen et al., 2012; Reinders et al., 2012),

which are involved in loading the phloem through either a symplastic or

apoplastic pathway (Lalonde et al., 2004). The capacity of a sink-tissue to

compete for carbon import is defined as sink-strength. It has been shown

that the cleavage of Suc by invertase (INV) is essential to establish sink

strength in young developing tissues, e.g. flowers and fruits. The increase in

the carbon consuming processes of cell division and expansion in those

tissues drives sink strength (Bihmidine et al., 2013), thereby promoting

growth.

Sucrose degradation

The consumption of Suc in both source and sink tissues is regulated by the

catalytic activities of INV and Suc-synthase (SUS). Suc-cleavage by INV,

which may occur in the apoplast, cytosol or vacuole, is conducted by several

isoforms of the enzyme. INV activity results in the formation of the reducing

sugars, Glc and Fru, in a given compartment. While Glc and Fru may be

imported and stored in the vacuole, they are readily phosphorylated by

hexokinase (HXK) or fructokinase (FK) in the cytosol, thereby being directed

towards glycolysis or biosynthetic processes. Especially in photosynthetic

tissues it is assumed that during the light period the cytosol is depleted of

free Glc and Fru (Deuschle et al., 2006; Wingenter et al., 2010). Suc-cleavage

through SUS activity results in the generation of Fru and UDP-Glc. UDP-Glc

is an important precursor for the production of cell wall components,

sulfolipids and glycoproteins (Kleczkowski et al., 2010). In addition, it serves

as substrate for the trehalose-6-phosphate-synthase (TPS) in the formation

of trehalose-6-phosphate (T6P), an indispensable sugar signalling molecule

(Schluepmann et al., 2003; Tsai and Gazzarrini, 2014).

4

Starch production and degradation

Starch, which is synthesised in the chloroplast, represents the major

transient carbon-storage form in leaves. Both the production and

degradation of starch are complex processes, being controlled by a multitude

of enzymes. Starting with the Calvin-Benson-cycle-derived Fru-6-P, the

combined action of PGI, PGM and ADP-glucose-pyrophosphorylase

(AGPase), results in the formation of ADP-Glc, a direct starch precursor. The

polymerisation of ADP-Glc molecules to form starch involves the enzymes

starch synthase (SS), branching enzyme (BE) and debranching enzyme

(DBE). Their activities result in a simple, but compact structure, visible as

starch granules in the chloroplast (Streb and Zeeman, 2012).

Starch degradation is controlled by kinases [e.g. glucan water dikinase

(GWD)], exo-amylases [e.g. β-amylase 3 (BAM3)] and endo-amylases [e.g. α-

amylase 1 (AMY1)], phosphatases [e.g. starch excess 4 (SEX4)], debranching

enzymes [e.g. Isoamylase 3 (ISA1)], α-glucan phosphorylases (e.g. PHS1) and

diproportionating enzymes (e.g. DPE1) (Streb and Zeeman, 2012). This

battery of enzymes promotes the gradual cleavage of longer and shorter

oligosaccharide chains, which are finally degraded to maltose (Mal) and Glc

units. Mal, the major degradation product, is transported to the cytosol via

the Mal exporter MEX1 (Niittylä et al., 2004), and subsequently converted

into Glc and hexose-phosphates (hexose-P) units, which are introduced into

the hexose-P pools.

Maintenance of carbon homeostasis

The production of both Suc and starch serves to supply sufficient carbon to

continuously sustain growth and development. While Suc-transport ensures

the provision of carbon to heterotrophic sink-tissues, the transitory starch in

the leaves serves as carbon storage, which, upon carbon-release, enables the

maintenance of carbon-export, energy homeostasis and growth during the

night.

The ultimate requirement for carbon storage - an assimilation rate which

exceeds carbon consumption during the light period - is guaranteed through

the adjustment of starch production to a broad range of photoperiods (Gibon

et al., 2009). As the starch synthesis rate is adjusted to the perceived length

of the photoperiod, sufficient carbon is stored to meet the predicted

consumption during the following night. Furthermore, starch degradation in

the dark is set to retain small amounts of starch at the end of the night

(Smith and Stitt, 2007). A complete exhaustion of the carbon reserves during

the night would result in growth inhibition, which would not immediately be

5

reversed during the subsequent light period (Gibon et al., 2004; Bläsing et

al., 2005).

Starvation

The state of complete exhaustion of carbon reserves is defined as C-

starvation. Short-term starvation, caused by a cloudy morning, leads to

temporary growth inhibition mediated by phytohormones such as gibberellin

(GA) (Paparelli et al., 2013), induced photosynthesis and adjustment of the

starch synthesis rate in the following light period (Gout et al., 2011). In

contrast, long-term starvation leads to a loss of the CH-homeostasis. The

subsequent response involves growth inhibition, the induction of autophagy

and protein and lipid catabolism (Osuna et al., 2007; Usadel et al., 2008;

Stitt et al., 2010). These processes ensure the continuous carbon-supply for

respiration to maintain energy homeostasis and survival.

Physiological responses to altered carbohydrate homeo-stasis

The induction of endogenous sugar accumulation through the blockage of

specific metabolic or regulatory pathways or the exogenous application of

sugars to plants may trigger a vast variety of physiological, morphological

and developmental responses leading to distinct phenotypes.

Development and growth in mutants impaired in carbohydrate

metabolism

While the genetic manipulation of some enzymes in sugar metabolism, e.g.

SUS or TPT has little or no impact on growth phenotypes (Schneider et al.,

2002; Bieniawska et al., 2007; Barratt et al., 2009; Schmitz et al., 2012),

mutants impaired in the maintenance of CH-homeostasis, such as pgm1,

adg1, sex1 (GWD), adg1/tpt2, spsa1/spsc, cinv1/2, suc2/pho-3, nana, tic

and gin2.1 (Caspar et al., 1985; Lin et al., 1988; Gottwald et al., 2000; Yu et

al., 2001; Ritte et al., 2002; Moore et al., 2003; Barratt et al., 2009;

Heinrichs et al., 2012; Paparelli et al., 2012; Sanchez-Villarreal et al., 2013;

Volkert et al., 2014), may exhibit rather strong growth phenotypes visible in

stunted developement. For the pgm1 and gin 2-1 mutants, it has been shown

that growth repression is almost invisible in long day conditions or low light

but gets more pronounced the shorter the length of the photoperiod or the

higher the light intensities (Caspar et al., 1985; Moore et al., 2003; Gibon et

al., 2004; Gibon et al., 2009). The cause for the reduced growth of the pgm1,

6

adg1 and sex1 can be found in a sugar starvation-dependent inhibition of

gibberellin biosynthesis during the night time, which results in a diminished

leaf elongation (Paparelli et al., 2013).

The dwarf phenotype, as a consequence of an imbalanced CH-homeostasis,

illustrates the negative correlation between growth and CH-availability,

especially accumulation of transitory starch (Smith and Stitt, 2007;

Yazdanbakhsh et al., 2011; Sanchez-Villarreal et al., 2013). Interestingly,

effects of an impaired CH-balance are not restricted to growth but also

visible in developmental phenotypes such as the delay in the transitions from

juvenile-to-adult phase (Matsoukas et al., 2013) and the delayed flowering

induction (Gottwald et al., 2000; Yu et al., 2000; Paparelli et al., 2012). Both

phenotypes are a consequence of sugar-dependent regulation of transition

processes, which depend on allocation of carbon from photosynthesis (Wahl

et al., 2013; Yang et al., 2013; Yu et al., 2013). The application of exogenous

Suc or Glc to the growth media results in re-establishment of wild-type (wt)

growth and flowering in some of the mutants (Gottwald et al., 2000;

Heinrichs et al., 2012; Paparelli et al., 2012; Schmitz et al., 2012), indicating

that the applied sugar is taken up and incorporated into the plant system to

restore growth.

Development and growth in response to exogenous sugars

The physiological response to exogenous sugar treatment depends highly on

the sugar species and concentration applied. While low concentrations of

Suc, Glc, Fru, arabinose (Ara) and xylose (Xyl) induce root growth, fucose,

galactose, rhamnose and mannose inhibit this process (Stevenson and

Harrington, 2009). The induction of root and rosette growth by relatively

low sugar concentrations (up to 20 mM) is in contrast to a decreased

hypocotyl and root elongation in A. thaliana seedlings in response to

increasing sugar concentrations (100 mM and more; Jang et al., 1997;

Solfanelli et al., 2006; Stevenson and Harrington, 2009). In comparison to

mutants impaired in the CH-balance, similar inhibition or delay of seed

germination, juvenile-to-adult-transition as well as of flowering induction

can be observed in response to increasing exogenously applied

concentrations of Suc, Glc, and trehalose (Ohto et al., 2001; Dekkers et al.,

2004). Phenotypic alterations of growth and development in response to

high sugar concentrations overlap with effects assigned to different

phytohormones, such as auxin, ethylene, abscisic acid (ABA) and GA. This

overlap is partially caused by the sugar-dependent regulation of hormone

metabolism as reported for auxin (Sairanen et al., 2012), GA (Paparelli et al.,

2013) and ABA (Cheng et al., 2002; Matiolli et al., 2011).

7

Carbohydrate regulation of photosynthesis and stress response

In addition to growth and developmental phenotypes, high sugar

concentration (>100 mM) might also trigger an increase in the content of

anthocyanin in the leaves, which is related to the Suc-dependent

transcriptional regulation of anthocyanin biosynthesis enzymes (Teng et al.,

2005; Solfanelli et al., 2006; Stevenson and Harrington, 2009).

Accumulation of soluble sugars in the leaf inhibits photosynthesis, a

phenomenon further analysed through the characterisation of Glc-

dependent gene expression of photosynthesis-related genes (Jang and

Sheen, 1994; Koch, 1996; Jang et al., 1997). The process of photosynthesis

feeds into the sugar-pools during day-time under constantly changing light

intensities. Long-term exposure of a plant to high-light conditions triggers

redox imbalances as well as metabolic changes, such as the accumulation of

anthocyanin and soluble sugars (Schmitz et al., 2014). Using a comparative

metabolomics and transcriptomics study for the high-light sensitive

genotype adg1-1/tpt2, it could be distinguished that CH and redox/ROS

signalling are crucial for the retrograde control of nuclear gene expression,

which accompanies the high-light acclimation response (Schmitz et al.,

2014).

Sugar perception and signal integration

The availability of sugar promotes growth and development. This is due to

sugars acting both as important substrates within the primary and

intermediary metabolism but also as potent signalling molecules. The

perception of a sugar and its integration with environmental and

developmental information is essential to ensure a plant´s fitness and

survival. The broad spectrum of physiological, developmental and growth

responses to an impaired CH-homeostasis indicates that plants possess the

ability to monitor the sugar signal and specifically respond to alterations in a

coordinated fashion. Screening for mutants impaired in sugar responses has

enabled the identification of a large variety of sugar hyper- or hyposensitive

mutants, some of them being impaired in sugar sensing or signalling

(Ramon et al., 2008).

Sugar perception

The signalling molecule

A biological signal or stimulus contains information, which can be related to

the state of a cell environment or interior. In multicellular organisms, cells

8

within a tissue exchange a broad spectrum of different signals (e.g.

hormones, ions, peptides or metabolites) in response to a diversity of stimuli

(e.g. light, temperature or wounding). The signal quality and quantity are

often subject to a time-dependent oscillation, which fine-tunes simultaneous

responses to different signals. Besides their role as energy source, sugars

were shown to act as signalling molecules in plants, thereby modulating

physiological and molecular processes such as the expression of sugar-

responsive genes (Koch, 1996). While sugar signals may transmit the

information on the state of carbon supply or its flow through metabolism,

the explicit nature of the signal-giving sugar molecule has hardly been

proven. Despite the fact that basically all sugars and their derivatives could

act as sugar signals, high signalling potential has so far been assigned only to

the disaccharide Suc (Koch, 1996; Wind et al., 2010; Tognetti et al., 2013),

the hexoses Glc (Jang et al., 1997; Moore et al., 2003) and Fru (Cho and Yoo,

2011; Li et al., 2011) and their derivatives G1P, G6P (Nunes et al., 2013b) and

T6P (Halford and Hey, 2009; Zhang et al., 2009). Furthermore it is still

debated whether the sole presence or accumulation of a given sugar molecule

or its flux through the metabolism act as a momentum giving signal (Koch,

1996).

Signal generation: intracellular pools of signalling sugar species

The constant production, transport and metabolisation of CH give rise to a

multitude of sugar-molecules, which may act as distinct sugar signals.

Virtually all of the sugar metabolising enzymes are encoded by several genes,

each of them possessing a unique pattern of expression and subcellular

targeting within different organs and developmental stages of the plant

(Tiessen and Padilla-Chacon, 2012). Only the co-localisation of the sugar

with the sugar-metabolising enzyme allows for the formation of the

signalling molecules. Consequently, the potential for the generation of

putative signalling molecules underlies a spatiotemporal distribution both

within the cell and the whole organism.

In autotrophic plant cells, fixed carbon is either stored as starch in the

chloroplast or immediately transported further to other compartments

(Fig.1). The exchange of carbon between the different organelles and its

subsequent metabolisation result in the formation of several cellular pools of

the same species, such as the plastidic, cytosolic and vacuolar Glc-pool

(Fig.1). The generation of the Glc-, Fru-, G1P- and G6P-pools may occur in

several cellular compartments (Morandini, 2009; Stitt et al., 2010; Tiessen

and Padilla-Chacon, 2012). However, it has been suggested that the cytosolic

Glc- and Fru-pools are almost non-existing, as those metabolites are either

immediately phosphorylated by cytosolic HXK to feed into the hexose-

9

phosphate pools, or allocated to the vacuole (Deuschle et al., 2006). Still, the

presence of cytosolic Glc has proven to be essential for Glc-signalling

(Wingenter et al., 2010).

Suc- and T6P-formation is mainly restricted to the cytosol, where T6P is in

close relation to the pool sizes of hexose phosphates and UDP-Glc (Paul et

al., 2008). The responsiveness of the rather small T6P-pool size to the

cytosolic hexose phosphate and UDP-Glc suggests a “second messenger”-like

function. Based on results from sugar-feeding experiments, it was

furthermore concluded that a mechanism for T6P-uptake into the

chloroplast exists, which mediates the T6P-dependent activation of starch

synthesis (via redox regulation of AGPase) and breakdown (Paul et al.,

2008). However, the required transporter protein remains unknown and

doubts have been raised about the importance of the redox regulation of

AGPase on starch contents (Li et al., 2012), and whether T6P signalling is

involved in this process (Lunn et al., 2014).

Fig.1: The flow of carbohydrates through the cellular metabolism determines the subcellular

pool-size of the sugar signals Suc, Glc, Fru, G1P, G6P and T6P. Their rates of production,

consumption, inter-conversion and transport highly depend on cell identity, developmental and

metabolic state of the surrounding tissue, and environmental cues. Red, blue and grey circles

represent specific transporters. Suc: sucrose, Fru: fructose, Glc: glucose, Mal: maltose, G1P: Glc-

1-phosphate, G6P: Glc-6-phosphate, UDP-Glc: uridine-diphosphate-glucose, T6P: trehalose-6-

phosphate, TP: triose-phosphate, OPP: oxidative pentose-phosphate pathway, ATP: adenosine-

tri-phosphate, NADP: nicotinamide-adenine-dinucleotide-phosphate.

10

Heterotrophic plant cells, which are dependent on Suc-allocation from

photosynthetic tissues, feed their cytosolic sugar-pools with Suc-, Glc- or

Fru, that are taken up from the extracellular pools (Fig.1). It can be assumed

that, similar to sugar-treated seedlings or cells, the uptake occurs fast and is

accompanied by a rapid metabolisation and allocation of the sugars to

different compartments (Deuschle et al., 2006; Häusler et al., 2014).

As a consequence of environmental and developmental changes, total pool

sizes of potential signals are subject to constant fluctuations. The light-

dependent carbon fixation rate in the chloroplast leads to changes in total

Glc- and Fru-quantities, in contrast to a rather constant Suc content (Gibon

et al., 2006). Stress conditions, which result in carbon-starvation, induce

fluctuations in Glc, Suc, G6P and T6P levels (Baena-González and Sheen,

2008; Paul et al., 2008). How the amplitude of the fluctuation is represented

in the sizes of specific subcellular sugar-pools is still unclear.

From a systems point of view, sugar metabolites represent fluctuating

variables, whose quantity or flux within the system determine the energy

state. Therefore sugar signals can be translated into information on excess

energy or starvation, states which have the potential to rapidly change in

response to long- and short-term environmental and developmental

alterations. The requirement to coordinate the response to an altered energy

homeostasis with physiological adjustments, especially growth regulation, is

covered by several sugar-signal transduction pathways involved in either

promoting or restricting plant growth.

Signal transduction

Signal transduction is the process by which signal recognition through ligand

binding leads to the creation of a cellular response, which is used to alter

physiology, morphology and development of the tissue, organ or whole

organism. The signalling function of sugars implies the presence of a signal

transduction machinery consisting of intra- or extracellular receptors

(sensors) and a variety of signal transduction elements transmitting the

signal to downstream effectors (Fig.2). So far, different putative sugar-

specific signalling pathways have been suggested to integrate sugar-derived

signals to adapt growth and development to a plant´s CH-status (reviewed

and described in Baena-González, 2010; Cho and Yoo, 2011; Li et al., 2011;

Eveland and Jackson, 2012; Granot et al., 2013; Tognetti et al., 2013;

Lastdrager et al., 2014; Sheen, 2014).

11

Hexose-signalling

HXK1, the Glc sensor

Hexose signalling has been proposed both for Glc and Fru. The hexokinase 1

(HXK1) was the first protein to be identified as Glc-sensor in Arabidopsis

thaliana (Jang et al., 1997). A variety of genetic, chemical, genomic and

proteomic approaches indicated that the sensing activity, which mediates the

Glc-dependent response, is independent of the catalytic activity (Jang et al.,

1997; Moore et al., 2003; de Jong et al., 2014). In Arabidopsis there are three

HXK and three HXK-like (HKL) proteins. The HXK proteins are divided into

an A- and B-clade. While the A-type-protein (HXK3) is localised to the

plastid, the B-type proteins (HXK1 and 2) localise to the cytosol, where they

are associated with the outer membrane of the mitochondria (Giegé et al.,

2003; Karve et al., 2008; Granot et al., 2013).

Fig.2: Sugar signalling in the cytosol of plant cells requires the availability of a functional

signal-transduction machinery. The generation of external or internal sugar-signalling

molecules (A) is sensed by a receptor which might be localised to the exterior or the interior of

the cell (B). Along a signal-transduction chain, consisting of several elements, the perceived

signal is transmitted to an effector, such as a transcription factor (C). The activity of the effector

determines the response to the signal, which may include the translocation of the transcription

factor to the nucleus and the induction of a sugar-dependent regulation of responsive genes (D).

R: receptor, ORF: open reading frame, T: sugar transporter, TF: transcription factor.

12

Even though the Glc-sensing function was shown for HXK1, HXK2, HXK3

and HKL1 (Karve and Moore, 2009; Zhang et al., 2010; Karve et al., 2012;

Granot et al., 2013), high importance was assigned to the cytosolic HXK1.

The assumption for a specific HXK1 role in Glc-signalling was supported by

molecular evidence for the requirement of HXK1-import into nucleus

(Yanagisawa et al., 2003) and the proof of Glc-dependent interaction of

HXK1-protein with the vacuolar H+-ATPase B1 (VHA-B1) and the subunit of

the 19S particle of the proteasome (RPT5B). This interaction results in the

formation of a complex which localizes to the nucleus and binds to the

promoters of responsive genes (Cho et al., 2006).

Overexpression and antisense-repression of HXK1 as well as the analyses of

the HXK-knockout lines, such as gin2.1, revealed a function of HXK-

dependent Glc-signalling in root elongation, cell proliferation, vegetative and

reproductive development, stomatal opening and regulation of the

expression of photosynthetic genes (Jang et al., 1997; Xiao et al., 2000;

Moore et al., 2003; Kelly et al., 2012; Granot et al., 2013). Thereby it became

apparent that HXK-dependent Glc-sensing is accompanied by an extensive

crosstalk with phytohormones-induced signalling, such as the ABA, auxin,

ethylene and cytokinin signalling pathways (reviewed in Rolland et al., 2006;

Granot et al., 2013). This points towards an important function of HXK in

the integration of environmental and metabolic signals.

HXK1-independent Glc-signalling

The Glc-signalling pathway involving the HXK1-sensing function is further

complemented by a HXK-dependent pathway, which requires the catalytic

activity of HXK1 (Xiao et al., 2000) and a HXK-independent pathway. The

latter is based on the extracellular perception of Glc through RGS1, a G-

protein-coupled receptor (Chen and Jones, 2004), that is subject to

endocytosis in response to Glc. The removal of the receptor from the

membrane activates the signal transduction through a G-protein pathway,

which regulates the expression of a small set of Glc-responsive genes

(Grigston et al., 2008; Urano et al., 2012).

Convergent Glc- and Fru-signalling

Even though Fru-specific sugar-signalling has been suggested, partially

based on the observation that the HXK-knockout mutant gin2.1 is sensitive

to Fru (Moore et al., 2003; Cho and Yoo, 2011), no Fru-sensor has been

identified so far. However, recently two independent studies identified

components of a putative Fru-specific signalling pathway (Cho and Yoo,

2011; Li et al., 2011). While the FINS1/FBP, a Fru-1,6-bisphosphatase

13

without metabolic activity, was proposed to act as a mediator of the Fru-

signal (Cho and Yoo, 2011), a potential regulation was also suggested for

ANAC089, a membrane-bound transcription factor which, upon release

from the membrane, regulates Fru-specific gene expression (Li et al., 2011).

Even though the Fru-signal transduction mediated by both components was

independent of the HXK1-Glc signalling, it showed similar crosstalk with

hormone-signalling, especially ABA. This suggests the potential of a

downstream convergence of hormone- and HXK-dependent Glc-signalling

and HXK-independent Fru-signalling (Cho and Yoo, 2011; Li et al., 2011).

Disaccharide-signalling

Despite the complexity of the emerging picture of signalling networks, both

Suc and T6P could be identified as potent signalling compounds. Recently, it

has been suggested that the metabolisation of Suc and T6P have a strong

impact on different sugar-signalling pathways which regulate metabolic and

developmental homeostasis (Yadav et al., 2014).

Suc, the transported signalling molecule

The disaccharide Suc is the major transport form of CH in A. thaliana. Suc

molecules are easily convertible to Glc and Fru, which themselves can trigger

signalling. Therefore Suc-specific signalling can be assumed only if Suc, but

not equimolar amounts of Glc and Fru, induce a Suc-response. Suc-specific

effects on gene expression of responsive genes (Ciereszko et al., 2001;

Gonzali et al., 2005; Teng et al., 2005; Solfanelli et al., 2006) as well as

organ morphology have been reported (Hanson et al., 2001), but no Suc-

sensor has yet been identified. Nevertheless, it could be shown that Suc

specifically represses the translation of the S1-group of bZIP transcription

factors (Wiese et al., 2005; Weltmeier et al., 2009). The translational

repression depends on a so-called upstream open reading frame (uORF)

positioned in the 5’-UTR of the bZIP mRNA (Wiese et al., 2005). The

formation of heterodimers between members of the S1- and C-group of bZIP

transcription factors enables a coordinated regulation of gene expression

throughout a variety of stress and developmental conditions (Alonso et al.,

2009; Weltmeier et al., 2009). Recently, a comparative study on metabolite

abundance and translation during the diurnal cycle revealed, that polysome-

loading of mRNA was more strongly correlated to Suc-availability than to

other metabolites (Pal et al., 2013). Although there is no unambiguous proof

that the Suc-moiety, and not a downstream metabolite, triggers the

underlying signalling cascade, the finding supports the evidence for a Suc-

dependent regulation of translation.

14

T6P, an essential signalling molecule

In the past years, T6P, a derivative of the Tre metabolism, emerged as a

signalling molecule with high correlation to Suc (Schluepmann et al., 2003;

Lunn et al., 2006; Paul et al., 2008; Zhang et al., 2009; Delatte et al., 2011;

Cho et al., 2012; Nunes et al., 2013a; Nägele and Weckwerth, 2014; Yadav et

al., 2014). In contrast to Suc, T6P is not transported and has to be

synthesised de-novo within each cell (Paul et al., 2008; Tsai and Gazzarrini,

2014). Based on the finding that T6P occurs in minute amounts in A.

thaliana and impacts a variety of physiological responses towards sugar

treatment and starvation, T6P was suggested to play a role as signalling

molecule rather than metabolite (Paul et al., 2008). Analysis of different

Arabidopsis mutants of trehalose-6-phosphate-synthase (TPS1) and

trehalose-6-phosphate-phosphatase, key enzymes in trehalose-formation,

revealed that T6P-biosynthesis is essential for growth induction and

development (van Dijken et al., 2004; Tsai and Gazzarrini, 2014). Despite

the observation of a large variety of T6P-specific effects on carbon-allocation,

embryogenesis, leaf growth and reproduction (Paul et al., 2008; Lunn et al.,

2014), no sensing-mechanism has yet been identified. T6P-treatment leads

to an inhibition of the Sucrose-Non-Fermenting Related Kinase 1 (SnRK1)

activity in crude plant extracts (Zhang et al., 2009). In contrast to the

frequent assumption that T6P potentially acts as a signal in the SnRK1-

signalling pathway, it has been recently hypothesised that T6P- and SnRK1-

signalling occur via two distinct partially overlapping pathways (Lunn et al.,

2014). This model attempts to rationalize the partially contradictory

experimental data gathered from sugar-treated and transgenic plants (Lunn

et al., 2014).

Energy-signalling

The overall energy state of a plant is continuously affected by constantly

changing factors. Maintaining a constant energy homeostasis, as a

precondition for growth and development, requires the ability to react

towards alterations with a coordinated response. Over the past decades, a

multitude of studies have revealed a far from complete, but more and more

detailed picture of a complex network of major signalling mechanisms that

sense and coordinate the long- and short-term adaptation to altered energy

homeostasis. Within those networks, the regulation is highly correlated with,

and partially controlled by, the availability of sugars, including Glc, Suc and

their derivatives G1P, G6P and T6P. Two important players within the

energy sensing network, the TOR (target of rapamycin) kinase and the

SnRK1, sense and respond to opposite energy levels, thereby acting as

antagonists in the coordination of growth promotion and inhibition.

15

TOR, an essential regulator of growth promotion

The TOR signalling pathway, as a central cellular node, senses the energy

state of the cell and integrates the information with environmental cues for a

balanced regulation of growth and development. In contrast to yeast and

mammals, plants possess only components of the TORC1 complex, which

consists of the TOR protein and its interactors RAPTOR and LST8 (Robaglia

et al., 2012). Genetic modification of any of the TOR-complex components or

known downstream target proteins leads to severe phenotypes, indicating a

high importance of the TOR-signalling pathway for the energy-dependent

regulation of growth, development (embryo-development, flowering and

senescence), inhibition of autophagy and coordination of carbon-allocation

and translation (Menand et al., 2002; Deprost et al., 2007; Liu and Bassham,

2010; Moreau et al., 2012; Xiong and Sheen, 2014). The primary localisation

of TOR expression to proliferating tissues (Menand et al., 2002), together

with the finding that root meristem activity is promoted through the Glc-

dependent activation of TOR-signalling (Xiong et al., 2013), indicates a role

of TOR in the regulation of organ and plant growth through the maintenance

of meristem activity. Based on the observations that in root tissue the

regulation of many G1- and S-phase genes occurs through the Glc-TOR

signalling, and that the E2 promoter binding factor (E2F) is a direct target of

the TOR kinase (Xiong et al., 2013), it can be assumed that TOR-signalling

directly impacts cell cycle progression in plants, thereby affecting organ

growth.

SnRK1-dependent energy signalling

SnRK1 is a serine-threonine protein kinase in plants, which is closely related

to the yeast Sucrose-non-fermenting 1 (SnF1) and the mammalian AMP-

activated protein kinase (AMPK) (Baena-González et al., 2007; Hardie,

2007). SnRK1 is a heterotrimeric complex, consisting of the catalytic α-

subunit (KIN10/11), and the regulatory β-subunit (KINβ1/2/3) and βγ-

subunit (KINγ/KINβγ) (Ghillebert et al., 2011). Generally, SnRK1-signalling

translates the information on nutrient deficiency into growth inhibiting

mechanisms, a conserved characteristic in eukaryotic organisms and hence

present in different plant species (O’Hara et al., 2013). In analogy to the

mammalian and yeast SnF1/AMPK-system, SnRK1 activation leads to

transcriptional reprogramming, which includes the activation of genes

involved in catabolism and photosynthesis, and the inactivation of genes

involved in biosynthesis and cell cycle progression (Baena-González et al.,

2007). The set of genes induced by SnRK1 is similar to the sets of genes

induced by starvation or different dark conditions. This expression profile,

which is opposite to the profile observed in Suc- or Glc-treated samples,

16

indicates a SnRK1-dependent activation of energy-conserving processes to

restore homeostasis and ensure growth and survival (Baena-González and

Sheen, 2008). The activity of plant SnRK1 is repressed by G1P, G6P and T6P,

with a synergistic effect of the combination of G1P and T6P on SnRK1

activity (Zhang et al., 2009; Nunes et al., 2013b). Interestingly, mathematical

modelling revealed that the metabolic function of HXK promotes the

generation of T6P from Suc, thereby strongly interacting with SnRK1-

signalling, as reflected in comparable phenotypes of HXK-overexpressor and

seedlings with induced SnRK1 activity (Nägele and Weckwerth, 2014).

The few identified direct target proteins of SnRK1 activity, e.g. the cell cycle

inhibitor KRP6 and KRP7 (Guérinier et al., 2013) as well as the FUS3 protein

(Tsai and Gazzarrini, 2012), further support the assumption of the SnRK1-

involvement in the regulation of cell cycle and development. In response to

the cellular T6P-availability, SnRK1 is assumed to induce the S1/C-group

bZIP-transcription factor- and miR156-signalling mechanism, leading to an

inhibition of the juvenile-to-adult and vegetative-to-reproductive phase

transition (Wahl et al., 2013; Tsai and Gazzarrini, 2014).

Sugars and gene expression

The analysis of sugar-dependent gene expression, as a direct consequence of

sugar signalling, aims at understanding the sugar responses on a cellular

level, as a basis for extrapolation to the whole organism response. According

to their role as signalling molecules, sugars can modulate the expression of

so-called “sugar-responsive” genes. Hundreds of such genes contribute to the

cellular adjustment to nutrient availability and resource allocation between

tissues and organs, thereby coordinating growth and development of a plant

(Koch, 1996; Smith and Stitt, 2007; Usadel et al., 2008). Sugar-modulated

gene expression can be both a consequence of, and a cause for, the

progression of physiological, morphological or developmental programs, as

is apparent in the sugar-dependent transcriptional regulation of core-clock

genes, which define circadian rhythms. These in turn affect the transcription

and catalytic activities of several sugar-metabolising enzymes (Bolouri

Moghaddam and Van den Ende, 2013).

Over the past decades, the analyses of sugar-dependent gene expression have

guided researchers in their work to increase understanding about the

coordination of metabolism and growth. In this context, a link between

carbon storage/ allocation and growth promotion could be established by the

discovery that a high percentage of genes that are regulated in response to

the diurnal and circadian rhythms are controlled in fact by fluctuating sugar

17

availability (Gibon et al., 2006; Usadel et al., 2008; Pal et al., 2013). To

investigate the underlying sugar-signalling mechanisms, marker genes

responsive to a specific sugar, such as CAB1 (also called LhcB1.3; At1g29930)

and MYB75/PAP1 (At1g56650), were identified and frequently used to test

sugar-specific effects both in wt and mutant plants (Jang et al., 1997; Moore

et al., 2003; Teng et al., 2005; Solfanelli et al., 2006; Baena-González et al.,

2007; Loreti et al., 2008; Zhang et al., 2009; Caldana et al., 2013).

Studying sugar-dependent gene expression – approaches and

challenges

Exogenous sugar application to test the induction of transcriptional

changes

Studies on sugar-dependent gene expression follow basically two major

approaches: (i) the exogenous application of different sugar species in a wide

spectrum of concentrations, ranging from 1 mM to over 100 mM (Jang and

Sheen, 1994; Gonzali et al., 2006; Osuna et al., 2007), and (ii) the correlating

of the endogenous sugar availability to gene expression patterns (Bläsing et

al., 2005; Gibon et al., 2006; Usadel et al., 2008). Depending on the sugar-

concentration used, exogenous sugar application has frequently been

considered as non-physiological, since high sugar concentrations might

induce osmotic effects or enhance the production of e.g. phytohormones,

which themselves trigger changes in gene expression (Matiolli et al., 2011;

Paparelli et al., 2013).

A short-term sugar treatment causes a fast perturbation of the CH-balance as

the interior and exterior of the cells are flushed with sugar (Gout et al. 2011,

Chaudhuri et al. 2008). Although this implies a drastic short-term increase

in the signalling molecule concentration, it restricts the clear distinction

between extra- and intra-cellular signal perceptions. Nevertheless, the

combination of sugar and related sugar-analogues [such as L-Glc, 2-

deoxyglucose (2dog), 3-O-methylglucose (3OMG), palatinose (Pal) and

turanose (Tur)], whose molecular characteristics restrict specific uptake and

further metabolisation (Fig.3), may indicate the localisation of signal-

perception (Jang and Sheen, 1994; Loreti et al., 2000; Fernie et al., 2001;

Lisso et al., 2013).

Even if it is known that the applied sugar is readily taken up into the tissue, it

is still unclear how fast and evenly it is distributed within the tissue and

cellular compartments, and how quickly the sugar is converted to related

sugar-species, which themselves may act as signal(s) (Chaudhuri et al.,

2008). Due to the spatiotemporal delay of sugar perception, caused by the

18

Fig.3: Analogues of Suc and Glc are restricted in their uptake and metabolisation. Due to their

molecular characteristics, Pal and L-Glc can not be transported over the plasma membrane.

While Tur and 3omg are taken up by the cell, they are not further metabolised. In contrast, 2dog

is both transported into the cytosol and metabolised by the HXK enzyme. Suc: sucrose, Tur:

turanose, Pal: palatinose, Fru: fructose, D-Glc: glucose, 3omg: 3-O-methyl-glucose, 2dog: 2-

deoxyglucose, D-G6P: glucose-6-phosphate, 2dog6P: 2-deoxyglucose-6-phosphate, HXK:

hexokinase, PM: plasma membrane.

time-dependent transport of the sugar, cells facing the exterior might

experience higher metabolite concentrations, resulting in divergent sugar-

responses between the cells of a tissue.

Correlation between transcriptome and metabolome

In comparison to exogenous sugar treatment, the systems-based correlation

of metabolomic and transcriptomic data may result in conclusions with

higher biological significance. Still, the methodology may not always give us

precise knowledge on the spatial and temporal distribution of metabolites

within a tissue and among the cellular compartments (Gibon et al., 2006;

Usadel et al., 2008; Hou et al., 2011; Kueger et al., 2012). This is an essential

prerequisite to understand the cellular sugar response. The present

understanding that transcription is a more comprehensive and linear

consequence of a signalling event, while changes in metabolite composition

underlie a more complex control by diverse levels of organisation

(Keurentjes et al., 2008), challenges the assumption of the existence of a

direct correlation between effective metabolite(s) and transcript availability

(Kueger et al., 2012).

19

Especially when analysing whole organs, a higher spatiotemporal resolution

of the transcript and metabolite distribution is required. Recent technical

advances, such as mass-spectrometry based methods, allowed for analyses of

metabolites at the tissue level, whose results indicate an uneven distribution

of single metabolites within the tissue (Kueger et al., 2012). Even though the

resolution of the spatial distribution of metabolites within a tissue could be

increased, intracellular compartmentation of metabolites is often still

disregarded. However, this could be partly approached through the

application of e.g. nuclear-magnetic-resonance (NMR) (Gout et al., 2011) or

fluorescence-resonance-energy-transfer (FRET) non-invasive techniques

(Deuschle et al., 2006; Chaudhuri et al., 2008). Unfortunately, although

being very specific and precise, thereby giving high resolution data, the

functionality of these techniques restricts their application to a rather small

amount of metabolites. Nowadays, non-aqueous fractionation (NAF) in

combination with metabolite analysis techniques such as enzyme assays and

MS-based methods are methods of choice for metabolite analysis, when

information about metabolite compartmentation is required (Kueger et al.,

2012).

Plant and organ cell composition affects the sugar-response

Another level of complexity is added by the architecture and cell composition

of the plant material analysed. The expression level of a gene within a cell is

defined by (i) the surrounding tissue [e.g. TOR expression in the

meristematic tissue (Menand et al., 2002)], (ii) the identity of the cell within

the tissue (Brady et al., 2007; Kalve et al., 2014), and (iii) the developmental

stage of the tissue (Birnbaum et al., 2003; Brandt, 2005; Brady et al., 2007).

These factors, which also influence cellular metabolic fluxes, cause a high

spatiotemporal variation of both transcript and metabolome within a tissue,

as observed both in leaf and in root (Brady et al., 2007; Kalve et al., 2014).

Therefore, common approaches for global gene expression studies, to

analyse whole organs/tissues (Tab.1), reveal an average tissue effect which

does not allow for the observation of responses, restricted to a small number

of specific cells. However, these single-cell responses might be essential for

the function of the organ, as shown by the comparison of whole-organ gene

expression analysis versus cell-type-specific analysis conducted for

Arabidopsis roots (Schmid et al., 2005; Iyer-Pascuzzi et al., 2012). To better

understand developmental and signalling patterns and networks through the

analysis of sugar-dependent gene expression, analysis of cell-specific profiles

are essential (Rogers et al., 2012).

20

Tab. 1: Overview of whole genome expression studies conducted within the past 10

years. Special attention was given to information on the plant material used and the

conditions/treatments applied.

Sugars and cell division

Cell cycle

Plant growth, morphology and development are determined by cell

elongation/expansion, spatially organised cell division and differentiation

during the cell cycle. The cell cycle consists of four phases: the replication of

the genetic material (DNA) during the S-phase (synthesis), sets the stage for

the segregation of the duplicated chromosomes and the formation of two

daughter cells through cytokinesis during the M-phase (mitosis). Two

intermediate G-phases (gap) separate the S- and the M-phase. The transition

from G1- (between M- and S-phase) and G2-phase (between S- and M-

phase) into the respective subsequent cell cycle phases is subject to tight

control through the finely modulated activity of cyclin-dependent-

21

kinase/cyclin complexes (Perrot-Rechenmann, 2010). The G1/S- and G2/M-

transitions thereby serve as check-points to ensure that the previous phases

have been successfully accomplished and that the prerequisites for cell cycle

progression are fulfilled.

Sugar control of phase transition

The progression of a cell through all phases of the cell cycle is controlled in

response to environmental and nutritional information derived from the

surrounding tissue. Together with phytohormones, sugars are potent signals

within a complex signalling network, affecting enzyme activity, chromatin

modification and wavelike gene expression patterns of the cell-cycle-control

genes (Francis and Halford, 2006; Bertoli et al., 2013; Wang and Ruan,

2013; Desvoyes et al., 2014). Sugar-dependent regulation is especially

evident for the regulation of cyclin D, CDKA;1 and CDKB;1, genes, which are

involved in the G1/S-phase transition (Riou-Khamlichi, 1999; Riou-

Khamlichi et al., 2000; Menges et al., 2005; Skylar et al., 2011). Although

Suc seems most important for the transcriptional regulation, there is

evidence that also Glc and Fru are indispensable for the progression of the

cell cycle, especially the progression from G2 to M-phase (Skylar et al., 2011;

Peng et al., 2014). The Suc-dependent induction of cell cycle progression is

actually frequently used to partially synchronise cell division in plant cell

culture (Menges et al., 2005). In contrast to the progression of the cell cycle

through the G1, S and G2 phases, sugar´s impact on the M-phase

progression and the actual process of cytokinesis remains undefined.

Cell division (M-phase)

Plant cell division occurs during the M-phase. It includes cytokinesis as the

last step in mitosis, which is defined as the physical separation of two

daughter cells through the establishment of a cell plate and subsequent cell

wall formation. Cell division can be divided into six phases. During the pre-

prophase the pre-prophase band (PPB) is formed, which defines the future

plane of division. The subsequent prophase is characterised by the

condensation of the chromosomes, the formation of the mitotic spindle (MS)

and the narrowing of the PPB at the cortical division zone (CDZ). The

breakdown of the nuclear envelope (NEB) during the metaphase allows for

an alignment of the chromosomes along the metaphase plate. Chromosome

segregation can be observed during the anaphase, which is guided by polar

microtubules. The telophase is characterised by the formation of the

phragmoplast, which is defined as a unique array of short parallel

microtubules at the cortical division site (CDS). Finally the new cell plate is

22

formed during cytokinesis, which finalises the separation of the new

daughter cells.

The course of cell division is accompanied by an extensive re-arrangement of

the endomembrane system and the cytoskeleton (Müller et al., 2009;

Rasmussen et al., 2013). The spatial re-arrangement of the cytosol through

re-organisation of the nucleus and vacuoles and through re-association of

the nucleus with the endoplasmic reticulum (Seguí-Simarro et al., 2004;

Seguí-Simarro and Staehelin, 2006) is coordinated with the temporal

formation of the specific microtubule-based structures. The latter ones

include the pre-prophase band (PPB), the mitotic spindle (MS) and the

phragmoplast (Rasmussen et al., 2013). These structures are essential for the

definition and organisation of the region of the future cell plate. The

significant spatial and temporal appearance of the PPB, MS and

phragmoplast makes them valuable markers for the M-phase progression.

Sugar-control of cell division

Given that successful progression through cell division involves a load of

biosynthetic processes to rearrange existing structures and complement

them with newly established structures, one can assume that cell division is a

highly energy-consuming process. Accordingly, recurring cell division

increases sink strength of the meristems, thereby making sufficient sugar

supply indispensable for cell division (Wobus and Weber, 1999). The

influence of sugars on cell division due to their role as energy source might

be mediated through the major integrating signalling networks, such as TOR

and T6P/SnRK1 networks (Baena-González et al., 2007; Wahl et al., 2013;

Xiong et al., 2013). Based on the findings that sugars could rescue cell

division arrest in stimpy, a mutant impaired in the homeobox gene WOX9,

or in elo3 mutant, which is affected in DNA replication, it was demonstrated

that metabolic sugars are necessary but not sufficient to establish meristem

activity during the initiation of postembryonic growth (Skylar et al., 2011;

Skylar et al., 2013). Furthermore, mutants of genes involved in the

progression of the cell division [e.g. clasp, (Ambrose et al., 2007); rbr,

(Gutzat et al., 2011); gso1/gso2, (Racolta et al., 2014)] possess dwarfed

phenotypes, which partially or fully could be recovered through the

application of exogenous Suc or Glc. This might suggest either direct or

indirect impact of sugar regulation on cell division; however, the exact

mechanism underlying this process remains unclear. Aside from

transcriptional effects on cell cycle control genes and rescue of growth

through sugar application, direct sugar effects on protein-activities of cell

division marker genes [involved e.g. in the establishment of PPB or CDS,

23

such as TPLATE, CLASP, TAN, MAP65 or TON1 (reviewed in Müller et al.,

2009)] have not been shown yet.

24

Aim of Study

Despite the vast number of studies on physiological adaptations of plant

growth and development in response to sugar availability, and despite the

formulation of putative sugar signalling pathways, it was frequently unclear

what the exact nature of actual signal(s) regulating gene expression was. In

addition, studies on sugar signalling, using intact plants or plant tissues,

have frequently been hampered by tissue heterogeneity, uneven sugar

transport, sugar interconversions and metabolisation, etc. In order to

address these problems and to obtain an integrated understanding of the

cellular and molecular responses of plants to different sugars, I formulated

the following questions: (A) Can plant cells distinguish between the

interconvertible sugars Suc, Glc and Fru to regulate global gene expression

responses? (B) Does sugar responsiveness of a gene require transport

and/or metabolisation of a specific sugar to generate the actual signalling

molecule?; and (C) How do different sugar signals control physiological

responses, such as growth alteration through the regulation of cell division?

To address these questions, I developed a novel biological system, based on a

heterotrophic A. thaliana cell culture. This system facilitated the observation

of sugar-type specific expression changes, while minimising crosstalk with

e.g. tissue derived signals. Furthermore, I used transgenic plants impaired in

carbohydrate metabolism to address the question of the nature and origin of

the actual sugar signal regulating gene expression. Finally, I optimised a

long-term real-time live-cell imaging method, which was applied to the

established cell culture system, thereby enabling me to test the sugar type

specific impact on growth regulation, especially cell division.

25

Results and Discussion

The focus of this thesis touches different aspects of general and specific

effects of CH on cultured plant cells and seedlings. Within the scope of

sugar-modulated gene expression and cell division, special attention was

directed to the distinction between the dual function of sugar as essential

energy source and potent signalling molecule. In the following section, main

results and conclusions, which form the basis of this thesis, are introduced

and discussed according to their contribution to the present understanding

of CH signalling and subsequent effects on gene expression and cell division.

The development of a novel biological system, based on a stem-cell-like

Arabidopsis thaliana cell culture supplied with Xyl as sole carbon source,

allowed for the identification of a set of genes, rapidly and specifically

responsive to low concentrations of the soluble sugars Suc, Glc and Fru,

respectively (Paper I). Detailed characterisation of the sugar-responsive

expression of selected genes in cell culture and young seedlings enabled me

to functionally define the signalling sugar molecule with respect to its

specific nature, potential site of perception and requirements for its

generation within the cell (Paper I and II). Interestingly, several of the

selected genes were subject to a transcriptional regulation which, both in

plant cell culture and whole seedlings, was especially sensitive to a perturbed

CH-homeostasis (Paper I and II). Evidence, based on the function of the

identified sugar-responsive genes, pointed towards a direct relationship

between distinct sugar signals and the regulation of the cell cycle progression

through the M-phase (Paper I and III). The long-term real-time live-cell

imaging of microtubule-based structures, marking the progression of cell

division during the M-phase, indicated a sugar-species specific regulation of

the cell division rate, efficiency and synchronisation by Suc, Glc and Xyl

(Paper III). However, the complete inhibition of cell division and root

growth by the sugar analogue 2dog highlighted the importance of the

availability of sufficient energy supply for the progression of the cell cycle

and consequently organ growth through cell division (Paper I and III).

Xyl-grown Arabidopsis thaliana cell culture - A novel biological

system

With the aim to extend our understanding of growth regulation as a

consequence of sugar-specific transcriptional regulation and cell division, I

applied a combined analysis of both cell suspension culture (Paper I and

III) and young seedlings (Paper I, II and III) of the model organism

Arabidopsis thaliana.

26

Processes contributing to growth and development of multicellular

organisms like plants are subject to a strict coordination at the single cell

level. Highly linked regulatory signalling pathways integrate information

from both extrinsic and intrinsic signals, such as phytohormones and sugars

(Hartig and Beck, 2006; Wang and Ruan, 2013). Sugars effectively

contribute to organ/plant growth through the regulation of cell division and

gene expression (Usadel et al., 2008; Skylar et al., 2011; Sulpice et al., 2013;

Schuster et al., 2014; Xiong and Sheen, 2014). However, the observation of a

cell-specific gene expression response is frequently hampered by the

architecture and cell composition of a tissue/organ, which is very

heterogeneous with respect to the age, developmental stage, identity and

spatial distribution of the cells present (Brady et al., 2007; Kalve et al.,

2014). Consequently, gene expression and growth data derived from a whole

organ reflect a merged image of diverse cell specific responses (Brandt,

2005; Kalve et al., 2014) and might obscure the identification of

decisive/important cell specific effects. In contrast, a cell suspension culture

consists of a rather homogenous cell population, which is easily accessible by

different exogenously applied signalling compounds that are assumed to

trigger a uniform cellular response. Furthermore, triggered changes in

cellular processes, such as cell proliferation and differentiation can directly

be observed at a single cell level, as cell suspension culture is easily

applicaple for a variety of microscopy techniques, such as live cell imaging.

This simplified experimental model not only facilitates the generation of

“bulk data”, like transcriptomic data, which requires a substantial amount of

starting material (Paper I), but also allows to test assumptions derived from

bulk-data analysis by monitoring single cell behaviour, i.e. sugar-dependent

cell-division (Paper III).

Xylose – an alternative CH-source in A. thaliana cell suspension

culture

In Paper I, I describe the development of a new biological system, based on

a habituated heterotrophic A. thaliana cell suspension culture (Pesquet et

al., 2010). This cell culture was used to monitor and test transcription and

cell division as a consequence of sugar-type specific signalling in response to

Suc, Glc and Fru. For this purpose, cell culture growth was adapted to the

supplementation of the growth medium with D-xylose (Xyl) as single CH-

source.

Among 17 alternative CH-sources tested (including different mono-, di- and

polysaccharides), the pentose Xyl has proven to promote growth of the cell

population (Paper I, Fig. 1A, Fig.S1A). Depending on the plant species

and experimental system, different effects of Xyl on growth have been

27

observed in a variety of plant species (Bhagyalakshmi et al., 2004; Kato et

al., 2007; Stevenson and Harrington, 2009; Yaseen et al., 2013; Xiong and

Sheen, 2014). A similar growth-promoting Xyl-effect was shown for Vigna

angularis cell suspension culture (Kato et al., 2007) and root growth of A.

thaliana seedlings (Paper III, Fig. S3; Stevenson and Harrington, 2009).

As Xyl constitutes the major component of hemicellulose (Bar-Peled and

O’Neill, 2011), it is not surprising, that enzymes necessary for the uptake and

metabolisation of exogenously supplied Xyl are present in A. thaliana. In

fact, the three sugar transporter proteins STP1, PMT1 and PMT2 have been

shown to support Xyl-transport over the plasma membrane (Sherson et al.,

2003; Klepek et al., 2010). The subsequent intracellular metabolisation of

Xyl through the action of a xylose isomerase (At5g57655) and a xylulokinase

(At2g21370, At5g49650) presumably channels the sugar towards the non-

oxidative part of the pentose-phosphate pathway to sustain the pivotal

formation of precursors for glycolysis and biosynthetic pathways (Carpita et

al., 1982) (Fig.4).

Long-term culture growth in Xyl-containing medium in comparison to Suc-

containing medium is characterised by a decreased cell division rate (Paper

Fig.4: Scheme of the potential Xyl-metabolisation pathway in Xyl-grown A. thaliana cell

suspension culture. D-Xyl: D-xylose, ATP: adenosine-tri-phosphate, ADP: adenosine-di-

phosphate, D-Xylulose-5-P: D-xylulose-5-phosphate, Fru-6-P: fructose-6-phosphate, Glc-1-P:

glucose-1-phosphate, Glc-6-P: glucose-6-phosphate, UDP-Glc: uridine-diphosphate-glucose,

GA-3-P: glyceraldehyde-3-phosphate, Suc: sucrose.

28

III, Fig. 5A and 6) and, accordingly, an increased doubling time of the cell

population (Paper I, Fig.1A). Considering that the limited supply of energy

from Xyl-catabolism still permits an accumulation of internal soluble sugars

(Paper I, Fig. 1B), cells most likely adapt their entire metabolism, similar

to plants subjected to a low carbon regime (Smith and Stitt, 2007; Gibon et

al., 2009). Despite the observation of culture growth and cell division within

3 days (Paper I, Fig. 1A; Paper III, Fig. 8A), it cannot be entirely

excluded that 7 day old cells experience C-starvation. A further comparative

expression analysis of the established starvation marker genes Din6, ATG8,

At1g76410 and TPS8 (Baena-González et al., 2007; Liu and Bassham, 2010;

Paparelli et al., 2013) in both Suc- and Xyl-grown cells should test the

assumption of a non-starved metabolic state of the cell material. In any case,

the a priori absence of potential signalling compounds (both growth factors

and the soluble sugars Suc, Glc and Fru) from the Xyl-containing growth

medium reduces the number of factors/signals, affecting a cellular response.

This simplified experimental set-up clearly represents an advantage for the

analysis of direct cellular sugar-responses, independent of the heterogeneity

of a tissue and the overlap with other signals.

Growth on Xyl permits sugar-responses

The induction of sugar-dependent transcriptional changes, as shown for the

known sugar-responsive marker gene BT2 (BTB and TAZ domain protein 2)

(Mandadi et al., 2009), confirmed a susceptibility of Xyl-grown cells for low

amounts of the sugar signals Suc, Glc and Fru (Fig. 5) (Paper I, Fig.2). In

contrast to cells in whole organs/plants, cells in suspension culture are easily

accessible by exogenously applied signalling molecules, inducing a direct

response, independent of the transport/diffusion properties of the molecules

through the tissue. This also allowed for the application of sugar at low

concentrations suitable for a signalling molecule, which rapidly and

effectively repressed BT2 expression and induced cell division, even at low

concentrations (Fig. 5) (Paper I, Fig.2; Paper III, Figs. 5 and 6). The

application of sugar at rather low concentration prevents both osmotic

effects and the induction of plant hormone biosynthesis through sugar-

signals (Matiolli et al., 2011; Paparelli et al., 2013). Furthermore, the low

contribution to the overall intra-cellular sugar content (Paper I, Fig. S2)

minimizes the generation of signals derived from huge changes in the

metabolic state, and is more likely to mimic the small differences/changes a

cell experiences within the different plant tissue contexts during the diurnal

cycle (Bläsing et al., 2005; Gibon et al., 2006; Brauner et al., 2014).

29

Fig.5: The sugar-dependent repression of BT2 is sensitive to low Suc and Xyl concentrations (A

and B), being saturated already at 5 mM sugar (A). 7 day old Xyl-grown A. thaliana cell culture

was treated for 3 h in the dark with ascending sugar concentrations. A: Quantification of BT2

expression relative to the expression of the reference gene PP2A using qRT-PCR. B: Sugar-

concentration dependent BT2 expression in A. thaliana cell culture as revealed by semi-qRT-

PCR. The data shown represent 1 biological repeat from a preliminary study on the sugar-

responsiveness of the established cell culture system.

Complementary experimental systems – solving a conceptual

problem

The cultured A. thaliana cell, generated from root cells, possesses the stem-

cell-like properties to divide independently from growth factors and to

differentiate into at least 3 different cell types (personal information from E.

Pesquet; Pesquet et al., 2010). The concept of studying sugar-specific cellular

responses in a heterotrophic dark-grown undifferentiated cell culture system

bears the advantage of identifying direct responses, independent of the

heterogeneity of the surrounding tissue and overlapping signals potentially

derived from it, as mentioned before. However, within a plant tissue, cells do

experience both cell-autonomous and non-cell autonomous signals, which in

combination define the cellular response, e.g. growth, in a physiological

context (e.g. Schuster et al., 2014). Despite the use of the heterotrophic cell

culture system for identification of sugar-responsive genes, their expression

was not restricted to one tissue type in whole seedlings (Paper I, Fig. 5, 6,

S5 and S9; Paper II, Fig. 2, 3 and 4). Furthermore, spatial distribution of

expression of the selected Glc-responsive gene bZIP63 (Paper I, Fig. 3;

Matiolli et al., 2011) appeared dependent on metabolic states

(autotrophic/heterotrophic; sink/source) of a tissue, but not the tissue

identity itself (Paper II, Fig. 4 and S1). In general, it can therefore be

assumed that within whole plants, signals primarily related to differentiation

and metabolic state of the surrounding cells/tissue determine if selected

genes are expressed or not. However, once a gene is expressed it might still

remain sugar-responsive within the spatial expression profile, such as shown

for bZIP63 (Paper II, Fig. 4). While the identification of genes possessing a

30

basal potential for sugar-responsiveness is facilitated in the established cell

culture system, the assessment of their biological function has to be tested

under physiological conditions within a plant context.

Sugar-modulated gene expression in A. thaliana cell culture and

seedlings

Plant growth is constantly adjusted to the cellular CH-availability (Smith and

Stitt, 2007; Sulpice et al., 2013; Sulpice et al., 2014), partly mediated through

a general sugar-dependent regulation of gene expression (Koch, 1996;

Bläsing et al., 2005; Usadel et al., 2008; Gibon et al., 2009; Stitt et al.,

2010). However, depending on the observed developmental stage,

environmental state or tissue identity (i.e. sink or source), different sugar-

types might possess unequal regulatory importance. In A. thaliana, the

soluble sugars Suc, Glc and Fru have been suggested to regulate

physiological responses to sugar availability through both separate and

overlapping signalling pathways (see introduction; signal transduction). This

suggestion is mainly based on detailed investigation of signalling

mechanisms underlying the specific sugar responses using specific marker

genes, such as A. thaliana chlorophyll binding protein (CAB), dark-inducible

6 (DIN6/ASN1), proline-dehydrogenase 2 (ProDH2) or barley alpha-amylase

(AMY) (Jang et al., 1997; Loreti et al., 2000; Gonzali et al., 2006; Baena-

González et al., 2007; Hanson et al., 2008). However, in comparison to the

number of genes in the whole A. thaliana genome and the number of

supposedly sugar-responsive genes (Bläsing et al., 2005), so far, only few

marker genes have been described and used to increase the understanding of

underlying mechanisms of the sugar-type-specific resonses.

The identification of a direct relation between gene expression and one

explicit sugar type is complicated by the interdependency of the Suc, Glc and

Fru pools. However, earlier described evidence on sugar-type-specific

signalling pathways indicates that distinct sugars contain different

information. Assuming that for the plant cell it might be important to

integrate the distinct information both in overlapping and specific response,

the following questions arise: (I) Is global gene expression brought about by

independent sugar-type specific signalling pathways?; (II) How extensive is

the overlap between the distinct responses?; (III) What defines this overlap

– the nature of the signalling molecule or the signal transduction pathway?

To address these questions, whole genome expression analysis in Xyl-grown

A. thaliana cell culture (Paper I) was combined with a more detailed

characterisation of the sugar-type dependent expression profiles of selected

genes in Xyl-grown cells (Paper I) and seedlings (Paper I and II).

31

Distinct sugar types differentially modulate gene expression

The whole genome expression analysis of Xyl-grown A. thaliana cell culture

treated for 1 h with 1 mM Suc, Glc or Fru, respectively, identified sugar-

specific differential expression of 290 genes (Paper I, Dataset 1). The

classification of those 290 genes, based on their response to a specific sugar

type, resulted in 7 classes of genes showing distinct or overlapping responses

(Paper I, Fig. 3 A).

Given that constant subcellular compartmentation maintains stable cytosolic

Suc-concentrations, while Glc and Fru are almost depleted from the cytosol

during the day (Deuschle et al., 2006; Kueger et al., 2012), it is tempting to

speculate that, under the assumption of cytosolic sugar sensing mechanisms,

those sugars have a quantitatively different impact on the regulation of

global gene expression. However, the number of genes selected to be

responsive to the specific sugars was comparable. This suggests an equal

effect of all three sugar types on the overall regulation of gene expression

(Paper I, Fig. 3A and B), indicating that the sugars tested possess similar

signalling power and equally account for sugar related transcription changes.

Furthermore, exogenous sugar-treatment might result in a fast, but in our

case small (Paper I, Fig. S2), temporary accumulation of internal sugars in

the cytosol (Deuschle et al., 2006; Gout et al., 2011), where the sugars might

contribute to the Suc, Glc and Fru pools through fast interconversion.

Detailed expression analysis of selected genes using qRT-PCR revealed that a

high number of genes was responsive to more than one specific sugar

(Paper I, Fig. 3C and S7), which pointed towards a prevalence of a general

sugar- or hexose-specific response of the genes selected. Nevertheless, for

several genes tested, sugar-specificity in the transcriptional response was

partly visible in quantitatively stronger expression changes upon treatment

with the sugar identified to potentially act as the specific signal (Paper I,

Fig. 3C and S7). These quantitative differences in expression might be

meaningful for the adjustment of the strength of physiological effects, like

the observed modulation of cell division rates in response to Suc and Glc

(Paper III).

Comparative analysis of the expression profiles of the selected 290 genes

within publically available datasets focusing on the transcriptional response

to modulated CH-availability in A. thaliana seedlings or rosettes (Bläsing et

al., 2005; Osuna et al., 2007; Usadel et al., 2008), revealed for 20% of the

genes an overlapping response in C-starved seedlings exogenously supplied

for 3 h with 15 mM Suc (Paper I, Fig. S3, Dataset 1). The common

expression profiles of the overlapping genes, whose responsiveness was not

restricted to Suc in cell culture (Paper I, Fig. S3), indicate that either

32

degradation of the applied Suc to Glc and Fru determines the sugar-

responses of the majority of the genes or that several of the genes selected

are regulated by convergent signal transduction. Furthermore, the

expression profiles of the 20% overlapping genes show a time-dependency of

the sugar-response, affecting both the strength and direction of the

expression change, as seen for the expression of CASP (Paper III, Tab. S1).

The time-dependency of the response might indicate that either further

processes are required to generate the signalling molecule (e.g. transport or

metabolisation of the sugar applied) or that the change in gene expression

involves e.g. the sugar-dependent synthesis of effector proteins. However,

the different strength or directions of the expression change, observed in the

datasets analysed, might also indicate a potential regulatory impact of Xyl,

which was used as control.

Xylose – an additional sugar signal in A. thaliana?

The pentose Xylose was earlier shown to support cell culture and seedling

growth (Kato et al., 2007; Stevenson and Harrington, 2009) and it was

selected as an alternative CH-source for growth maintenance of the A.

thaliana cell culture, forming the basis of the presented gene expression

study. With the intention to identify differential sugar-type specific gene

expression, Xyl – as a metabolisable sugar – was applied as control.

However, Xyl-addition affected the expression of selected sugar-responsive

genes (Paper I, Fig. 3 and S7) and the induction of cell division in cell

suspension culture, where it has proven to be even more efficient than Glc

(Paper III, Fig. 5 and 6). Assuming that the expression of a gene is

induced to a different extent by Suc and Xyl, the resulting ratio Suc/Xyl

would be lower than the ratio Suc/untreated (Fig.6). Therefore, one could

assume that this Xyl-effect possibly contributed to the observation of rather

small global expression changes in comparison to the expression changes

observed for whole plants in publically available datasets (Paper I, Fig. S4,

Dataset 1; Paper III, Tab. S1). However, the Xyl-effect did not impair the

identification of sugar-specific regulation of gene expression, as seen for the

genes TPS9 and ERF104 (Paper I, Fig. S7).

Given that the cellular metabolism is adjusted to growth on Xyl as sole CH-

source, it is pivotal for the cells to perceive signals derived from its

availability. Therefore, it can be hypothesised that Xyl, similar to Suc, Glc or

Fru, acts as sugar signal in A. thaliana cells, inducing comparable changes in

the expression of selected genes. However, it still remains to be investigated

whether the actual signal is related either to the identity of Xyl, or its

metabolisation or energy content.

33

Fig.6: Illustration of the effect of Xyl usage as control on the observation of differential gene

expression. The example assumes a Suc-inducible gene, which also shows a similar response

towards Xyl-treatment. The ratio between Suc and T0, which represents an untreated sample,

will be much larger than the ratio between Suc and Xyl. This restricts the observation to

apparently small expression changes but supports the identification of genes responsive to

different sugar types.

Dissection of sugar signal(s) – nature, perception and

generation

To further define the requirements and properties of both the signalling

sugar and the potential signalling pathway, I assessed the expression profiles

of a small number of genes both in cell culture and in whole seedlings of A.

thaliana.

The group of genes further analysed included, among others, the basic

leucine zipper transcription factor 63 (bZIP63, At5g28770), the RING-type

Zinc finger protein At5g22920 (putative E3 ligase), the monogalactosyl-

diacylglycerol synthase MGD2 (At5g20410), the trehalose-6-phosphate

synthase 9 (TPS9, At1g23870) and the previously selected marker gene BT2

(BTB and TAZ domain protein 2, At3g48360). In A. thaliana cell culture, the

transcriptional signature of these five genes was characterised by their sugar

responsiveness, which was comparable between all three sugars tested

(Paper I, Figs. 2, 3 and S7). The expression patterns of those genes proved

to be truly sugar-responsive in planta, as it followed the diurnal sugar-

availability in A. thaliana seedlings of the ecotypes Col-0, Ler-0 and Be-0

(Paper I, Figs. 6 and S9; Paper II, Figs. 1, 2 and 3). Furthermore, under

diurnal conditions these patterns were not affected in mutants impaired in

enzymes of the CH-metabolism and signalling, such as tpt2, sus1/2/3/4,

sus5/6, pgm1, adg1, sex1, gin2.1 and hxk1 oe3.2 (Paper I, Figs. 6 and S9;

Paper II, Figs. 2 and 3). Despite the mutations, the seedlings maintained a

comparable CH-homeostasis, as reflected in generally comparable soluble

34

sugar contents in the mutants and their respective wt (Paper I, Fig. 6;

Paper II, Fig. 1), and they had comparable growth in long-day condition

(Caspar et al., 1985; Jang et al., 1997; Schneider et al., 2002; Moore et al.,

2003; Barratt et al., 2009; Heinrichs et al., 2012; Paparelli et al., 2013).

In cell culture, low Suc-concentrations were sufficient to saturate the signal

perception involved in the transcriptional regulation of the selected genes,

which therefore showed no sensitivity to small differences between low Suc-

concentrations (Fig. 5 and 7) (Paper I, Fig. 2). This suggests that, under

constant diurnal growth conditions, the available sugar is sufficient to

completely trigger repression/induction of the selected genes; a suggestion

supported by the finding that bZIP63, MGD2, At5g22920, TPS9 and BT2

were previously identified as “early” – or “slightly later”-responsive during

the diurnal cycle (Usadel et al., 2008). Consequently, the selected genes

appear insensitive to the small differences in sugar content observed

between the mutant and wt plants (Paper I, Fig. 6; Paper II, Fig. 1),

which makes it difficult to identify in planta the nature and origin of the

actual signal-giving molecule using the above mentioned mutant lines.

However, it could be suggested that the regulation of bZIP63, MGD2,

At5g22920, TPS9 and BT2 expression is responsive to changes in the CH-

balance within the tissue observed.

Fig.7: The responsiveness of gene expression of bZIP63, At5g22920, TPS9, BT2 and MGD2 is

saturated at low Suc-concentrations. 7 day old Xyl-grown A. thaliana cells were treated in the

dark with 0, 1 and 5 mM Suc for 1 and 3 hours, respectively. The overall repression/induction of

the genes was independent of the concentration applied, but was enhanced in a time-dependent

manner. Data represent the average expression ratio of a given gene between Suc-treated vs.

untreated samples. Error bars represent standard deviation (n=3).

35

Generation of the signal requires sugar-uptake and metabolisa-

tion

As suggested from analysis of intracellular sugar concentrations using NMR-

and FRET-based technologies, exogenously fed sugars are readily taken up

into the plant cell/tissue and undergo a fast metabolisation and subcellular

compartmentation (Deuschle et al., 2006; Gout et al., 2011; Kueger et al.,

2012). These processes result in an alteration of the nature and the

localisation of a signal-giving molecule. Even though the experimental set-up

(1 mM sugar, 1h incubation) aimed to reduce the impact of the applied sugar

on the diverse intracellular sugar pools (Paper I, Fig.S2), I could

demonstrate through the use of the sugar analogues Tur, Pal, L-Glc and 2dog

(Fig.3), that generation of effective signalling molecules, modulating the

expression of bZIP63, At5g22920, MGD2, TPS9 and BT2 may depend on

fast uptake and conversion of the sugar applied (Paper I, Figs. 4 and 6,

Tab. S2). Despite a general responsiveness of the selected genes to Suc, Glc

and Fru, 2dog-induced transcriptional changes suggested a Glc-dependent

regulation of the bZIP63, At5g22920 and MGD2 expression, involving the

action of the HXK protein, while Tur induced repression of TPS9, At5g22920

and BT2, suggesting a Suc-dependent regulation of expression (Paper I,

Tab. S2). Both Glc- and Suc-dependent signalling for the selected genes is

most likely localised to the cytosol.

Transcriptional regulation of bZIP63, At5g22920, TPS9 and BT2

through the energy-signalling network

In contrast to the expression of MGD2, sugar-responsiveness has been

earlier postulated for the C-type bZIP63 (Weltmeier et al., 2009; Matiolli et

al., 2011), At5g22920 (Hanson et al., 2008), TPS9 (Gibon et al., 2006; Paul

et al., 2008; Yadav et al., 2014), and BT2 (Mandadi et al., 2009), which all

were responsive to the exogenous application of Suc to young seedlings

(Osuna et al., 2007) and possessed an “early” or “slightly later”

responsiveness during the diurnal cycle (Usadel et al., 2008). In addition, it

has been suggested that the regulation of those genes is mediated by the

opposing activities of the TOR and SnRK1 kinases (Baena-González et al.,

2007; Delatte et al., 2011; Xiong and Sheen, 2012), with At5g22920 and BT2

being also targets of the S1-type bZIP11, which mediates SnRK1-dependent

Suc-responses. Furthermore, it has been shown that bZIP63 partially

mediates the SnRK1 dependent regulation of the starvation marker gene

DIN6 (Baena-González et al., 2007) and potentially interacts with the S-type

bZIP1 during the transduction of Glc-derived signals (Kang et al., 2010).

These findings support the interpretation that the transcriptional regulation

of those genes is adjusted in response to general CH-homeostasis, and that

36

the signal might derive from the convergent cellular sugar metabolism

(indicated by the 2dog effect) or convergent sugar/energy signalling

pathways, such as the bZIP11/SnRK1/TOR signalling network.

Perturbation of carbohydrate homeostasis is required for the

observation of differential expression in planta

Plants possess high flexibility to maintain CH-homeostasis, even in mutants

impaired in important metabolic enzymes (see above). This flexibility

ensures plant survival and growth under a variety of different conditions

(Gibon et al., 2009; Sulpice et al., 2014). However, this flexibility prevents

the observation of expression changes of genes, responsive to perturbations

in the CH-homeostasis, such as bZIP63, At5g22920, MGD2, TPS9 and BT2.

Consequently, the observation of changes in bZIP63, At5g22920, MGD2,

TPS9 and BT2 expression requires sudden and dramatic changes in the

cellular CH-availability. This assumption was strengthened by monitoring

the pbZI63::GUS expression in stably transformed A. thaliana seedlings

(Matiolli et al., 2011), which showed strong expression changes only upon

heavy perturbation of the intracellular CH-homeostasis (Paper II, Fig. 4).

This, in the case of bZIP63, furthermore suggests that the specific Glc-

responsiveness, indicated by the 2dog-effect in A. thaliana cell culture, is

related to a general state of cytosolic Glc-availability, feeding into the energy-

metabolism rather than being related to the specific sugar molecule.

Conditions causing a fast perturbation of the CH-balance include the

exogenous application of sugars to C-depleted seedlings. Under these

conditions, HXK activity is of utmost importance, as it mainly contributes to

the channelling of the imported sugar towards CH-metabolism, supporting

glycolysis and biosynthetic processes. From the analysis of the HXK1 null

mutant gin2.1 and the ectopic gain-of-function mutant hxk1_oe3.2, it could

be concluded that, in response to exogenous Glc-feeding, repression of

bZIP63, At5g22920 and BT2 involved the catalytic activity of HXK1 for

signal generation, triggering changes in gene expression (Paper II, Fig 5).

TPS9 repression appeared also susceptible to the HXK1 involvement, though

to a milder extent.

An interdependency of HXK and SnRK1 activity has recently been

hypothesised based on mathematical modelling of the in silico effect of HXK

overexpression (Nägele and Weckwerth, 2014). This model describes a

strong impact of the HXK activity on the Suc and T6P metabolism under

perturbation conditions, which is most likely related to the size of the

cytosolic UDP-Glc pool. Further analysis of the T6P and UDP-Glc content in

the sugar-treated hxk1_oe3.2 seedlings would provide experimental evidence

for the validity of this assumption. The potentially effected T6P-balance,

37

however, directly relates to a changed SnRK1 activity (Paul et al., 2008;

Nunes et al., 2013b), which in turn will affect the expression of bZIP63,

At5g22920, TPS9 and BT2.

bZIP63, At5g22920, TPS9 and BT2 expression potentially

determines growth

Previous knowledge and evidence from this study can be summarised in a

revised model for the sugar-dependent regulation of bZIP63, At5g22920,

TPS9, BT2 and MGD2 (Fig. 8).

Fig.8: Simplified model for the putative signalling pathways controlling the transcriptional

regulation of bZIP63, TPS9, BT2, At5g22920 and MGD2. The illustration combines

experimental evidence from the present study and previous knowledge from available

publications (cited in the text). Red lines indicate inhibition, while blue lines indicate induction.

Suc: sucrose, Xyl: xylose, Fru: fructose, Glc: glucose, HXK: hexokinase, G6P: Glc-6-phosphate,

G1P: Glc-1-phosphate, UDP-Glc: uridine-diphosphate-glucose, T6P: trehalose-6-phosphate,

SnRK1: Suc-non-fermenting-related kinase 1, TOR: target of rapamycin.

38

In this model, regulation of all genes requires metabolisation of the applied

sugar, independently of the sugar species, towards a sugar-phosphate. This

explains the effect of Xyl on the expression of these genes, since the

postulated metabolisation pathway (Fig. 4) includes formation of molecules

contributing to the sugar-phosphate pools. Based on knowledge about their

putative function, mutant phenotypes and potential/confirmed localisation

of their expression and products, it can be speculated that the genes studied

are important for regulation/progression of growth and developmental

processes, such as cell division/cell cycle progression (At5g22920, BT2 and

bZIP63), germination (bZIP63, MGD2) and pollen-/flower development

(MGD2). The general response to CH-homeostasis is reflected in the diurnal

pattern of the gene expression within the plant. Given that the genes studied

were selected in a system where Xyl-grown cells, characterised by a low CH-

status, were re-supplied with a small amount of sugar, obviously perceived as

a change/increase in the intracellular CH/energy-status, it is tempting to

speculate that the genes mediate the adjustment of the progression of the cell

cycle, and thereby growth, to the availability of CH. This hypothesis is

furthermore supported by the recent findings that (i) the expression of

TPS9, bZIP63 and At5g22920, potentially localised to meristematic tissues

(Winter et al., 2007; Weltmeier et al., 2009), is controlled via the

transcription factors Hecate1 (HEC) and Wuschel (WUS), both essential for

determination of stem cell fate and induction of stem cell proliferation in the

SAM (Schuster et al., 2014); and (ii) that TOR signalling regulates the Glc-

dependent expression of bZIP63, At5g22920, TPS9 and BT2 in root

meristems (Xiong et al., 2013).

In contrast to bZIP63, At5g22920, TPS9 and BT2, less previouse knowledge

is available for the expression and importance of MGD2. Still, it has been

shown that MGD2 expression, which is mainly restricted to developing

flowers and pollen (Shimojima and Ohta, 2011), is regulated in response to

cytokinin-treatment and environmental stress, which is accompanied by

nutrient deprivation (Shimojima and Ohta, 2011; Kobayashi et al., 2012).

Especially phosphate deficiency signalling, which is known to be partly

mediated by sugar signalling (Karthikeyan et al., 2007), affects MGD2

expression. This supports the assumption of a transcriptional regulation of

MGD2 in response to sugar-availability or general CH-balance, which may

be signalled through convergent or distinct signalling pathways. Assuming a

strong tissue-dependent MGD2 expression, restricted to the inflorescence in

mature plants (Awai et al., 2001), no conclusion can be drawn on the

requirement of HXK1 catalytic activity for the transcriptional regulation, as

the presented analysis was restricted to non-flowering 13 day old seedling,

which might possess already strongly reduced MGD2 expression (Awai et al.,

2001). As reported earlier, the loss-of-function mutant of HXK1 (gin2.1) does

39

show an impaired flowering phenotype (Moore et al., 2003), which might

involve the alteration of MGD2 expression. However, this remains to be

tested.

Long-term real-time live cell imaging – an upgrade of the

toolbox

Besides the indication for sugar-dependent regulation of cell division and

development through the expression of bZIP63, At5g22920, TPS9, MGD2

and BT2, functional annotation of genes selected from the bulk

transcriptomic data generated from sugar-treated Xyl-grown A. thaliana cell

culture (Paper I) further pointed towards a sugar-types specific expression

of genes implicated in the cell cycle progression, especially during the M-

phase (Paper I, Fig. S6; Paper III, Fig. S1, Tab. S1).

Even though bulk data analyses, like transcriptomics data, provide powerful

means for the generation of novel hypotheses, the real cause-effect

relationship between gene activity and phenotypic adaptation has to be

confirmed to conclude on biological relevance (Kell and Oliver, 2004). Bulk

transcriptomics data generally represent a snap-shot of a steady state, which

indicates a given signal response at the transcriptional level, but does not

allow for inference on physiological adaptation on e.g. enzyme activity levels.

This was highlighted by analyses in A. thaliana seedlings, where diurnal

regulation of gene expression was not accompanied by subsequent changes

in the respective enzyme activities, which in fact determine the long-term

phenotypic adaptation (Gibon et al., 2006; Weckwerth, 2008). To test the

transcriptomics-based hypothesis of a cell cycle regulation by specific sugar

molecules (Paper III, Fig. S1, Tab. S1; Riou-Khamlichi et al., 2000), I

analysed cell cycle progression on a single cell level within A. thaliana cell

populations using an optimised long-term real-time live-cell imaging

methodology (Paper III).

Similar to the application of fluorescent fusion proteins both in planta and in

seedlings (Azimzadeh et al., 2008; Buschmann et al., 2011), the live cell

imaging method was based on monitoring of a TUA:GFP fusion protein over

a 72 h period to follow the time-dependent progression of cell division within

a stably transformed A. thaliana cell population. This fusion protein marked

the distinct microtubule structures PPB, MS and phragmoplast, and helped

to identifying the subsequent stages of the M-phase (Paper III, Fig. 2)

during the extensive rearrangement of the endomembrane system and the

cytoskeleton (Müller et al., 2009; Rasmussen et al., 2013). Using this system,

I could define that on average the processing through the M-phase took

about 3-4 h (Paper III, Fig. 7), which represented 6-10% of the average cell

40

cycle duration time of 19-48 h within different A. thaliana accessions

(Beemster et al., 2002). This percentage, however, might be an

overestimation, as the time necessary to progress through the cell cycle,

indicated by the observation of 2 subsequent related division events, could

not be estimated during the live cell imaging. Furthermore, Xyl-grown cell

culture was characterised by a doubling time of approximately a week

(Paper I, Fig. 1A), indicating that cell cycle completion of Xyl-grown cells

most likely requires more than 72 h.

Based on the microtubule-structures, I observed that the majority of cell

divisions in the vacuolated A. thaliana cell culture occurred in an

asymmetric manner resulting in two daughter cells of different size and

shape (Paper III, Fig. 4). The (a)symmetry of plant cell division directly

controls the size of the resulting daughter cells and their future function and

fate (Knoblich et al. 2008; Ten Hove et al. 2008; Dong et al.2007).

Therefore, it is an essential component in the establishment of tissue growth

and development (Roeder et al. 2012). Based on transcriptome analyses,

there was no clear indication for the assumption that the A. thaliana cell

culture represents one specific tissue (Paper I, Figs. 5, 6, S5 and S9;

Paper II, Figs. 2 and 3). Despite that, the observation of a high percentage

of asymmetric cell division might indicate that the A. thaliana cell

population does represent a specific tissue/cell type, characterised by

asymmetric cell division, e.g. cells from the stem cell niche. To test this

assumption, it would be interesting to express fluorescent markers under the

control of tissue (e.g. meristem) specific promoters, such as the pWUS,

pATML1, pLFY, pANT, pTPST, pRGF1/2/3, pSCR or pWOX5 (Grandjean et

al., 2004; Aichinger et al., 2012). Furthermore, the absence of extrinsic

factors in single cell suspensions, which are essential to define e.g. cell

polarity and orientation within the tissue, might affect cell division

geometry. Plant cells in cell suspension may both form aggregates but also

remain as single cells. In contrast, long-term live-cell imaging revealed, that

cells, which were not subject to the mechanic forces generated by the

shaking, mainly remained connected after successful cell division (Paper

III, Fig. 3, optimal). It might be interesting to investigate, whether this

connection allows for the establishment of cell polarity, which might provide

spatial information. Based on the live cell imaging analysis, I could also show

that the postulated rules stating that symmetric cell division represents the

general cell division mechanism, in which the microtubules guide the

positioning of the future cell plate at the shortest diameter of the cell (Besson

and Dumais 2011), did not fully apply for my observation. General

positioning of the cell plate, affecting (a)symmetric cell division, is crucial for

pattern formation, e.g. the vascular pattern during embryo development (De

Rybel et al., 2014), a process which is highly dependent on the balance of

41

cytokinin and auxin signals or other extrinsic information (Aichinger et al.,

2012; De Rybel et al., 2014). However, for the established Xyl-grown cell

culture system, one has to presume that all effects related to phytohormones

are caused by endogenous hormone-biosynthesis. Knowledge about the

extent of the exchange of those signals between cells as well as the effect of

exogenously applied hormones on the symmetry of cell division in the

versatile cell culture are therefore crucial for further speculation on the

biological meaning of my observations. To further investigation on the effect

of hrmones on cell division geometry, the combination of the fluorescent A.

thaliana cell line and the established live cell imaging method should prove

to be highly valuable.

Sugar-type specific progression of the cell cycle is determined by

the length of the interphase

Cell division is a highly energy consuming process, which depends on the

availability of sugars. They are essential for the control of the progression of

the cell cycle through the different phases (Wobus and Weber, 1999; Francis

and Halford, 2006). Several mutants which are impaired in either the CH-

metabolism (Barratt et al., 2009; Heinrichs et al., 2012; Paparelli et al.,

2013) or the progression of cell division (Ambrose et al., 2007; Gutzat et al.,

2011; Skylar et al., 2011; Skylar et al., 2013; Racolta et al., 2014) possess

dwarfed phenotypes which can be partially recovered through the exogenous

application of sugar. Root elongation is induced in response to moderate

sugar concentrations (Paper III Fig. S3; Stevenson and Harrington, 2009).

These growth inductions, which could be both caused by differential cell

division (Skylar et al., 2011; Skylar et al., 2013; Peng et al., 2014) or affected

cell elongation (Paparelli et al., 2013), did not allow for a distinction between

the impacts of different sugar-types or an effect on the cell cycle progression.

It has been reported that especially Suc impacts cell cycle progression

through the transcriptional regulation of important cell cycle control genes

(CDK and cyclins), regulating the transitions between G1-to-S and G2-to-M

phase (Riou-Khamlichi, 1999; Riou-Khamlichi et al., 2000; Menges et al.,

2005; Hartig and Beck, 2006; Skylar et al., 2011; Skylar et al., 2013).

However, the impacts of specific sugar-species, especially on progression

through the M-phase, are still undefined.

Using the newly established long-term real-time live-cell imaging technique,

I could confirm the hypothesis of a sugar-type dependent impact on the

efficiency of cell division, which relates to the number of cell division events

observed in a given time (Paper III, Figs. 5, 6 and S1, Tab. S1). The sugar

species-related differences were indicated through a higher cell cycle

synchronisation and efficiency when cells were supplied with Suc over Xyl

42

and Glc in the growth medium (Paper III, Figs. 5 and 6). As the

processing time through the M-phase stages, hallmarked by distinct

microtubule-based structures, was unaffected by different sugars (Paper

III, Fig. 7), it could be concluded that the length of interphase is decisive for

the sugar-type dependent control of cell division efficiency. This conclusion

is supported by the previous identification of Suc-effects on the G1-S and G2-

M- phase transitions (Riou-Khamlichi, 1999; Riou-Khamlichi et al., 2000;

Menges et al., 2005; Hartig and Beck, 2006; Skylar et al., 2011; Skylar et al.,

2013). On the basis of the experimental evidence from expression analysis of

selected genes, e.g. bZIP63, AT5g22920, TPS9 and BT2, as well as analysis of

cell division efficiency, it can be assumed that within the short-term range,

distinct sugars are perceived as a signal for the induction of cell division,

which was visualised by the low, but observable, induction of cell division by

2dog (Paper III, Fig. 8A and S2). However, under long-term observations,

the energy contained in a given sugar appears to define the maintained

efficiency of cell cycle progression (Paper III, Fig. 5). Together, it

furthermore can be speculated that, once a cell is committed to enter the M-

phase, it will divide independent of the metabolic state. This attaches great

importance to the previously described sugar-control of the phase-

transitions prior to the M-phase (see Introduction), to guarantee that cell

division only occurs when sufficient energy is provided.

2dog-effects indicate a perturbation or collapse of the cellular

carbohydrate/energy homeostasis

During this study, the Glc-analogue 2dog has been applied to estimate

underlying mechanisms of Glc-related cellular responses, both in gene

expression (Paper I) and cell division (Paper III). Within this context,

2dog was assumed to indicate the involvement of the enzyme HXK in the

mediation of the respective responses, as reported earlier (Jang and Sheen,

1994; Umemura et al., 1998; Riou-Khamlichi et al., 2000; Angeles-Núñez

and Tiessen, 2012; Yim et al., 2012). Due to its molecular properties, 2dog

affects the primary CH-metabolism, thereby strongly inhibiting UDP-Glc

and hexose-P formation in A. thaliana seedlings and Chenopodium cell

suspension culture (Klein and Stitt, 1998; Yadav et al., 2014). The inhibition

of UDP-Glc- and hexose-P- formation suggests substrate limitation for the

cellular energy metabolism, accompanied by a decreased ATP/ADP ratio

within short time, as observed both in 2dog-treated Chenopodium cells (6 h

treatment) and Xyl-grown A. thaliana cells (1 h treatment) (Paper III, Fig.

8B; Klein and Stitt, 1998). Furthermore, long-term 2dog-treatment of Xyl-

grown A. thaliana cells led to dramatic effects with an immediate arrest of

cell division followed by cell death (Paper III, Fig. 8A). Despite the

suggestion that 2dog inhibits Suc-biosynthesis in Chenopodium cells (Klein

43

and Stitt, 1998), it was shown that both Suc and T6P levels were unaffected

in 2dog treated A. thaliana seedling (Yadav et al., 2014). Furthermore,

treatment with both Suc and 2dog resulted in Suc-accumulation which was

comparable to that in Suc–treated seedlings (Yadav et al., 2014). However,

this combined treatment only non-significantly induced cell division in A.

thaliana cell culture (Paper III, Fig. 8C, light gray bar), suggesting that

Suc, which is essential for the progression of the cell cycle towards cell

division (Skylar et al., 2013), is necessary but not sufficient. This

assumption, needs further support by live-cell imaging on Suc-grown A.

thaliana cell culture treated for 48 h with or without 2dog. This would

provide the resolution of the effect on cell division effiviency that could not

be distinguished by the measurement of dryweight. The rapid growth

recovery observed after 2dog-removal from Suc-containing media (Paper

III, Fig. 8C) indicates that the cells survived and Suc is properly

metabolised to restore the CH-balance. On a whole plant level, 2dog inhibits

germination (Paper III, Fig. S4; Pego et al., 1999) and prevents TOR-

dependent root meristem activation (Xiong et al., 2013). Most likely through

the induction of cell death in the meristem, 2dog irreversibly stops root

elongation (Paper III, Fig. 9 and S4; Baskin et al., 2001). Interestingly, the

removal of 2dog from the growth media, allows for the formation of

lateral/adventitious roots from the obviously unaffected hypocotyl area

(Paper III, Fig. 9 and S4). This represents an example of the high

flexibility of plants to activate new meristems, a process most likely involving

Glc-TOR signalling (Xiong et al., 2013). Given that the TOR-induced

activation of meristems requires Glc from photosynthesis (Xiong et al.,

2013), it further can be speculated that the 2dog treatment affected either (i)

the production/transport of photosynthate, (ii) its perception in the

hypocotyl area or (iii) TOR gene-expression, as slightly indicated in the

2dog-treated cell culture (Paper I, Fig. S8).

Generally, 2dog-effects on growth and development are likely to be mediated

through a SnRK1/TOR-signalling pathway (Baena-González et al., 2007;

Wahl et al., 2013; Xiong et al., 2013). The 2dog-induced alteration of the

overall energy state might therefore explain the observed induction of

transcriptional regulation of e.g. bZIP63, At5g22920 and MGD2 in A.

thaliana cell culture (Paper I, Fig.4, Tab. S2). However, the direction of

the expression changes within 1 h were similar to sugar-induced responses,

indicating that 2dog is perceived as sugar, which potentially signals energy,

not perturbation of the energy homeostasis, a discrepancy, which might have

its reason in the chronological sequence of the early 2dog-response. To

further conclude on the effects caused by 2dog, a higher temporal resolution

of the 2dog response leading to cell death is necessary.

44

Conclusion and Future Perspective

The survival of a plant and its population is highly dependent on its ability to

respond to environmental influences through the modulation of

physiological processes, which contribute to the adjustment of growth and

development. Growth is a highly demanding process, which requires the

availability of energy, e.g. in the form of carbohydrates (CH). In order to

integrate information on the metabolic state and environment of a plant,

sugar(s) control specific responses though complex signalling networks.

Within the scope of this thesis I analysed different aspects of the

mechanisms underlying CH-specific regulation of gene expression and cell

division. The establishment and application of a novel biological system,

based on Xyl-grown stem-cell-like Arabidopsis thaliana cell culture,

confirmed the assumption of a rapid gene expression response which

distinguishes between the soluble sugars Suc, Glc and Fru, respectively.

Detailed characterisation of the expression of the selected Glc-responsive

genes bZIP63, At5g22920, MGD2 and the Suc-responsive genes TPS9 and

BT2, led to the formulation of a model, which included the finding that gene

regulation requires a cytosolic localisation of the sugar signal, which is

generated through sugar metabolisation. Furthermore, using mutants

impaired in the CH-metabolism, I could show that the generation of the

signal regulating bZIP63, At5g22920 and BT2 expression, involves the

catalytic activity of HXK1, resulting in a regulation which was especially

sensitive to a perturbed CH-homeostasis both within plant cell culture and

whole seedlings. The application of an optimised protocol for long-term real-

time live-cell imaging to the Xyl-grown A. thaliana cell culture enabled me to

prove that plant cells distinguish between the sugar signals Suc, Glc and Xyl

in their adaptation of cell division. This adaptation, which results in a

differential cell division rate in response to the specific sugars, is mainly

caused by a prolonged interphase. Furthermore, using the Glc-analogue

2dog, I could show that the sugar signal itself might be sufficient to induce or

prepare for cell division. However, progression of cell division and

consequently organ growth over long-time requires the availability of the

energy contained in the sugar.

The results of this study and the conclusions drawn from it open up a wide

range of further perspectives. First of all, the application of the highly

versatile biological system, which is based on the A.thaliana cell culture,

grown in a hormone-free Xyl-containing media, can be further extended to

research on signalling processes which might interact with the CH-

45

availability, e.g. investigation of a variety of molecules, which either

separately or in combination are potent promoters or inhibitors of cellular

responses or the detailed characterisation of a variety of signalling pathways

using transgenic cell lines. However, these studies should be accompanied by

a detailed analysis of the metabolic signature of the underlying cell system.

Furthermore, combination of the Xyl-grown A. thaliana cell culture with

live-cell imaging allows to further assess the impact of small changes in the

metabolic state on other cellular processes, e.g. autophagy, cell wall

formation, cell differentiation, establishment of cell polarity and cell

communication. Analysis of the identity of the cells using specific marker

genes, together with the ectopic expression of fluorescent proteins indicating

polarity (e.g. PIN proteins) could help to clarify to which extent the cell

culture system resembles meristematic cells.

Based on my results from the analyses of sugar-type specific regulation of

expression of bZIP63, At5g22920, TPS9, MGD2 and BT2, I revised a model

of the underlying signalling pathway and the putative involvement of those

genes in the regulation of plant growth. The hypothesis of a potential

function of these genes in mediating the sugar-dependent cell division and

plant growth needs further support from e.g. detailed localisation studies,

mutant phenotypes, the analysis of gene expression and protein localisation

in mutants impaired in cell cycle progression and cell plate formation, the

analysis of the impact of the proteins on the cell cycle machinery and the

identification of potential interactors.

In contrast to the sugars Suc, Glc and Fru, appeared 2dog as a strong

inhibitor of cell division and root growth. The fact that cell culture may

recover from 2dog treatment and that plants can initiate adventitious root

formation in response to 2dog treatment indicate that the 2dog and its

derivatives are not directly toxic and they are not transported over long

distances through the plant. Still, it remains unclear whether the growth

arrest is only a consequence of death of the meristematic cells or of the whole

root. Further studies on sugar-dependent cell division, in response to Suc,

Glc, Fru, Xyl but also 2dog, should include the analyses of cell vitality and

cell division dynamics in roots, using e.g. propidium-iodine staining, to

support both the hypothesis of sugar type specific cell division in plants and

to further dissect the 2dog-effect on root growth.

Altogether, future work, based on evidence derived from this study, will

broaden our view on the regulation of plant growth as a consequence of a

plant sensing its metabolic state, which in the long run assures growth and

survival of the population.

46

Acknowledgements

First of all, I would like to thank everybody who accompanied me during the

past years at UPSC. You all contributed to create this great working

atmosphere that allowed me to develop both scientifically and personally.

I would like to thank my supervisor Leszek A. Kleczkowski for giving me

the opportunity and the space to pursue a doctoral degree. I’m greatful for

your kindness and your help with all the writing of the manuscripts and the

thesis. I would also like to thank my Co-supervisor Edouard Pesquet for all your scientific impact on my work, which you supported by a never ending flow of ideas and critical thoughts. I appreciate your help in communicating my ideas and understanding of how science works.

Special thanks to Daniel D., for all your help in the lab and all the

discussion, be it about science, politics or society. I very much enjoyed

having your positive attitude and humour around.

Agnieszka Z., Krisztina Ö., Anna P., Marta D. and Delphine G.,

thank you so much for all your help, advice and fruitful discussions, which

clearly contributed to improving my lab work.

Thanks to all my former and present office mates, Peter K., Paulina S.,

Agnieszka G., Daniel D., Nico B. and Daria C. for creating a warm and

inspiring atmosphere. Especially Nico B., thank you for your interest in my

work, for taking the time to critically discuss results and all the constructive

criticism on my presentations and writing.

Special thanks to Daniela L. for critical reading of the thesis, and the ability

to see the positive side of everything.

Christian K., Paulina S. and Daniel D., thanks for sharing the teaching

experience with all the pleasant and challenging moments.

Many thanks to Matt G., Jane LG. and Elisabeth F., for your welcome

and support during my stay in the US. I learned so much during this short

time!

47

Thanks to all the people that keep this place running, especially Janne K.

for all help with technical problems and Siv S. and Rosi F. for your help in

cleaning and autoclaving all my glassware. Furthermore, I would like to thank everybody, who encouraged me in times of doubt. Especially Rebecca H., Krisztina Ö., Nico B., Nathaniel S., Kathryn “Tiggi” R., Judith F., Anke C. and Daniela L., your words gave me the strength to continue on the path.

I would like to thank Agnieszka G. and Adele W. for sharing all the

excitement during the first year here in Sweden. I miss you guys a lot!

Nico B. and Olivier K., thanks for supplying me with a huge variety of

music, which stroke another string in my head and clearly helped to focus.

Thanks to all my friends back in Germany. Especially Maja B., Rico W. and

Anja L., who over the years supported me with never ending phone-calls

and a huge number of “Care-packages”.

Special thanks to my parents, Angelika and Stephan K., who supported

me in all my decisions and spared neither trouble nor expense to help me on

my way!!!

Albrecht R., even though you never wanted to live here in Sweden, with

your decision to come to Umeå and stay with us, you gave me the

possibilities and freedom I needed for my work. I’m deeply grateful for that.

Finally, I want to thank Louise K., my little sunshine and anchor in life.

Deine Sicht auf die Welt, deine Neugierde, Unbeschwertheit und dein

Lächeln zeigen mir jeden Tag aufs Neue, welche Dinge im Leben wirklich

wichtig sind.

48

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