Drought

salt

Heat

Cold

Cellular Signal Transduction Pathways under abiotic stress in

plants & its applications

Chavan Neha

Securing food with less land

13 billion hectares (Earth’s surface)

1.5 billion 3.5 billion hectares ( agriculture) (meadowland pasture.)

seven million hectares of agricultural land are lost (every year)

Need of four billion hectares of land.

As a result of population growth, agricultural production must increase by around two percent per year .(FAO statistical division ,2008)

What is stress???

‘’A biological stress is an adverse

force or a condition, which inhibits the normal functioning and well being of a biological system such as plants .’’

(jones et.al ,1989)

Stress elicitors

• cell response is initiated by interation of extracellular material with plasma membrane protein.

• The extracellular molecule is called as ligand and the protein with which it will interact is called as receptor.

Abiotic 1. Cold (chilling and frost)2. Heat (high temperature)3. Salinity (salt)4. Drought (water deWcit condition)5. Excess water (Xooding)6. Radiations (high intensity of ultra-violet and visible light)7. Chemicals and pollutants (heavy metals, pesticides, and aerosols)8. Oxidative stress (reactive oxygen species, ozone)9. Wind (sand and dust particles in wind)10. Nutrient deprivation in soil

Biotic 1. Pathogens (viruses, bacteria, and fungi)2. Insects3. Herbivores4. Rodents

(Mahajan & Tuteja, 2005)

Major cause of loss in Crop production

stress

•Stress reduces harvests dramatically•Abiotic factors are responsible for the lion’s share of harvest losses, however.

(Bayers crop science magazine,2008)

How abiotic stress affects the growth and development of crop

(Vicers et.al ,2009)

Biotechnological approches to study stress response in plants

(Climente et.al,2013)

Overview of signal transduction pathway

(Mahajan & Tuteja, 2005)

Crosstalk of signal transduction pathways

(Knight & knight ,2001)

Complexity

(Wangxia Wang et al.,2003)

Types of signal transduction pathways

Ionic &Osmotic stress signaling

Signaling to co-ordinate cell division

& expansion

Signaling for detoxification .

(Jian –kang Zhu ,2011)

Cellular Homeostatis Control and repair cell damage due to stress

To level suitable stress condition

(Miransari et al ,2013)

Contd..,

Major signal transduction pathways under abiotic stress(drought ,salt and osmotic stress )

• ABA (dependent and independent)signaling

• MAPK mediated signaling • SOS signaling • Phospholipid signaling.

ABA biosynthesis

(Zhu et al ,2005)

Pyrophosphate +glyceraldehyde 3 phosphate

IPP(isopentenyl pyrophosphate)

Farnesyl pyrophosphate,GGPP,B-carotein

Beta carotein

Zeaxanthin

ZEP

Violaxanthin

NCED

Neoxanthin

Xanthoxin

ABA aldehyde Xanthoxinic acid ABA

Phaseic acid

Abscisic alcohol

ABA conjugation • ABA can be inactivated at C1 ,by forming different conjugate . • One of this conjugate is ABA –GE

• Over-expression of UGT71B6 leads to an increased ABA-GE content in Arabidopsis. The incresed ABA-GE will be stored in vacuoles.

• What happens is that under dehydration condition the GE gets separated out from ABA by the enzyme Betaglucosidase(BG)

‘’Recently 2 BG ,BG1 and BG2 identified in Arabidopsis’’

(Danquah et al , 2013)

Free ABA

ABA Transport :

Two main transporters of ABA – 1.AtABCG25 2.AtABCG40

‘’ The stomata of atabcg40mutants close more slowly in response to ABA, resulting in reduced drought tolerance.’’

(Danquah et al , 2013)

Early events in ABA signaling

(Nakashima & shinozaki ,2013)Identification of SnRK2

ABA dependent and Independent pathway

Mitogen activated protein kinase pathway

(Danquah et al , 2013)

Recent updates

• ZmMkk1-Chilling stress & pathogen defence

(Cai et al ,2013 )• ZmMkk3- mediates osmotic stress and

gives signal for ABA (Cai et al ,2013 )• ZmMpk5-salt stress in maize (Zang et

al ,2013)• MAPK3 – confers U.V and Heat tolerance

(Raina et al ,2013 )

(Danquah et al , 2013)

ABA induced activation of MAPK

ABA perception in guard cell

activate SnRK2 kinase (OST 1)

Phosphoraylation of NADPH oxidase Rhof

Leads to ROS accumilation

Activate 2 MAPKs,MPK9/12

SLAC activation

Stomal opening

SLAC-s type ionic channel

PYR-Enhanced transpiartional loss

SOS pathway under salt stress

(Mahajan & Tuteja ,2005)

Osmotic stress, cold, and ABA activate several types of phospholipases that cleave phospholipids to generate lipid messengers (e.g., PA, DAG, and IP3), which regulate stress tolerance partly through modulation of gene expression. FRY1 (a 1-phosphatase) and 5-phosphatase-mediated IP3 degradation attenuates the stress gene regulation by helping to control cellular IP3 levels.

Phospholipid signaling

(jian-kang Zhu ,2002)

PLD and PA in response to H2O2PLD , is activated in response to H2O2 and the resulting PA functions in amplification of

H2O2 -promoting antiPCD

Stress stimulates production of H2O2 that activates PLD associated with the plasma membrane. Potential activators: Ca2+ and oleic acid. This increases PLD affinity to its substrates, stimulating lipid hydrolysis and PA production. PA may bind to target proteins, such as Raf-like MAPKK, that contain a PA binding moti, leading to the .) activation of MAPK cascades. PA may also function by modulating membrane trafficking and remodeling. These interactions modulate the cell's ability to respond to oxidative stress and decrease cell death. Dashed lines - hypothetical interactions.

•Knockout of PLD renders Arabidopsis plants more sensitive to the reactive oxygen species H2O2 and

to stresses •H2O2 activates PLD , and PLD -derived PA functions

to decrease the promotion of cell death by H2O2.

These results suggest that both PLD and its product PA play a positive role in signaling stress responses •PLD and its derivative PA provide a link between phospholipid signaling and H2O2-promoted cell

death. PLD and PA positively regulate plant cell survival and stress responses.

PLD and PA

(Laxalt and Munnik,2002)

Transcriptional regulatory network under abiotic stress responses

(Hirayama & shinozaki ,2010)

Signaling under heat stress

(Bokszczanin & fragcostefanakis ,2013)

Signaling under cold stress

Cold stress signaling with secondary messenger

Plants may sense low temperature through changes in the physical properties of membranes, because membrane fluidity is reduced during cold stress

plasma membrane rigidification raised by a membrane rigidifier, dimethyl sulfoxide (DMSO),

Induction of COR gene

Second shock

Increase in ca+ Regulation of COR gene expression

Ca sensors

CBF

calmodulinCDPKs

CCaSK

Positive regulation of cold stress Negative regulation of

cold stresss

CAMAT

Miura and Furumoto,2013

CBF dependent signaling

Miura and Furumoto,2013 ICE1

Applications

Case study 1

Experimental protocol and results

1.Overexperssion of SOS1 in transgenic plants:

A. A.thaliana plants were transformed with a construct containing the SOS1 cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter.

B. Screening is done on M.S. agar medium cantaining 40mh/l kanamycin.C. Presence confirmed by PCR (primer specific for 35s promoter and SOS1gene )

2.RNA gel Blot:

A. A.Thaliana plant grown on M.S.medium under continous light.B. For salt treatment

10 days old seeding

Whatman filter paper soaked with 100mM Nacl &200 mM Nacl

Whatman filter paper soaked in M.S. medium

(stress) (Control)

Total RNA isolation and Nouthern analysis

Control

transgenic

Stress given 0mM,1oomM,200mM

Resisitant lines (ST-4,ST-8) analysised for RNA gel Blot

Same lines S-4 ,S-8 grown on medium (M.S),cantaning different conc. of Nacl

Physiological analysis of plants

Root growthChlorophyll content

Total protein level

figure 4. :Reduced Na+ accumulation in plants overexpressing SOS1.

control ST -8 ST-4

Figure 3. Enchanced salt tolerance of SOS1-overexpressing plants

Control 50 mM 150mM

Figure 5. Calli overexpressing SOS1 are more tolerant of NaCl.

Case study 2

Results

High expresssion was found in P58 and P1142

Fig. 2 Growth characteristics of the AtDREB1A plants and the cultivar BR16 under control (C-dark bars) and moderate water stress (DS-grey bars) conditions in the greenhouse. Differences were not statistically significant (Duncan 5 %) (n = 6)

Transpiration of Atderb1A plants an cultivator BR16 ,A - water stress ,B-in greenhouse

Greenhouse

phytotron

Conclusion • Abiotic stress signaling is an important area with

respect to increase in plant productivity. Therefore, the basic understanding of the mechanisms underlying the functioning of stress genes is important for the development of transgenic plants. Each stress is a multigenic trait and therefore their manipulation may result in alteration of a large number of genes as well as their products. A deeper understanding of the transcription factors regulating these genes, the products of the major stress responsive genes and cross talk between different signaling components should remain an area of intense research activity in future.

Discussion

References• Knight,H.and knight,M.R.2001.Abiotic stress signaling

pathways:specificity and crosstalk.Trends in Plant sci.,6:262-267

• Zhu,J.K.2002.Salt and Drought stress signal transduction in plants.Ann.Rev.Plant Biology,53:247-273.

• Danguah,A.,Zelicourt,A.,colcombet,J.and Hirt,H.2013.The role of ABA and MAPK signaling pathways in Plant abiotic stress response.Biotechnological Adv.,

• Hirayama,T and Shinozaki,k.2010.Research on Plant abiotic stress response in post genome era:past,present& future.The Plant journal,61:1041-1052

• Mahajan,s and Tuteja,N.2005.cold,salinity & drought stress:An overview .Archi.Biochem.biophysics,444:139-158

References• Miura,k and Furumoto,T.2013. Cold Signaling and Cold Response in

Plants.Int.J.Mol.Sci.,14:5313-5337.

• Shinha,A.k.,Jaggi,M.,Raghuram,B.and Tuteja,N.2011.Mitogen activated protein kinase signaling in plants under abiotic stress.Plant signaling and behaviour,6:196-203

• Nakashima,K.and shinozaki,K.Y.2013.ABA signaling in stress response & seed development.Plant cell Rep,32:959-970

• Shi,H.,Lee,B.Wu,S.and Zhu,J.2002.Overexpression of plasma membrane Na+/H+ antipoeter gene improves salt tolerance in arabidopsis .Nature Biotechnology,21

• Bokszczanin ,K.L., and Fragkostefanakis ,S.2013.Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance.Frontiers in Plant Sci.,4:135

Thank you…..