SATYA PRAKASHMSC( BOTANY) , SEM IV(2013)

Abscisic acid signaling in guard cell movement

Jung et al.,2002

Presentation by : Satya Prakash Chaurasia Department of Botany, University of Delhi, satya.prakash101991@gmail.com www.facebook.com/91satya www.twitter.com/Satyapr101991h ttp://www.linkedin.com/pub/satya-prakash chaurasia/65/a76/a25

Mob: +919654814497

INTRODUCTION

• It is important to study ABA signaling. Why?

It is a key endogenous messenger in plants’ responses to biotic and

abiotic stresses [Christmann, A. et al. (2006); Adie, B.A. et al. (2007);

Ton, J. et al. (2009)]. ( e.g drought , salanity pathogen attack etc.)

Beside this also involved in seed dormancy , maturation, germination

embryo development and many other developmental processes.

Intro…continued….

Stomatal closure requires release of solutes from guard cells where K+ seems to be the most important as more than 85% of it is released during stomatal closure( Talbott and Zeiger., 1998).

How ABA acts to induce the loss of solutes in guard cells??

ABA Receptors in gurad cells

Previous studies have identified several types of putative receptors that may mediate ABA functions (McCourt and Creelman, 2008; Cutler et al., 2010).

Most recently, a family of START domain proteins, known as PYR/PYLs-RCARs, were shown to function as ABA receptors (Fujii et al., 2009; Ma et al., 2009; Melcher et al., 2009; Miyazono et al., 2009; Park et al., 2009).

Melcher et al., 2010

PYL2

Binding of ABA with START family proteins shows interesting Gate-Latch-Lock Mechanism!

Type 2C protein phosphatases(PP2C)

Genetic studies have identified several PP2C genes that are required for ABA signaling in Arabidopsis. Of these members, a group A PP2C, including ABI1, ABI2, HAB1, HAB2, AGH1, and PP2CA, generally function as negative regulators of the ABA response (Merlot et al., 2001; Nishimura et al., 2004; Saez et al., 2004; Kuhn et al., 2006; Rubio et al., 2009).

Studies have shown that PYL/RCAR proteins interact with PP2C -type family members and inhibit their activities (Ma et al., 2009; Park et al., 2009; Lee at al.,2010).

Lee et al., 2010, demonstrated the interaction between the two by Yeast two hybrid system.

Physical Interactions among PP2CA and RCARs. Yeast two-hybrid assay of interactions between PP2CA and RCARs. Growth on the selection medium (SC-ALTH) was used as an

indicator of interaction (left row) and with 10µM ABA (middle row).

Lee S C et al. Mol. Plant 2013;6:528-538

© The Author 2012. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

10µM ABA

• ABA receptor of START family physically interacts with negative regulator of ABA induced stoamatal closure, protein phosphatase 2C.

OST1(Snrk2) activity Is required for proper stomatal closure

Geiger et al., 2009 , compared stoamta of WT Arabidopsis with the stomata of ost1-2 mutant Arabidopsis plant and showed that ost1–2 stomata during day-night transition close much slower than those of WT plants.

They also proposed that ost1 promotes guard cell closure by activating outward anion channel SLAC1.

Source:- Takashi Hirayama and Taishi Umezawa., 2010

Some targets of SnRK2 kinases:-• Transcription factors• Guard cell anion channels• Inward K+ ion channels• NADPH oxidase for ROS production• Nitrate Reductase for NO

production

Patch clamp

• Erwin Neher and Bert Sakmann in early 1980s.• Currents of single ion channels for the first time.• Nobel Prize in Physiology or Medicine in 1991 for this work.

• Electrode• Micropipette• Membrane ( patch)• Amplfier ( clamp)• Cell protoplast

phospholipase D-α 1(PLD)

PLD and its lipid product PA are involved in ABA signaling pathway that leads to stomatal closure(Gosti et al.,1999; Jacob et al.,1999 ; Schroeder et al., 2001;Sang et al., 2001a; Zhang et al., 2004; Mishra et al., 2006 ;Zhang et al., 2009).

Jacob et al., 1999; treated epidermal peels of vicia faba phosphatic acid( product of PLD) and butanol ( inhibitor of phosphatic acid production fron PLD).

The effects of PtdOH and buOH on stomatal aperture.

Jacob T et al. PNAS 1999;96:12192-12197

Abbreviations:

buOH> butanol

DAG>diacylglycerol

PtdOH>phosphatidic acid

LysoPtdOH>lysophosphatidic acid

• ABA signaling pathway which leads to stomatal closure recruites phosphatic acid.

Jacob T et al. PNAS 1999;96:12192-12197

• PLD produced PA binds to ABI1-PP2C and promotes ABA signal resulting in stoamatal closure.

• PLD and PA interact with Gα subunit of heterotrimeric G protein to mediate ABA inhibition of stomatal opening. Mishra et al., 2006

Bright et al., 2009

• H2O2 and NO are required for ABA induced stomatal closure.

PTIO:- 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxideSNP:- sodium nitroprusside

H2O2 and NO activate Ca2+ ion channel, MAPK .

NO also activates cGMP

• Calcium dependent protein kinases(CDPK), CPK3 and CPK4 function in ABA and cytosolic Ca2+ dependent activation of S-type anion channels.

E

F

Mori et al., 2006

ABA

START Proteins

PP2C

SnRk2

PLD

PA

[ K+ ] in

NADPH oxidase

ROS(H2O2)

TF

Ca2+ channel

[ Ca2+]cytOutward Anion channelsCPK3,4

NR

NO

Stomatal closure

Membrane depolarization

Efflux of pottasium ions

Efflux of water

Loss of guard cell volume

closure

pH

[ K+ ] out

cGMP

THANK YOU!!

ROS mediated aba signaling• Hydrogen peroxide (H2O2) is one of the major reactive oxygen species

(ROS) and plays an important role as a second messenger in ABA-induced stomatal closure (Pei et al., 2000; Murata et al., 2001; Zhang et al., 2001; Kwak et al., 2003; Bright et al., 2006; Miao et al., 2006).

• Treatment with exogenous catalase (CAT) reduced H2O2 accumulation and inhibited ABA-induced stomatal closure (Zhang et al., 2001; Munemasa et al., 2007).

• Direct application of ROS to guard cells is known to inhibit inward K+

channels (Pei et al., 2000; Murata et al., 2001; Zhang et al., 2001).• Pei et al. (2000) demonstrated that treatment of Arabidopsis guard cells

with ABA induced a rapid burst of H2O2 production that resulted in stomatal closure. These findings have subsequently been reported in guard cells of Vicia faba (Zhang et al., 2001c) and pea also.

• NO is also an essential signal mediating ABA-induced stomatal closure (Neill et al., 2002a).

• Exogenous NO (applied as NO donors) induced stomatal closure and reduced transpiration in Vicia faba, Salpichroa organifolia and Tradescantia sp. (Garcia-Mata and Lamattina, 2001).

• Application of PTIO (or cPTIO), a NO scavenger, inhibited ABA-induced stomatal closure, indicating the involvement of endogenous NO (Desikan et al., 2002; Garcia-Mata and Lamattina, 2002; Neill et al., 2002°.

• Burnett et al., 2000 , reported that MAPK(s) mediate both ABA and H2O2 induced stomatal closure.

• H2O2 induces the activation of a MAPK in cell cultures, leaves and protoplasts of Arabidopsis (Neill et al., 2002b).

• Interestingly, treatment with the NO donor SNP also induces the transient activation of a similar kinase.

• Two MAPK genes, MPK9 and MPK12 are preferentially and highly expressed in guard cells.The two genes are functionally redundant as mutation in any one of them does not produce an altered phenotype. If the both MPK9 and MPK12 transcripts are silenced the ABA induced stomatal closure is impaired. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. Abscisic Acid and H2O2 treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.

Jammes et al. ,2009