SATYA PRAKASHMSC( BOTANY) , SEM IV(2013)Abscisic acid signaling in guardcell movementJung et al.,2002Presentation by : Sat...
INTRODUCTION• It is important to study ABA signaling. Why? It is a key endogenous messenger in plants’ responses to bioti...
Intro…continued…. Stomatal closure requires release of solutes from guard cellswhere K+ seems to be the most important as...
ABA Receptors in gurad cells Previous studies have identified several types of putativereceptors that may mediate ABA fun...
Melcher et al., 2010PYL2Binding of ABA with START family proteins shows interesting Gate-Latch-LockMechanism!
Type 2C protein phosphatases(PP2C) Genetic studies have identified several PP2C genes that are required for ABAsignaling ...
Physical Interactions among PP2CA and RCARs. Yeast two-hybrid assay of interactionsbetween PP2CA and RCARs. Growth on the ...
OST1(Snrk2) activity Is required forproper stomatal closure Geiger et al., 2009 , compared stoamta of WT Arabidopsiswith ...
Source:- Takashi Hirayama and Taishi Umezawa., 2010Some targets of SnRK2 kinases:-• Transcription factors• Guard cell anio...
Patch clamp• Erwin Neher and Bert Sakmann in early 1980s.• Currents of single ion channels for the first time.• Nobel Priz...
phospholipase D-α 1(PLD) PLD and its lipid product PA are involved in ABA signaling pathway thatleads to stomatal closure...
The effects of PtdOH and buOH on stomatal aperture.Jacob T et al. PNAS 1999;96:12192-12197Abbreviations:buOH> butanolDAG>d...
Jacob T et al. PNAS 1999;96:12192-12197
• PLD produced PA binds to ABI1-PP2C and promotes ABA signal resulting in stoamatalclosure.• PLD and PA interact with Gα s...
Bright et al., 2009• H2O2 and NO are required for ABA induced stomatal closure.PTIO:- 2-(4-carboxyphenyl)-4,4,5,5 tetramet...
• Calcium dependent protein kinases(CDPK), CPK3 and CPK4 function in ABA andcytosolic Ca2+ dependent activation of S-type ...
ABASTART ProteinsPP2CSnRk2PLDPA[K+ ] inNADPH oxidaseROS(H2O2)TFCa2+ channel[Ca2+]cytOutward Anion channelsCPK3,4NRNOStomat...
THANK YOU!!
ROS mediated aba signaling• Hydrogen peroxide (H2O2) is one of the major reactive oxygen species(ROS) and plays an importa...
• NO is also an essential signal mediating ABA-induced stomatal closure (Neill etal., 2002a).• Exogenous NO (applied as NO...
• Two MAPK genes, MPK9 and MPK12 are preferentially andhighly expressed in guard cells.The two genes arefunctionally redun...
 ( Dissertation) Abscisic acid signaling in guard cell movement.
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( Dissertation) Abscisic acid signaling in guard cell movement.

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Now on the way to understand the aba signaling , the initial task is to identify and uderstand the proteins that receive the aba signal and initiate signaling cascade. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure.

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  • Reported in many papers refrences here are a few only.in responcce to the wter deficiency ABA production is increased in guard cells(apoplat). This elevted amount of aba triggers a squence of events that results in closure of stomata. Thus pereventing the water loss.This plant harmone is a major player in adapting the plant to various stresses.
  • Plants close their stomata to conserve water in response to a water-deficit condition. Plant hormone abscisic acid (ABA) plays a key role in the adaptation to water-deficit conditions through regulatory pathways that control gene expression and stomatal closure (Luan, 2002; Zhu, 2002;Wasilewska et al., 2008). The levels of ABA in plant tissues increase under drought conditions, and ABA down-regulates guard cell turgor pressure and thus triggers stomatal closure by modifying activities of a number of ion channels (Schroeder et al., 2001). The ion channels targeted by ABA signaling include K+ channels and anion channels, which control ionic fluxes across the plasma membrane and tonoplast, thereby adjusting guard cell turgor pressure (Schroeder et al., 1987; Schroeder and Hagiwara, 1989; Lemtiri-Chlieh and MacRobbie, 1994;Negi et al., 2008; Vahisalu et al., 2008). The process starts with an increasing level of ABA activating anion efflux through anion channels thus inducing depolarization of guard cell plasma membrane. Depolarization drives K+ efflux through outward-rectifying K+ channels leading to water efflux, which reduces guard cell volume and leads to stomatal closure (Ache et al., 2000; Li et al., 2000; Ward et al., 2008).Aba signaling pathway is a very complex pathway including many signaling molecules , redundant componenets etc.
  • Now on the way to understand the aba signaling , the initial task is to identify and uderstand the proteins that receive the aba signal and initiate signaling cascade. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure..These receptors transmit the ABA signal through PP2C. (PYrabactin Resistance/PYrabactin Resistance-Like/RegulatoryComponent of Abscisic acid Receptor) proteinsthe ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. 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).Park et al.,2009 , identified ABA receptors using ABA agonist, pyrabactin
  • They identified a conserved gate-latch-lock mechanism underluing ABA signaling.
  • identified by genetic screen. Mutant pp2c(abi) can not bind aba recepters.abi1-1 mutant PP2C constitutively inactivated SRK2E even in the presence of ABA. Probably because aba cud not bind it .ABI1, ABI2, HAB1, HAB2, AGH1, and PP2CA, generally function as negative regulators of the ABA response
  • Open stomata 1 is an snrk2 type kinase. Reduction in transpiration of leaves after transition from light to dark is delayed in mutant. So stomata during transition close much slower in mutant. (perfomed using gas exchange method).Showing wilting of eexcised ost1 leaves in light. Same leaves are shown at indicated time points.Comment:- distance between leaf base and hieght have been plotted against the time since leaves were excised. Leaves of mutnat plant undergone wilting very early due to rapid loss of water.Comparison of time course of wilting of mutant and wild type. Position of leaf tip relative to leaf base was plotted against time. they showed that slac1 is regulated by abi1/osti pair i.e drought signaling phosphatase/ kinase pair.SLAc is regulated via reversible protein phosphorylation including aba signaling omponents.Downstream to receptors pp2c and snrk2 are the ion channels that control the stomatal movement.
  • So far I have discuused one of the major ABA signaling pathway and this is the schematic representation of the same. Under normal condition PP2c constantly interacts with aBa-activated SnrK2 and inactivates its kinase activity by dephosphorylation. When the soluble aBa receptor recognizes the aBa molecule, it binds PP2c and inhibits its phosphatase activity. then SnrK2 is activated and phosphorylates its targets, and the aBa signal is consequently turned on.HAB1, HAB2, AHG1 and AHG3/AtPP2CA. Arabidop. Has 14 different members of ABA receptors, 9 pp2c( redundent components)..receptors seem to have different selectivity to isomers of ABA ..Interections verified by yeast two hybrid experiments.
  •  The micropipette is pressed against a cell membrane and suction is applied to assist in the formation of a high resistance seal between the glass and the cell membrane (a "gigaohm seal“ A chlorided silver wire is placed in contact with this solution and conducts electric current to the amplifier. A patch clamp recording reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom).
  • Based on these and some previous data available they concluded…. Bnds to galpha subunit of heterotrimeric g protein and convets it into inactive GDP form thus promoting
  • I have put h2o2 and no saparatly but recent studies on spatial and temporal production of these milecules claim close interrelationship b/w them, where ,perhaps , the no synthesis is somehow regulated by h2o2.
  • All these things they support hat H2o2 , it has role in aba induccedstomatall closure. And h2o2producton seems to be essential for aba induced stomatalclosur. There are many physiological process where h2o2 and no are required.stomatal closure in response to ABA also appears to require the tandem synthesis and action of both these signalling molecules.NO donor SNP
  • Findings that we just discuused were soon followed by the discovery that NO is also an essentail signal mediating aba induced stomatal closure. Exogenous application of no induced stomatal closure vicia and some other sp and this process was time ,dose dependent and reversible .
  • First anion channels are activated by aba resultibg in loss of anions through channels it results itodepolarisation of plasma membrane that drives k+ ions out of the guard cell through outward rectifying k+ channels. Leading to water effulx followed by reduction in torgour and stomatal closure .
  • ( Dissertation) Abscisic acid signaling in guard cell movement.

    1. 1. SATYA PRAKASHMSC( BOTANY) , SEM IV(2013)Abscisic acid signaling in guardcell movementJung et al.,2002Presentation by : Satya Prakash ChaurasiaDepartment of Botany,University of Delhi,satya.prakash101991@gmail.comwww.facebook.com/91satyawww.twitter.com/Satyapr101991h ttp://www.linkedin.com/pub/satya-prakash chaurasia/65/a76/a25Mob: +919654814497
    2. 2. INTRODUCTION• It is important to study ABA signaling. Why? It is a key endogenous messenger in plants’ responses to biotic andabiotic 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, germinationembryo development and many other developmental processes.
    3. 3. Intro…continued…. Stomatal closure requires release of solutes from guard cellswhere K+ seems to be the most important as more than 85%of it is released during stomatal closure( Talbott andZeiger., 1998). How ABA acts to induce the loss of solutes in guard cells??
    4. 4. ABA Receptors in gurad cells Previous studies have identified several types of putativereceptors that may mediate ABA functions (McCourt andCreelman, 2008; Cutler et al., 2010). Most recently, a family of START domain proteins, known asPYR/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).
    5. 5. Melcher et al., 2010PYL2Binding of ABA with START family proteins shows interesting Gate-Latch-LockMechanism!
    6. 6. Type 2C protein phosphatases(PP2C) Genetic studies have identified several PP2C genes that are required for ABAsignaling in Arabidopsis. Of these members, a group A PP2C, includingABI1, ABI2, HAB1, HAB2, AGH1, and PP2CA, generally function as negativeregulators of the ABA response (Merlot et al., 2001; Nishimura et al., 2004; Saez etal., 2004; Kuhn et al., 2006; Rubio et al., 2009). Studies have shown that PYL/RCAR proteins interact with PP2C -type familymembers and inhibit their activities (Ma et al., 2009; Park et al., 2009; Lee atal.,2010). Lee et al., 2010, demonstrated the interaction between the two by Yeast twohybrid system.
    7. 7. Physical Interactions among PP2CA and RCARs. Yeast two-hybrid assay of interactionsbetween PP2CA and RCARs. Growth on the selection medium (SC-ALTH) was used as anindicator 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 associationwith Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.10µM ABA• ABA receptor of START family physically interacts with negative regulator ofABA induced stoamatal closure, protein phosphatase 2C.
    8. 8. OST1(Snrk2) activity Is required forproper stomatal closure Geiger et al., 2009 , compared stoamta of WT Arabidopsiswith the stomata of ost1-2 mutant Arabidopsis plant andshowed that ost1–2 stomata during day-night transition closemuch slower than those of WT plants. They also proposed that ost1 promotes guard cell closure byactivating outward anion channel SLAC1.
    9. 9. Source:- Takashi Hirayama and Taishi Umezawa., 2010Some targets of SnRK2 kinases:-• Transcription factors• Guard cell anion channels• Inward K+ ion channels• NADPH oxidase for ROS production• Nitrate Reductase for NOproduction
    10. 10. 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
    11. 11. phospholipase D-α 1(PLD) PLD and its lipid product PA are involved in ABA signaling pathway thatleads to stomatal closure(Gosti et al.,1999; Jacob et al.,1999 ; Schroeder etal., 2001;Sang et al., 2001a; Zhang et al., 2004; Mishra et al., 2006 ;Zhanget al., 2009). Jacob et al., 1999; treated epidermal peels of vicia faba phosphatic acid(product of PLD) and butanol ( inhibitor of phosphatic acid production fronPLD).
    12. 12. The effects of PtdOH and buOH on stomatal aperture.Jacob T et al. PNAS 1999;96:12192-12197Abbreviations:buOH> butanolDAG>diacylglycerolPtdOH>phosphatidicacidLysoPtdOH>lysophosphatidic acid• ABA signaling pathway which leads to stomatal closure recruites phosphatic acid.
    13. 13. Jacob T et al. PNAS 1999;96:12192-12197
    14. 14. • PLD produced PA binds to ABI1-PP2C and promotes ABA signal resulting in stoamatalclosure.• PLD and PA interact with Gα subunit of heterotrimeric G protein to mediate ABA inhibition ofstomatal opening.Mishra et al., 2006
    15. 15. 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 nitroprussideH2O2 and NO activateCa2+ ionchannel, MAPK .NO also activatescGMP
    16. 16. • Calcium dependent protein kinases(CDPK), CPK3 and CPK4 function in ABA andcytosolic Ca2+ dependent activation of S-type anion channels.EFMori et al., 2006
    17. 17. ABASTART ProteinsPP2CSnRk2PLDPA[K+ ] inNADPH oxidaseROS(H2O2)TFCa2+ channel[Ca2+]cytOutward Anion channelsCPK3,4NRNOStomatal closureMembrane depolarizationEfflux of pottasium ionsEfflux of waterLoss of guard cell volumeclosurepH[K+ ] outcGMP
    18. 18. THANK YOU!!
    19. 19. 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-inducedstomatal 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 andinhibited ABA-induced stomatal closure (Zhang et al., 2001; Munemasa etal., 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 cellswith ABA induced a rapid burst of H2O2 production that resulted instomatal closure. These findings have subsequently been reported inguard cells of Vicia faba (Zhang et al., 2001c) and pea also.
    20. 20. • NO is also an essential signal mediating ABA-induced stomatal closure (Neill etal., 2002a).• Exogenous NO (applied as NO donors) induced stomatal closure and reducedtranspiration in Vicia faba, Salpichroa organifolia and Tradescantia sp. (Garcia-Mata and Lamattina, 2001).• Application of PTIO (or cPTIO), a NO scavenger, inhibited ABA-induced stomatalclosure, 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 inducedstomatal closure.• H2O2 induces the activation of a MAPK in cell cultures, leaves and protoplasts ofArabidopsis (Neill et al., 2002b).• Interestingly, treatment with the NO donor SNP also induces the transientactivation of a similar kinase.
    21. 21. • Two MAPK genes, MPK9 and MPK12 are preferentially andhighly expressed in guard cells.The two genes arefunctionally redundant as mutation in any one of themdoes not produce an altered phenotype. If the both MPK9and MPK12 transcripts are silenced the ABA inducedstomatal closure is impaired. Furthermore, ABA andcalcium failed to activate anion channels in guard cells ofmpk9-1/12-1, indicating that these 2 MPKs act upstream ofanion channels in guard cell ABA signaling. Abscisic Acidand H2O2 treatments enhance the protein kinase activityof MPK12. Together, these results provide genetic evidencethat MPK9 and MPK12 function downstream of ROS toregulate guard cell ABA signaling positively.Jammes et al. ,2009

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