Abscisic Acid (ABA) PDF

Abscisic Acid (ABA) The Plant Cell: Teaching Tools ABA is one of the most important hormones that governs plant development in response to the environment Most of its effects are associated with water relations Main roles of ABA during plant development and adaptation: Se...

Abscisic Acid (ABA) The Plant Cell: Teaching Tools ABA is one of the most important hormones that governs plant development in response to the environment Most of its effects are associated with water relations Main roles of ABA during plant development and adaptation: Seed development and dormancy Root growth Guard cell responses (stomatal closure) Vegetative dehydration responses Senescence Abiotic stress responses (especially drought responses) Biotic stress responses  ABA controls seed maturation, dormancy and desiccation Seed dormancy and desiccation tolerance is correlated with ABA synthesis and accumulation. ABA Embryo devp Embryo growth and Desiccation and Dormancy and patterning reserve accumulation developmental arrest Seed maturation and desiccation protect the seed when released in dry environment while dormancy prevents its germination until favourable conditions Germination involves catabolism of ABA and synthesis of GA GA ABA Mutants in ABA biosynthesis show reduced dormancy and vivipary (ability to germinate on the plant) Seeds of mutants of ABA biosynthesis or signalling are not desiccation tolerant and germinate prematurely Zea mays vp1 mutant Arabidopsis snrk2.2/2.3/2.6Triple triple mutant SnRK2 mutant (snrk2.2, 2,3, 2.6) MaCarty et al., (1989) Plant Cell, 1:523-532 Fujii, H., and Zhu, J.-K. (2009) Proc. Natl. Acad. Sci. USA 106: 8380-8385.  ABA promotes root elongation and suppresses lateral root branching Well watered conditions Drought conditions Lateral root branching allows Lateral root branching inhibited water uptake from the top soil Primary root elongates and goes deep in search of water  ABA promotes root elongation and suppresses lateral root branching Well watered Drought (surface water present) (No surface water) Well-watered conditions Drought conditions Lateral root branching allows Lateral root branching inhibited water uptake from the top soil Primary root elongates and goes Plants invest in LR growth deep in search of water ABA promotes root elongation and suppresses lateral root branching Drought stress suppresses lateral root growth while Lateral roots primary root resume growth elongation is upon rewatering maintained Water stress and ABA promote root growth at the expense of shoot growth Increasing water stress ABA promotes stomatal guard cell closure in response to water stress - Stomatal pores allow exchange of gases, particularly CO2 as well as water vapour. - During photosynthesis, CO2 is taken up while water is lost through transpiration. - When the environment becomes dry and the vapour pressure deficit increases, the stomata close to prevent excess loss of water. (photosynthesis cannot be continued at the cost of drying) (VPD = vapour pressure difference between the leaf and atmosphere) - This closure is controlled by ABA  Guard cells are the portals through which CO2 enters and H2O exits CO2 C  OPEN CLOSING ABA Guard cells responding to ABA Marin, E., Nussaume, L., Quesada, A., Gonneau, M., Sotta, B., Hugueney, P., Frey, A., and Marion-Poll, A. (1996). Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.EMBO J 15: 2331 – 2342. ABA-induced stomatal closure is extremely rapid and involves changes in ion channel activities ABA triggers an increase in cytosolic calcium (Ca2+), which activates anion channels (A-) allowing Cl- to leave the cell. ABA activates channels that move potassium Cl- out of the cell (K+out) and inhibits channels that move potassium into the cell (K+in). A- channel The net result is a large movement of ions out of the cell. As ions leave the cell, so does water (by K+in channel osmosis), causing the cells to lose volume and close over the pore. K+ H2O Adapted from Kwak JM, Mäser P, Schroeder JI (2008) The clickable guard cell, version II: Interactive model of guard cell signal transduction mechanisms and pathways. The Arabidopsis Book, ASPB. doi: 10.1199/tab.0114. ABA mediates responses to abiotic stresses such as 1. Drought 2. Salinity Wild-type tobacco aba2 mutant 3. Cold Marin, E., Nussaume, L., Quesada, A., Gonneau, M., Sotta, B., Hugueney, P., Frey, A., and Marion-Poll, A. (1996). Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.EMBO J 15: 2331 – 2342. How are ABA-related responses regulated during each of these processes? 1. Control over levels of ABA A. Biosynthesis B. Degradation C. Conjugation 2. Intercellular Transportation (movement across cells) 3. Signaling A. ABA receptors (PYR/PYL/RCARs) B. Protein phosphatases (PP2C) C. Protein kinases (Ca++ dependent and independent) (SnRKs/CDPKs/CPK) 4. Transcription factors  How are components of ABA related responses (biosynthesis, signaling and response) identified?  Zea mays vp1 mutant Mutant screens based on ABA related effects: 1. Level of dormancy (or appearance of vivipary) eg. vp1, vp14 in maize insensitive insensitive Wildtype wild type 2. Sensitivity to ABA in germination and root hypersensitive hypersensitive length eg. aba2, abi1, abi3, 4, 5, ahg1, etc ABA 3. Stomatal effects (ability or inability to withstand drought stress) Mutant Mutant wild type Control 4. Thermal imaging (based on leaf temperature which increases in plants with closed stomata Wild type and decreases in plants with open stomata) ost1-1 eg. ost (lower leaf temp due to open stomata) ost1-2 1. Control over levels of ABA A. Biosynthesis B. Degradation C. Conjugation ABA is synthesized in the plastid and cytoplasm and is derived from zeaxanthin, a plant pigment (a component of the carotenoid pathway) Zeaxanthin (40 C compound) ABA Zeaxanthin is abundant in green tissues but can be limiting for ABA synthesis in roots ABA2  Zeaxanthin epoxidase (ZEP) converts zeaxanthin to violaxanthin Zeaxanthin Antheraxanthin All trans-Violaxanthin Schwartz, S.H., Qin, X., and Zeevaart, J.A.D. (2003). Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol. 131: 1591-1601.  ZEP mutants are ABA deficient and lose water rapidly Wild-type tobacco mutant Closed stomata Open stomata ZEP levels are limiting in non-photosynthetic tissues but not in leaves ZEP is also under circadian control decreasing at onset of darkness Marin, E., Nussaume, L., Quesada, A., Gonneau, M., Sotta, B., Hugueney, P., Frey, A., and Marion-Poll, A. (1996). Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana.EMBO J 15: 2331 – 2342. All-trans-violaxanthin rearranges to form 9-cis-epoxycarotenoids 9-cis-epoxycarotenoids Han, S.-Y., Kitahata, N., Sekimata, K., Saito, T., Kobayashi, M., Nakashima, K., Yamaguchi-Shinozaki, K., Shinozaki, K., Yoshida, S., and Asami, T. (2004). A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants. Plant Physiol. 135: 1574-1582. NCED cleaves 9-cis-epoxycarotenoids (40 carbon) to produce xanthoxin (15 carbon) NCED, 9-cis-epoxycarotenoid dioxygenase, is a major regulatory step in ABA biosynthesis xanthoxin Xanthoxin is transported to the cytosol NCED belongs to the group of enzymes known as carotenoid cleavage dioxygenases - identified from several plants The first gene to be identified was VP14 in the maize vp14 (viviparous) mutant based on precocious (early) germination and rapid loss of water In tomato, the notabilis mutant is affected in the LeNCED1 gene and shows a wilty phenotype (plants wilt very fast) In Arabidopsis, mutations in AtNCED3 (like vp14) also primarily affect seed germination NCEDs are part of a large family, but only some are involved in ABA synthesis Cluster of NCED genes thought to be involved in ABA synthesis. Some are primarily expressed in seeds, and others in vegetative tissues. NCED genes are induced during seed maturation and by drought stress Seed maturation - increase in NCED expression levels and then decrease Leaf detachment - Increased mRNA levels within 30 minutes Increased NCED levels = increase in ABA Expression of NCED in transgenic plants leads to an increased synthesis of ABA This indicates that NCED is a major rate limiting step in the biosynthesis of ABA Substrate 5000 1 B 1000 2 Rate limiting C 100 3 D 300 4 E 200 5 Product 170 Conversion of xanthoxin to ABA requires two enzymes and occurs in the cytosol Short chain alcohol dehydrogenase/reductase (SDR) Abscisic aldehyde oxidase  Conversion of xanthoxin to ABA requires two enzymes and occurs in the cytosol WTWT aba2 Short chain alcohol dehydrogenase/reductase (SDR) aba2 aba2 aba2 mutants germinate mutants germinate even under under high salt inappropriate aao3 mutants conditions conditions are wilty WT aao3 Abscisic aldehyde oxidase Gonzalez-Guzman, M., et al. (2002). The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde. Plant Cell 14: 1833-1846. Schwartz, S.H., Qin, X., and Zeevaart, J.A.D. (2003). Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol. 131: 1591-1601. Seo, M., et al. (2000). The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves. Proc. Nalt. Acad. Sci. USA 97: 12908-12913. Sterio-specificty of ABA adds another level of regulation ABA exists as cis-trans isomer and all trans isomer The cis-trans isomer is the biologically active form of ABA while the all trans has much reduced activity Cis-trans isomer is converted to all trans isomer by UV light The equilibrium between both isomers may decide the effects Different receptors may have different specificities for the two isoforms Please be careful when preparing plates with ABA (for germination studies) (Do not keep in laminar where others may put on the UV light and convert the ABA to the all trans form in the plate) ABA levels are also controlled by inactivation pathways Rehydration Developmental signals [ABA] Leaf dehydration causes ABA accumulation while rehydration causes ABA levels to drop ABA ABA levels accumulates decrease Leaf Rehydration by detachment immersion in Rehydration leads to drying water leads to and ABA ng/g fresh wt decrease in ABA accumulation levels ABA What happens to ABA? Krochko, J.E., Abrams, G.D., Loewen, M.K., Abrams, S.R., and Cutler, A.J. (1998). (+)-Abscisic Acid 8'-Hydroxylase Is a Cytochrome P450 Monooxygenase. Plant Physiol. 118: 849-860. Zeevaart, J.A.D. (1980). Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol. 66: 672-678. ABA is irreversibly deactivated by conversion to phaseic acid ABA ABA accumulates converted to phaseic acid Leaf detachment Rehydration by Phaseic leads to drying immersion in Acid and ABA water leads to accumulation decrease in ABA Rehydration levels ABA Krochko, J.E., Abrams, G.D., Loewen, M.K., Abrams, S.R., and Cutler, A.J. (1998). (+)-Abscisic Acid 8'-Hydroxylase Is a Cytochrome P450 Monooxygenase. Plant Physiol. 118: 849-860. Zeevaart, J.A.D. (1980). Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol. 66: 672-678. Deactivation of ABA is brought about by ABA-8′-hydroxylases (CytP450 enzymes) encoded by CYP707A (encoded by four genes, A1-A4) Low humidity High humidity (less need for ABA) CYP707A genes are up-regulated upon transfer to high humidity Okamoto, M., Tanaka, Y., Abrams, S.R., Kamiya, Y., Seki, M., and Nambara, E. (2009). High humidity induces abscisic acid 8'-hydroxylase in stomata and vasculature to regulate local and systemic abscisic acid responses in Arabidopsis. Plant Physiol. 149: 825-834. ABA inactivation by 8′-hydroxylase is necessary for seed germination Loss-of-function of one CYP707A gene copy Wild type reduces germination, and loss-of two copies nearly abolishes it. Single mutants Double mutant Okamoto, M., Kuwahara, A., Seo, M., Kushiro, T., Asami, T., Hirai, N., Kamiya, Y., Koshiba, T., and Nambara, E. (2006). CYP707A1 and CYP707A2, which encode abscisic acid 8'-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis. Plant Physiol. 141: 97-107. ABA can also be reversibly inactivated by glucosylation β-glucosidase ABA glucosyltransferase CO2-Glu (+)-S-ABA glucosylester β-glucosidase ABA glucosyltransferase Reprinted by permission from Macmillan Publishers Ltd: Kushiro, T., Okamoto, M., Nakabayashi, K., Yamagishi, K., Kitamura, S., Asami, T., Hirai, N., Koshiba, T., Kamiya, Y., and Nambara, E. (2004). The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA catabolism. EMBO J 23: 1647-1656 copyright 2004. ABA- glucosyl ester (ABA-GE) is an inactive storage and transfer form AtBG1 CO2-Glu (+)-S-ABA glucosylester β-glucosidase ABA glucosyl Reprinted from Schroeder, J.I., and Nambara, E. (2006). A quick release mechanism for abscisic acid. Cell 126: 1023-1025 with permission from Elsevier. Beta glucosidase mutants (atbg1) that cannot recover ABA from ABA-GE are ABA deficient (and show greater water loss) atbg1 mutant WT Mutant complemented X with AtBG1 CO2-Glu (+)-S-ABA glucosylester β-glucosidase ABA glucosyl Reprinted from Lee, K.H., Piao, H.L., Kim, H.-Y., Choi, S.M., Jiang, F., Hartung, W., Hwang, I., Kwak, J.M., Lee, I.-J., and Hwang, I. (2006). Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell 126: 1109-1120  ABA accumulation and homeostasis are tightly controlled NCED 9-cis- xanthoxin Rehydration expoxycarotenoids Developmental signals Water stress [ABA] Developmental signals ABA movement – between organs and cells  Two transporters, AtABCG25 (an efflux transporter) and AtABCG40 (an influx transporter) help in movement of ABA across membranes between vascular tissues and guard cells 10 μM ABA ABA- AtABCG25 is expressed in veins and encodes an ABA exporter AtABCG25pro::GUS expression in veins (vascular tissues) Kuromori, T., Miyaji, T., Yabuuchi, H., Shimizu, H., Sugimoto, E., Kamiya, A., Moriyama, Y., and Shinozaki, K. (2010) ABC transporter AtABCG25 is involved in abscisic acid transport and responses. Proc. Natl. Acad. Sci. USA 107: 2361-2366.  Two transporters, AtABCG25 (an efflux transporter) and AtABCG40 (an influx transporter) help in movement of ABA across membranes between vascular tissues and guard cells 10 μM ABA ABA- AtABCG25 is expressed in veins and encodes an ABA exporter AtABCG25pro::GUS expression in veins (vascular tissues) ABA- AtABCG40 is expressed in guard cells and encodes an ABA importer AtABCG25 expressing plants show reduced water loss in detached leaves Guard cells in loss-of-function abcg40 mutants are less sensitive to ABA and the mutants are more susceptible to drought stress. Kuromori, T., Miyaji, T., Yabuuchi, H., Shimizu, H., Sugimoto, E., Kamiya, A., Moriyama, Y., and Shinozaki, K. (2010) ABC transporter AtABCG25 is involved in abscisic acid transport and responses. Proc. Natl. Acad. Sci. USA 107: 2361-2366. Kang, J., Hwang, J.-U., Lee, M., Kim, Y.-Y., Assmann, S.M., Martinoia, E., and Lee, Y. (2010) PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid. Proc. Natl. Acad. Sci. USA 107: 2355-2360. Transport of ABA mediated by AtABCG25 and AtABCG40 Epidermis Palisade Tissue Vascular tissue Mesophyll cells Guard cells Once ABA becomes available to the cell how is it sensed? What are the components of the signal cascade that mediate its effects? Protein phosphorylation/dephosphorylation form an important component of ABA signal transduction PP2Cs (Kinases) SnRKs -P Ion -P channels TFs -P ABA responses Protein phosphatase PP2Cs – primary regulators of ABA signaling Function: Dephosphorylation of Ser/Thr phosphates from phosphorylated protein kinases Loss of function mutants show hypersensitivity to ABA (increased ABA responses leading to strong inhibition of germination) In what way do PP2Cs mediate ABA signal? Are they positive or negative regulators? Global negative regulators of ABA signaling Arabidopsis has 76 PP2Cs. Only clade A (with 9 PP2Cs) participates in ABA signalling AHG1 -AHG1 AHG3 At5g59220 HAI1 At1g07430 HAI2 At2g29380 HAI3 HAB2 HAB1 ABI1 ABI2 All the clade A proteins (white) have a confirmed role in ABA signalling. AHG1, AHG3 (ABA HYPERSENSITIVE GERMINATION 1 and 3) ABI1, ABI2 (ABA INSENSITIVE1, ABA INSENSITIVE 2) HAB1, HAB2 (HOMOLOGY TO ABI1, HOMOLOGY TO ABI2) HAI1, HAI2, HAI3 – (HIGHLY ABA INDUCED1-3, in black) show reduced ABA effects as single mutants but show germination effects as triple mutants. PP2Cs are functionally redundant at the molecular level (ie they can complement each other). Specificity occurs due to distinct expression patterns i.e. AHG1 and 3 are expressed predominantly in seeds ABI1 is expressed in many tissues ABI1 localized to nucleus and cytoplasm, AHG1 and 3 only in nucleus Function conserved amongst different plants What are the targets of PP2Cs?  PP2Cs interfere with the action of protein kinases specifically SnRK2 (SNF1 related protein kinase2) type NO ABA In the absence of ABA, PP2Cs dephosphorylate SnRK2 protein kinases and inhibit their activity. ABI1 SnRK2 ABA inactivates PP2Cs to activate SnRK2 kinases P NO ABA RESPONSES SnRK2s are Ca++ independent protein kinases that promote ABA responses by phosphorylation of targets P SnRK2 P P TF Ion P channel ABA RESPONSES They phosphorylate transcription factors as well as ion channel proteins (anion channels, potassium channels) and NADPH oxidase (involved in ROS signaling) SnRK2s are part of a large protein kinase super family of Ca++ dependent and independent PKs The CDPK-SnRK superfamily of protein kinases Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker- Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. SnRK2s are part of a large protein kinase super family of Ca++ dependent and independent PKs The CDPK-SnRK superfamily of protein kinases Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker- Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. SnRK2s are part of a large protein kinase super family of Ca++ dependent and independent PKs SnRK3 (CIPKs) The CDPK-SnRK superfamily of protein kinases SnRK1 SnRK2 The SnRK2 subfamily (10 members -3 classes) Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker- Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. All 10 members of SnRK2 show distinct activation patterns by ABA and osmotic stress Subclass I : strongly activated by osmotic stress within 1 min but not by ABA Subclass II: activated by osmotic stress and weakly by ABA Subclass III: activated by osmotic stress and strongly by ABA (SnRK2.2, SnRK2.3, SnRK2.6) SnRK2.6 /ost1 functions mainly in guard cells; SnRK2.2 and 2.3 function in tissues other than guard cells such as seeds and vegetative tissues  OST1 (OPEN STOMATA 1/SNRK2.6) is expressed in guard cells and vascular tissues The ost1 mutant (identified by thermal imaging) shows reduced stomatal closure (open stomata), reduced leaf temperature and greater water loss ost1-1 Wild type ost1-2 Wild ost1-1 type ost1-2 Two other homologues of SnRK2.6 identified: SnRK2.2 and SnRK2.3 The snrk2.2 snrk2.3 double mutants lack a few ABA responses But the snrk2.2snrk2.3snrk2.6 triple mutant lacks almost all ABA responses Mustilli et al., (2002) Arabidopsis OST1 Protein Kinase Mediates the Regulation of Stomatal Aperture by Abscisic Acid and Acts Upstream of Reactive Oxygen Species Production Plant Cell 14:3089-3099 The snrk2.2/2.3/2.6 triple mutants show nearly complete elimination of ABA responses Triple mutant plants are nearly completely insensitive to ABA, show vivipary and can germinate on even 10 mM ABA! This indicates that Class III SnRKs govern almost all ABA responses through their targets (form a central hub for ABA 10 mM ABA responses) Fujii, H., and Zhu, J.-K. (2009). Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc. Natl. Acad. Sci. USA 106: 8380-8385. Fujita et al., (2009) Three SnRK2 Protein Kinases are the Main Positive Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis. Plant Cell Physiology 50:2123-32 Other members of the large protein kinase super family are also involved in ABA signaling SnRK3 (CIPKs) The CDPK-SnRK superfamily of protein kinases SnRK1 SnRK2 Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker- Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. Some SnRK3/CIPKs (CBL Interacting Protein Kinases) act as Ca++ dependent negative regulators of ABA signaling (CBL = Calcineurin B like – a Calcium sensor protein) CIPK3 negatively regulates ABA signaling and cold and salt-induced gene expression in seed germination but not stomatal responses CIPK15 and its interacting protein CBL9 negatively regulate ABA signaling (mutants are hypersensitive to ABA). Affects seed germination and stomatal responses AtERF7 (a repressor type of ERF) interacts with CIPK15 and represses ABA responses CIPK23 negatively regulates ABA signaling in stomata but not seed germination  Calcium-dependent protein kinases (CDPKs/CPKs) participate in ABA signaling especially in abiotic stress responses There are 34 CDPKs in Arabidopsis. A few have been confirmed to transduce the ABA signal. Hrabak, E.M., Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker- Simmons, K., Zhu, J.-K., and Harmon, A.C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680.  Drought tolerance is correlated with activity of some CDPKs ABA Control Control Drought (Mild) CDPK Drought (severe) ABA RESPONSES WT cpk4 CPK4-over- cpk11 WT expression Less CPK activity, Less More CPK activity, More Drought tolerance drought tolerance drought tolerance Zhu, S.-Y., Yu, X.-C., Wang, X.-J., Zhao, R., Li, Y., Fan, R.-C., Shang, Y., Du, S.-Y., Wang, X.-F., Wu, F.-Q., Xu, Y.-H., Zhang, X.-Y., and Zhang, D.-P. (2007). Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell 19: 3019-3036.  Transcription factors are major targets of CDPKs and SnRK2s and regulate ABA signaling ABA SnRK2 P SnRK2 CDPK P TF ABA RESPONSES Target TFs mediate ABA responses in different tissues (seed, guard cell, root) or in response to development or stresses (seed desiccation, stomatal control, drought tolerance, salinity)  A group of bZIP type TFs (basic region–Leucine Zipper type) governs several ABA responses Leucine zipper = every seventh amino acid in that domain is a leucine residue Basic Region (DNA Leucine Zipper binding) (Dimerization) A bZIP protein binding to DNA ABA-responsive element (ABRE) ABRE - PyACGTGG/TC - identified as a conserved sequence in the promoters of many seed-specific and ABA-responsive genes ABRE-binding proteins are called ABFs (ABRE Binding Factors) or AREBs (ABA Response Element Binding proteins). Reprinted from Jakoby, M., Weisshaar, B., Dröge-Laser, W., Vicente-Carbajosa, J., Tiedemann, J., Kroj, T., and Parcy, F. (2002). bZIP transcription factors in Arabidopsis. Trends Plant Sci. 7: 106-111 with permission from Elsevier.  The Arabidopsis AREB/ABF/ bZIP subfamily has nine members (bZIP family in Arabidopsis = 78 members) AREB1/ABF2, AREB2/ABF4 and ABF3 induced by drought, high salinity and ABA treatment of vegetative tissues – master regulators of ABA stress responses AREB3 and EEL are involved in seed development and regulate expression of LEA proteins (Late Embryogenesis Abundant proteins) ABI5 associated with dormancy and seed maturation AREB1, AREB2, ABF3 are master regulators of drought stress The three form homo or heterodimers and function redundantly. All three interact with SnRK2.2 in the nucleus. All three require ABA for activation (effects of over-expression seen only in presence of ABA) The triple areb1areb2abf3 (or abf2abf4abf3) is strongly drought susceptible. The triple snrk2.2/2.3/2.6 mutant and the triple areb1areb2abf3 have 75% target genes common indicating that the three SnRKs function by regulating the three ABFs. The expression of PP2Cs (AHG1, AHG3, HAI1, HAI2 and HAI3) is up-regulated by the three TFs (negative feedback regulation?). All nine PP2Cs are down-regulated in triple snrk mutant too. Wt abf2 abf4 abf3 abf2/4 abf3/4 abf2/3 abf2/3/4 Before Dehydration 1 week after Rehydration Survival 52% 57% 37% 38% 37% 34% 22% 5% Yoshida et al. 2010 Plant Journal  Over-expression of ABF3 or ABF4 confers ABA hypersensitivity Over-expression of the ABF3 or ABF4 transcription factors causes ABA-hypersensitive germination (increased ABA responses) Over-expression of ABF3 or ABF4 also confers drought tolerance. Kang, J.-y., Choi, H.-i., Im, M.-y., and Kim, S.Y. (2002). Arabidopsis basic leucine zipper proteins that mediate stress- responsive abscisic acid signaling. Plant Cell 14: 343-357. ABI5 promotes ABA responses in dormancy (inhibits germination) abi5 mutants are insensitive to ABA VIVIPAROUS-1 and ABI3 encode seed-specific B3 domain transcription factors These transcription factors are highly expressed in seeds, and bind to the RY DNA element [CATGCA(TG)] that is enriched in the promoters of seed-expressed genes. Binds RY motif in seed sp prom Bind bZIP trac factors (ABI5, bZIP10,bZIP25) which bind G box or ABRE elements McCarty, D.R., Carson, C.B., Stinard, P.S., and Robertson, D.S. (1989) Molecular analysis of viviparous-1: An abscisic acid-insensitive mutant of maize. Plant Cell 1: 523-532 Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F., and Goodman, H.M. (1992). Isolation of the Arabidopsis ABI3 Gene by positional cloning. Plant Cell 4: 1251-1261. Mönke, G., Altschmied, L., Tewes, A., Reidt, W., Mock, H.-P., Bäumlein, H., and Conrad, U. (2004). Seed-specific transcription factors ABI3 and FUS3: molecular interaction with DNA. Planta 219: 158-166. ABI3 is regulated post-transcriptionally as well as post-translationally Post-transcriptional regulation: ABI3 has a long 5’UTR that destabilizes its transcript. Removal of the UTR increases ABI3 transcript levels. Post-translational regulation: ABI3 is proteasomally degraded by AIP2 (ABI3 Interacting Protein2) – a RING motif E3 ubiquitin ligase. Over-expression of AIP2 leads to reduced ABA responses while aip2 mutants show enhanced dormancy due to higher ABI3 levels ABI3 AIP2 dormancy ABI4 (an AP2/ERF type TF ) is another positive regulator of ABA responses in seed -Expressed with ABI3 from globular stage of embryo and regulates the expression of ABI5 -ABI3, ABI4 and ABI5 mediate several seed dormancy and germination responses as well as responses to sugar How do ABA responsive TFs recognise target genes? Cis elements recognized by ABA response mediating TFs - Ry element CATGCA(TG) in seed-specific genes bound by ABI3 and VP1 - G-box ABREs (PyACGTGG/TC) bound by bZIP TFs like ABI5 Some TFs also require an additional coupling element (CE) in combination with ABRE Some ABA responsive genes contain sites for MYC or MYB TFs  PP2Cs inactivate SnRKs by dephosphorylation Phosphorylated SnRKs activate transcription factors by phosphorylation TFs mediate ABA responses  PP2Cs inactivate SnRKs by dephosphorylation Phosphorylated SnRKs activate transcription factors by phosphorylation TFs mediate ABA responses Where does ABA come into the picture? How is it perceived? The PYR/RCAR ABA receptors were identified in 2009 and made it to Science magazines Top 10 list GFP:RCAR1 PYR/RCAR proteins are found in the cytoplasm and nucleus (arrow) RCAR1pro:GFP From Ma, Y., Szostkiewicz, I., Korte, A., Moes, D.l., Yang, Y., Christmann, A., and Grill, E. (2009). Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324: 1064-1068 reprinted with permission from AAAS. Pyrabactin is an ABA agonist and can mediate ABA responses PYR/PYL/RCAR proteins (ABA receptors that bind ABA and Pyrabactin) bind to PP2Cs in presence of ABA and inactivate them. This releases the inhibition on SnRK2 members by PP2Cs. PYRABACTIN RESISTANCE (PYR1), PYRABACTIN RESISTANCE-LIKE (PYLS) or RCAR (REGULATORY COMPONENT OF ABA RECEPTOR) proteins belong to a large family of proteins with 14 members in Arabidopsis. These are soluble ligand-binding proteins with a START domain PYR/PYL/RCAR proteins bind PP2Cs in presence of ABA to inactivate them -ABA +ABA WILD TYPE WILD TYPE + ABA PYR1 PYR1 ABA PP2C PP2C P Kinase Kinase P P P TF NO ABA ABA RESPONSES RESPONSES  aba insensitive1 mutant: The abi1 mutation in PP2C prevents ABA binding and is not inactivated by ABA and constantly inhibits SnRKs WILD TYPE WILD TYPE + ABA abi1 mutant + ABA PYR1 PYR1 PYR1 ABI1 (PP2C) ABI1 (PP2C) abi1 P Kinase Kinase Kinase P P TF P P NO ABA ABA NO ABA RESPONSES RESPONSES RESPONSES Mechanism of the PYR/PYL and SnRK2 interactions with PP2C: Molecular mimicry Fen-Fen Soon, et al. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases Science 335, 85 (2012)  There are many genes encoding PYR/RCARs Number of The 14 PYR/RCARs in Arabidopsis Common Name Species genes Soybean Glycine max 23 Corn Zea mays 20 Populus Western poplar 14 trichocarpa Rice Oryza sativa 11 Grape Vitis vinifera 8 Sorghum Sorghum bicolor 8 Barrel medic (a Medicago 6 model legume) truncatula Arabidopsis Arabidopsis 14 thaliana Klingler, J.P., Batelli, G., and Zhu, J.-K. ABA receptors: the START of a new paradigm in phytohormone signalling. J. Exp.Bot. 61: 3199-3210 by permission of Oxford University Press; Raghavendra, A.S., Gonugunta, V.K., Christmann, A., and Grill, E. (2010) ABA perception and signalling. Trends Plant Sci. 15: 395-401. PYR/PYLs show unique and overlapping expression patterns (A) and (C) Embryos dissected from mature seeds imbibed for 24 or 48 h, respectively. (B) Dissected seed coat and endosperm imbibed for 24 h. (D) Primary root from 5-d-old seedlings. (E) and (F) Vascular tissue and guard cells in leaves of 15-d-old seedlings, respectively. Bars = 100 mm. Gonzalez-Guzman et al., Plant Cell (2012) 24: 2483-2496  The PYR/RCAR ABA receptors are necessary for ABA responses Wild-type plants fail to germinate on ABA- containing medium. Pyrabactin-insensitive mutants are ABA- insensitive and so germinate on ABA- containing medium. (PP2Cs constantly inhibit The ABA-insensitive SnRKs even in presence of mutant abi1 also ABA because of lack of germinates on ABA- receptors to bind ABA) containing medium. From Park, S.-Y., et al., and Cutler, S.R. (2009). Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324: 1068-1071 reprinted with permission from AAAS.  Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of germination and stomatal aperture Receptor mutant combinations studied here: PYR1 PYL1, 2, 4, 5, 8 Gonzalez-Guzman et al., Plant Cell (2012) 24: 2483-2496  Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of germination and stomatal aperture Pentuple (12458) and sextuple (112458) PYR/PYL mutants have higher germination and higher conductance (open stomata) like the Snrk2.2/2.3/2.6 triple mutant in presence and absence of ABA Stomatal conductance Gonzalez-Guzman et al., Plant Cell (2012) 24: 2483-2496 ABA signaling contributed to evolution of drought tolerance in land plants (higher numbers in more evolved land plants) Reprinted from Umezawa, T., Nakashima, K., Miyakawa, T., Kuromori, T., Tanokura, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2010). Molecular basis of the core regulatory network in ABA responses: Sensing, signaling and transport. Plant Cell Physiol. 51: 1821-1839 with permission from the Japanese Society of Plant Physiologists. Other players in ABA responses RNA mediated control of ABA responses Specific Cap binding proteins (ABH1) affect ABA responses AHG2 (ABA HYPERSENSITIVE AT GERMINATION2) is a polyA specific ribonuclease Several non-protein coding RNAs (almost 6000-8000 especially antisense RNAs) have been identified in seed germination transcriptomes HYL1 (HYPONASTIC LEAVES) involved in miRNA production affects ABA responses Chromatin remodelling: Some PP2Cs (eg HAB1) can interact directly with a homolog of the SWI3 component of SWI/SNF chromatin remodeling complexes thus blocking ABA responses. ABA treatment inhibits HAB1 and releases the repression Three histone chaperones, NUCLEOSOME ASSEMBLY PROTEIN1 also repress ABA responses Other proposed receptors of ABA G protein coupled receptor Loss-of-function alleles in the sole Arabidopsis G-alpha subunit gene (GPA1) show hypersensitivity to ABA at the level of germination and reduced guard cell sensitivity to ABA inhibition of stomatal opening, whereas they exhibit wild-type response to ABA-induced stomatal closure Overexpression of GCR1, the sole classical GPCR encoded by the Arabidopsis genome, reduces seed dormancy GPCR-type G proteins (GTG)1 and GTG2 bind GPA1 gtg1/gtg2 double mutants display reduced ABA sensitivity in seed germination, root growth, stomatal response, and gene expression ABA-response assays CHLH – a Mg++ chelatase- involved in Mg protoporphyrin formation Abiotic Stress responses pathway and role of ABA The abi1 mutation stabilizes the inhibitory effect of ABI1 WILD TYPE WILD TYPE + ABA abi1-1 + ABA PYR1 PYR1 PYR1 ABI1 abi1-1 ABI1 Kinase Kinase Kinase P P P P NO ABA ABA RESPONSES NO ABA RESPONSES RESPONSES  ABA-induced transcription can be reconstituted in vitro ABA SnRK2 TF PYR1 SnRK2 PP2C TF Using protoplasts as an assay system, expression of a TF (ABF2) and SnRK2 is sufficient for ABA-induced gene expression. Also adding PYR1 and ABI1 works too. Reprinted by permission from Macmillan Publishers Ltd: [ Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., Antoni, R., Park, S.-Y., Cutler, S.R., Sheen, J., Rodriguez, P.L., and Zhu, J.-K. (2009). In vitro reconstitution of an abscisic acid signalling pathway. Nature 462: 660-664 copyright 2009.  The Arabidopsis abi1-1 mutant has ABA-insensitive germination Wild type abi1-1 ABA ABA Dormancy NO Dormancy (Germination) NO ABA NO ABA NO Dormancy NO Dormancy (Germination) (Germination) From Koornneef, M., Reuling, G., and Karssen, C.M. (1984). The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiologia Plantarum 61: 377-383, with permission from John Wiley and Sons. abi1-1 mutants are ABA-insensitive in all their responses Germination is not inhibited on Root growth is ABA not inhibited on ABA ABI1 encodes a PP2C protein phosphatase Guard cells are not ABA- Wild responsive abi1 type Leung, J., Bouvier-Durand, M., Morris, P., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264: 1448-1452; Meyer, K., Leube, M., and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264: 1452-1455.  Transcriptional targets Stress and dehydration - Signaling genes induced genes A major output of ABA signaling is changes in transcription patterns. Many of the transcriptionally upregulated genes have functions in osmoprotection. Seed- Genes involved in specific genes ABA metabolism  ABA signaling - review PYR1 PYR/RCAR receptors ABA Phosphatase PP2C Protein phosphatases (including ABI1) Protein kinases (including Kinase SnRK2s and CDPKs) P P TF ABA RESPONSES  ABA / PYR1 binding sequesters PP2C and permits SnRK2 activity PYR1 PYR1, ABA and PP2C form a PP2C (ABI1) complex that inactivates PP2C. SnRK2 P This permits SnRK2 activation. SnRK2 Phosphorylation targets include SnRK2s, ion channels and P P transcription factors. TF Ion P channel ABA RESPONSES  ABA binding to an intracellular receptor initiates transcriptional responses PYL1 is an ABA receptor. When PYL1 binds ABA, it also binds the protein phosphatase PP2C, inhibiting its function. Reprinted by permission of Macmillan Publishers Ltd. Miyazono, K., et al. (2009) Structural basis of abscisic acid signalling. Nature 462: 609-614. atbg1 mutants show early germination Stomatal closure in dark is affected in atbg1 mutants Overexpression of AtBG1 increases ABA levels and leads to early stomatal closure even with minor changes in humidity ATBG1 functions as a polymer ATBG1 Is localized to the endoplasmic reticulum ABA-GE is stored in the vacuole How do the two come in contact? Is there another protein that shuttles ABA-GE to ER? ABA is believed to be synthesized in drying roots and translocated to leaves through the xylem Well-watered plant with Water-stressed open stomata plant with closed and high stomata and low transpiration transpiration rate rate Drought sensed even by a small portion of the roots leads to stomatal closure even when the leaf has not yet sensed drought. ABA is then synthesized de novo in leaves After water stress ABA accumulates in the veins and then guard cells A reporter construct made of an ABA-inducible promoter fused to luciferase was used to image ABA levels. Luciferase image Luciferase image merged with bright field image Christmann, A., Hoffmann, T., Teplova, I., Grill, E., and Muller, A. (2005). Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis. Plant Physiol. 137: 209-219.  SnRK2s were first characterized in wheat and Vicia faba AAPK is a guard cell specific PK from Vicia faba expressed in response to ABA, causes stomatal closure Phosphorylation PKABA1, an ABA- A dominant inducible SnRK2 negative form of protein kinase from AAPK interferes wheat, accumulates in with guard cell developing seeds response to ABA. Wheat Vicia faba SnRK2 activation requires the phosphorylated state of SnRK2 How is SnRK2 phosphorylated? Are any kinases known to phosphorylate SnRK2? No kinases have yet been identified for SnRK2 phosphorylation However, SnRK2 can autophosphorylate itself in vitro. In vivo experiments indicate that additional kinases may be required. Staurosporine (a kinase inhibitor) can inhibit SnRK2 autophosphorylation in vivo but cannot inhibit its activation in vivo. Therefore additional staurosporine insensitive kinases maybe involved in SnRK2 phosphorylation.  The bZIP transcription factors were identified both biochemically and through mutants Early studies of seed-specific and ABA- responsive genes identified a conserved DNA promoter sequence called the ABA- responsive element (ABRE). TF ABRE-binding proteins are called ABFs or AREBS.