Molecular Mechanisms of Abscisic Acid Action in Plants and Its Potential Applications to Human Health

Molecular Mechanisms of Abscisic Acid Action in Plants and Its Potential Applications to Human Health

ARCHANA JOSHI-SAHA, CHRISTIANE VALON AND JEFFREY LEUNG1

Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, Gif-sur-Yvette 91198 Cedex, France

I. II. III. IV. V.

VI. VII.

VIII. IX. X.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABA as a Positive and Negative Regulator in Plant Growth. . . . . . . . . . . . . ABA Circulation in the Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Transport in Guard Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ABA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A Family of Soluble Receptors PYR/PYL/RCAR..................... B. ABA Receptor in the Chloroplast Membrane .......................... C. Plasma Membrane-Localised ABA Receptors: The Link to G Proteins ................................................................. The Soluble PYR Signalling Complex is Part of a Short Phospho-Relay Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABA Controls Rapid Drought Adaptive Responses by Modification of Selective Transport Across the Plasma Membrane. . . . . . A. The Potassium Channels ................................................... B. The Anion Channels ........................................................ C. The P-Type Proton Pumps................................................. D. The Control of Downstream Targets by the Core ABA Signalling Complex in the Guard Cell ................................... Targets of SnRK2s in medium-term aba responses-gene expression and chromatin modelling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics in ABA Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitogen-Activated Protein Kinases in ABA Signalling . . . . . . . . . . . . . . . . . .

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Corresponding author: E-mail: [email protected]

Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.

0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00007-2

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XI. Root Growth in Response to Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. ABA is Conserved in Evolution and has Potential to Improve Human Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Drought tolerance actually embodies several protective mechanisms deployed by plants commensurate with the stress severity and duration. Against mild drought, one of the most rapid defensive measures is the closing of the stomatal pore, caused by shrinking of the flanking guard cells, in order to reduce transpiration that accounts for
90% of the water loss from plants. In contrast, the widening of the pore caused by expansion and bowing out of the guard cells is stimulated by light, permitting CO2 entry for carbon fixation. It is clear, therefore, that the two most decisive factors in plant growth—photosynthesis and water consumption—are directly influenced by guard cell regulation. Drought induces the synthesis of the ‘‘stress’’ hormone abscisic acid (ABA). Work within the past two decades has outlined the signalling pathway consisting of phospho-relay and ion transport across membranes that link ABA reception to stomatal closure. The threshold of the stomatal response to stress may also be set by more long-term processes that include gene expression and epigenetic regulation (‘‘stress memory’’), implying a feedback integration between rapid and slower protective mechanisms. In this chapter, some of the most recent, insightful, and exciting findings in the signalling network that orchestrate these ABA-dependent adaptive processes will be related. We will also extend our discussion in applying ABA research—not on the more obvious agricultural benefits—but as a novel and potent modulator of the immune system in human.

I. INTRODUCTION The seventeenth century psalmist George Herbert mused in his poem, ‘‘Man’’, after being inspired by his perception of congruity between man and his surroundings, that ‘‘Herbes gladly cure our flesh; because that they finde their acquaintance there’’. Indeed, biologists typing in a search for a string of codons from Arabidopsis will find hits in the mammals, flies, sculpin, fungus, and much more. With high-throughput technologies getting speedier by the day, and centralised databases easily accessible, large-scale discoveries are routinely being reduced to the effortless click of a button. We are already taking for granted that hundreds of thousands of genes in the plant genome have homologs in many other species, including humans (Jones et al., 2008). Perhaps with some reluctance, advances in science have also forced us to grapple with the sobering reality that, despite being the most evolutionary complex organisms, we are not even endowed with much more proteincoding capacity that the ‘‘lowly’’ Arabidopsis, whose life is surely less eventful

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than ours. The congruity in all life forms would have pleased Darwin, but these modern day insights seem to have already been an old truth for poets. The interconnectivity among all life forms is no less exemplified by adaptative responses to stress, an innate ability well known in both plants and animals. In the past years, many independent lines of anecdotal and experimental evidence converge on the startling conclusion that the ‘‘phytohormone’’ abscisic acid (ABA) is in fact a conserved signalling molecule that triggers protective functions in both the plant and animal kingdoms. We will return to this fascinating point later in the chapter. Land plants are subjected to continuously fluctuating microclimatic changes, particularly humidity, temperature and light quality. Being sedentary, they have evolved many elaborate mechanisms with built-in ‘‘redundancy’’ to optimise productivity in spite of adverse conditions. These adaptive responses have been operationally categorised as short term (e.g., stomatal closure in seconds to minutes), medium term (e.g., reprogramming of transcriptome in minutes to hours) and long term (morphological changes following days or weeks of stress, most notably, the root structure). A wealth of knowledge on the mechanistic nature of drought adaptation has already been distilled from imposed conditions in the laboratory, but still, all such controlled environments entail inadvertent biases to facilitate clear interpretations of the outcome. In nature, the climatic changes affect plant growth in more complicated ways. For example, temperature affects photosynthesis, but plants have considerable adaptive capacities enabling them to grow even at high temperatures providing that adequate water is available. Beyond a certain temperature, vapour pressure deficits of the air will be severe enough to heighten the transpiration rate from plant canopies, triggering stomatal closing and thereby suppressing growth. Increasing atmospheric CO2 (330–360 ppm) can also increase photosynthetic rates; but high CO2 will also stimulate stomata to close. It is not surprising, therefore, why most of the genes, when knocked out, do not necessarily lead to obvious visible phenotypes in the simplified and highly controlled laboratory conditions. Because of these apparent experimental limits, there has been a resurgent interest in exploring natural polymorphisms in plants, including Arabidopsis, to identify allelic variations across entire genomes that confer particular selective (dis)advantages (trade-offs). These complementary population approaches will enlighten us on quantitative traits, the nature of the genes across species or kingdoms that eventually fashion the developmental patterns of these plants for those environmental niches (‘‘Evolutionary Developmental Biology’’, or ‘‘evo-devo’’). There is a solid experimental proof that ABA is critical, in a dose-dependent manner (the underlying reason for which is still scarcely explored), for both normal plant development and stress

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adaptative capacity, especially to drought. The subject of how the ABA signal is transduced is a passionate scientific enquiry in itself, and with its obvious added value for enhancing agriculture for societal benefits, makes this topic truly vast as well as topical in view of climate threat, all of which cannot be dealt within the scope of a single chapter. At the time of writing, there are two recent and comprehensive reviews that provide excellent background on the initial steps of ABA signalling (Cutler et al., 2010; Raghaendra et al., 2010). This chapter will therefore focus on the more global advances made in the molecular aspects of the so-called short-, medium- and long-term adaptive responses in plants, with most of the mechanistic knowledge of the role of ABA derived from Arabidopsis thaliana. We hope that this chapter will be useful as a summary of a body of important work on molecular mechanisms of ABA signalling, but more so, a seemingly odd observation here and there would also ignite some latent ideas for further exploration, to perhaps even beyond plants. This is because an added intrigue that we put forth here concerns the origin of ABA, which seems ancient, as it also exists in model animals spanning the evolutionary scale from the most primitive to the most advanced—including humans. There is also tantalising clinical evidence that ABA is bioactive against certain ailments, most notably, type II diabetes, and could act as a powerful modulator of the immune system. This is ‘‘evo-devo’’ on a grand scale.

II. ABA AS A POSITIVE AND NEGATIVE REGULATOR IN PLANT GROWTH ABA is often viewed as a negative regulator because reduced growth under stress conditions is correlated with increased cellular ABA content, and that exogenously applied ABA (usually in micromolar) arrests seed germination and seedling growth. Several observations clearly indicate that this view is too simplistic, and that low levels of ABA can promote vegetative growth. ABA, while low in concentrations, can still be detected in extracts from aerial parts of wild-type plants grown even in well-watered conditions (Merlot et al., 2002). The basal level of ABA does not seem to be in passive storage, but studies carried out in tomato and Arabidopsis suggest that it is required to stem ethylene production (LeNoble et al., 2004; Sharp et al., 2000). Indirect in vivo imaging of ABA using reporter genes driven by ABA-sensitive promoters could also detect above background levels of ABA in some unstressed tissues, for example, the guard cells (Christmann et al., 2005). The concentration-dependent nature of ABA action is evident in root growth. Root elongation in Arabidopsis can in fact be stimulated significantly

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by exogenous ABA at 0.1 mM, while the hormone delivered at above 1.0 mM becomes inhibitory (Ghassemian et al., 2000). It seems that even in severe water stress, ABA is required to sustain growth in the root apex (3 mm-region) of maize, but inhibitory to cells (3–7 mm) proximal to the apex (Sharp et al., 2004), suggesting tissue-specific or developmental stage-dependent sensitivity to ABA. Indeed, suppressing ABA production in mutants or in transgenic plants was shown to result in developmental defects such as altered organisation of the mesophyll and stomatal morphogenesis (Barrero et al., 2005; Wigger et al., 2002). Thus ABA can act as both a promoter and an inhibitor of growth and development depending on its concentration and site of accumulation.

III. ABA CIRCULATION IN THE PLANT ABA is predominantly synthesised in bundle sheath cells (Marion-Poll and Leung, 2006) and then rerouted from there to all other tissues. These being the primary site of ABA synthesis is coincidental with expression of the biosynthetic genes AtNCED3, AtABA2 and AAO3 (Cheng et al., 2002; Koiwai et al., 2004; Tan et al., 2003) and the indirect detection of in vivo pools of ABA in these cells (Christmann et al., 2005; Wachter et al., 2003). The mechanisms by which ABA is rerouted from cell-to-cell is not known with precision, but it might circulate as an inactive glucose ester conjugate. The chemical properties of ABA glucose ester are well suited for its longdistance translocation in the xylem as it has low biomembrane permeability (Jang and Hartung, 2007). The ABA conjugate is stored in vacuoles or apoplastic space (Dietz et al., 2000), which is then released into the active form by apoplastic and endoplasmic reticulum b-glucosidases (Lee et al., 2006) in response to dehydration.

IV. MEMBRANE TRANSPORT IN GUARD CELLS How does ABA enter and exit cells? Previous pharmacological assays have hinted that ATP-binding cassette (ABC) transporters might have important roles in guard cell functions linked to environmental changes or hormone signalling (Leonhardt et al., 1997,1999). The ABC transporters are constituents of one of the largest gene families and are present in all taxa and assume diverse cellular functions, including detoxification of organic toxins, heavy metals and resistance against pathogens. More recent and direct evidence has now revealed the identities of some of these members in ABA transport,

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although there might still be others. In its acidic form, ABA can diffuse passively across biological membranes. This, of course, had begged the obvious question of whether ABA required at all active and vectorial transport, or by simple random diffusion coupled to other types of regulation such as controlled modification and/or cleavage of the hormone. Indeed, one essential component that facilitates active ABA expulsion into intercellular transport has now been identified by reverse genetic screens of mutants altered in ABA sensitivity (Kuromori et al., 2010). The gene corresponds to AtABCG25 (also known as AtWBC26), predicted to encode a so-called halfsize ABC transporter. One structural hallmark of ABC transporters is the presence of two conserved Walker A and B motifs within the nucleotidebinding-folds (NBF) which drive transport by ATP hydrolysis. Another diagnostic feature is the transmembrane domains (TMDs), each with 6–10 membrane-spanning a-helices with divergent sequences that determine substrate specificities. A full-size ABC transporter requires the concerted action of two NBFs and two TMDs, typically arranged in one contiguous polypeptide chain. AtABCG25 has a single copy of each TMD and NBF motifs, and thus qualified as a half-size ABC transporter. Insertion allelic mutants of abcg25 all display accentuated sensitivity to ABA. The protein is restricted to the plasma membrane, and it is expressed in cells close to the vascular tissues, presumably poised to export ABA into the intercellular space. Isotopically labelled 3H-ABA efflux activity can be detected in vesicles derived from Sf9 insect cell line expressing ABCG25. Overexpression of this gene in Arabidopsis conferred ABA resistance on seed germination, which is consistent with ABCG25 being an exporter or efflux factor of ABA. Stomatal response to rapid drought onset triggered by detaching leaves from these transgenic plants was found to be slower than that in the wild type, presumably because ABA was efficiently expelled from the guard cells (Kuromori et al., 2010), limiting their access to cytosolic targets that include receptors (see below). Simultaneously, a full-size ABC transporter functioning as an ABA importer was also identified. This latter discovery is particularly relevant in the context of stress response, whereby the increase in apoplastic pH would hinder the passive diffusion of the nonprotonated form of ABA across the plasma membrane; this situation logically necessitates active transport mechanisms to facilitate its delivery to the cytosolic receptors as primary targets. The plasma membrane-resident PDR12/AtABCG40, originally proposed to be a pump excluding lead or lead-containing toxic compounds (Lee et al., 2005), turned out to import ABA (Kang et al., 2010). 3H-ABA uptake showed time-dependent enhancement in the yeast mutant YMM12 or tobacco BY-2 cells expressing the AtABCG40. Uptake of 3H-ABA was sensitive to competition by the biologically active (S)-ABA, but not (R)-ABA, and also

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to inhibitors of ABC transporters such as glibenclamide, verapamil and vanadate. Conversely, Arabidopsis mutants disrupted in AtABCG40 (abcg40-1 and abcg40-2) showed reduced ABA uptake. It is noteworthy that, as compared to the wild type, the reduction of ABA uptake was greater for the knock-out mutants at higher pH, in which passive ABAH diffusion would be limited. The mutants also showed delayed expression of several ABA-inducible marker genes. Not surprisingly, these mutants are impaired in drought tolerance, and in a number of elementary developmental processes, including seed germination, that are known to be influenced by ABA. AtABCB14 (also known as AtPGP14 or AtMDR12) is expressed in several tissues as visualised by promoter fusion using the reporter uidA gene encoding b-glucuronidase. The stronger expression signals come from the guard cells (Lee et al., 2008). This transporter has a role in malate import from the apoplastic space into guard cells in response to CO2 (800 ppm), neither to high Ca2þ, ABA, nor to transition from light to darkness (Lee et al., 2008). Mutants disrupted for the AtABCB14 gene show significantly more reduced stomatal aperture than that of the wild-type guard cells exposed to malate and high CO2. Conversely, overexpression of the AtABCB14 transgene blocks stomatal closure to both of the above stimuli (Lee et al., 2008). The supposition that AtABCB14 is a malate importer comes from the fact that when the bathing solution contained only malate as the anion, stomatal opening was observed to be much faster in the AtABCB14 overexpressing lines than for the two mutants. This is consistent with malate acting as an osmoticum in guard cells to maintain stomatal opening. Another line of compelling evidence is that expression of AtABCB14 can restore growth of the Escherichia coli mutant defective in dicarboxylate transport on malate-containing medium. The combined results would thus link this particular ABC transporter to malate uptake in different cell types in response specifically to high CO2. However, in guard cells, high CO2 privileges stomatal closure by, at least in part, stimulating anions extrusion (and a decrease in osmotic pressure; Negi et al., 2008), the malate uptake by AtABCB14 may thus reflect a recycling mechanism. There are additional transporters whose mutations also lead to impaired ABA responses, but the underlying mechanisms are less obvious. Only some of the recent and more insightful studies are summarised here. Anion transporters that confer resistance to aluminium were first reported in wheat. These transporters were presumed to share a generalised function in excreting organic anions such as citrate or malate into the soil to chelate aluminium, thereby neutralising its toxicity. However, members of this transporter family may have functions more diverse than originally suspected. In Arabidopsis, the 13 homologous members of this gene family have been targets of

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physiological as well as molecular genetic studies. It seems that at least AtAML12 (At4g17970) is an anion transporter permeable to nitrate and chloride, and the corresponding mutant is impaired in guard cell regulation (Sasaki et al., 2010). The Arabidopsis mutant Atmrp5 is impaired in Ca2þ signalling and partial ABA-induced anion current activity as part of its pleiotropic phenotype (Klein et al., 2003; Suh et al., 2007). AtMRP5 (AtABCC5) turned out to transport inositol hexakisphosphate (IP6), a ubiquitous signalling molecule and the principle storage form of phosphorus in many plant species. Expression of the wild-type transgene restores the mutant’s sensitivity to ABAmediated inhibition of stomatal opening (Nagy et al., 2009).

V. THE ABA RECEPTORS A. A FAMILY OF SOLUBLE RECEPTORS PYR/PYL/RCAR

The passionate hunt for the ABA receptor(s) was ignited 25 years ago that began with three ABA-binding proteins detected biochemically in the plasmalemma of Vicia guard cells (Hornberg and Weiler, 1984). Subsequently, several other proteins with affinity to ABA were also reported, but either their molecular characteristics were not known, or they do not seem to work in the conventional pathways outlined by genetics and physiology. Owing to its accessibility to experimental manipulations, and most importantly, a simple physiological output (stomatal closure or opening), the guard cell dominated as the hunting ground over the next 15 years for ABA perception sites, whose presence were detected in the mid-1990s by various physiological means to be on both the cell surface as well as the ‘‘inside’’ of the cell (Leung and Giraudat, 1998). In 2009, the hunt culminated in the two independent molecular studies that converged simultaneously on the same candidates, which turned out to be the soluble ABA receptors. They are encoded by a gene family of 14 members sharing homologies with the steroidogenic acute regulatory related lipid transfer (START) proteins (Ma et al., 2009; Park et al., 2009; Fig. 1). The study headed by S. Cutler in the United States successfully exploited chemical genetics by selecting Arabidopsis mutants that can germinate on pyrabactin, a synthetic growth inhibitor of seed germination that can act as a selective agonist of ABA (Park et al., 2009). Pyrabactin and ABA trigger highly correlated transcriptional responses in seeds (although only moderately so in seedlings), suggesting that these two compounds share common targets. Even so, the mechanisms of pyrabactin and ABA in growth inhibition also seemed to implicate distinct

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SLAC

GORK1

−

K+

KAT1/KAT2 OST2 K+

H+

AtrbohF NADPH oxidase

A

H2O2 OH

COOH

Ca2+ (CPK21, PSK5)

H2O2

O

pH

PYR/PYL/RCAR

PP2C

SnRK2 b-ZIP transcriptional activators (ABF)

ABAR

WRKY

Gene expression

Fig. 1. The core complexes of the two better established ABA signalling pathways. The PYR/PYL/RCAR receptor is cytosolic/nuclear, whereas the ABAR is in the chloroplast. In the ground state (in the absence of ABA), PP2Cs in the clade A (ABI1, ABI2, HAB1, PP2CA, etc.) restrain the SnRK2 kinase activities (OST1, SnRK2.2, SnRK2.3) by dephosphorylation. The binding of ABA to PYR induces a structural change in the latter, creating a binding surface that sequesters ABI1, allowing OST1 and other SnRK2 kinases to phosphorylate downstream targets, which include at least three major b-ZIP transcriptional activators, and in the guard cell, plasma membrane transporters that are either activated (SLAC1) or repressed (KAT1), contributing to an overall decrease in osmotic pressure to bias stomatal closure. SLAC1 can also be directly dephosphorylated (and inactivated) by PP2CA independent of OST1. The second parallel ABA signalling pathway is highly unusual in that it starts in the chloroplast with the receptor ABAR (same as GUN5), a large 120-kDa protein that spans the envelopes. The binding of ABA allows ABAR to recruit, by as yet unknown mechanisms, at least three transcriptional repressors WRKY from the nucleus, relieving the suppression of gene expression. The differential sensitivity of the anion channel and the proton pump to Ca2þ is likely due to the action of Ca2þ-sensitive kinases (CPK21 and PSK5, respectively).

properties, because the founding member of the pyrabactin-insensitive mutants, pyr1 (also named as rcar11 in the other study headed by Grill in Germany), was isolated based on its resistance to pyrabactin and yet it was still wild type in sensitivity to ABA. It was in combining triple or quadruple knock-outs of the PYR, and some of its homologs (PYL), reduction in ABA sensitivity was finally revealed (Park et al., 2009). Conversely, overexpressing one of the members, PYL9/RCAR1 (for Regulatory Component of ABA Receptor), enhanced ABA sensitivity in virtually all elementary criteria such as root elongation, stomatal closure and seed germination (Ma et al., 2009). This suggests that PYR family members assume overlapping functions in

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ABA perception and that chemical genetics can by-pass functional redundancy that can sometimes hinder traditional genetic screens. It remains a curious observation that despite the fact that ABA and pyrabactin share very little similarity in their chemical structure, they both bind to PYR. Results from yeast two-hybrid screens, done independently by the two laboratories cited above, revealed that PYR interacted with several PP2Cs, including ABI1, ABI2, HAB1, and other closely related homologs, in the presence of ABA or pyrabactin, but not inactive ABA analogues or other hormones. Further, mutations in either PYR1 (S152L and P88S), or in other close homologs of HAB1, such as ABI2 (abi2-1) (Ma et al., 2009; Park et al., 2009) and ABI1 (abi1-1) (Ma et al., 2009), disrupted PYR–PP2C interactions in yeast. Importantly, the interaction was recapitulated in plant cells when selected PYR and PP2C members were coexpressed by transfection into Nicotiana benthamiana epidermal cells (Park et al., 2009), in Arabidopsis protoplasts (Ma et al., 2009; Szostkiewicz et al., 2010) or by in vitro pulldown assays using components expressed in E. coli (Park et al., 2009). Moreover, the protein PYR9/RCAR1 interacted with the PP2C in the cytosol and nucleus, the same subcellular compartments at the RCAR1 alone (Ma et al., 2009). Isotopically labelled ABA directly binds to PYR/PYL/RCAR, as ascertained by 15N-labelled PYR1 and PYR1P88S in heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments, which probe chemical shifts of protein amide-NH bonds in response to ligands (Park et al., 2009). The addition of (S)-ABA in the nanomolar range, and in the presence of PYR/PYL/RCAR, inhibits efficiently PP2C activities (Ma et al., 2009; Park et al., 2009; Szostkiewicz et al., 2010). No inhibition was observed if the PYR/PYL/RCAR was disrupted physically (Ma et al., 2009) or by mutations (Park et al., 2009). Thus, PYR/PYL/RCAR negatively regulates particular PP2Cs in response to ABA, which defines an unprecedented mechanism for ligand-mediated regulation of PP2C activity. Isothermal titration calorimetry revealed binding of (S)-ABA to PYL9/RCAR1 and ABI2 with an apparent binding affinity (Kd) of
64  8 nM ABA and a single binding site. The analysis for the binding of (S)-ABA to PYL9/RCAR1 yielded lower energy changes and a higher apparent Kd of
0.66  0.08 nM ABA (Ma et al., 2009). The considerably lower Kd value of the heteromeric protein (PP2C–RCAR1)-ABA complex was argued to reflect a ligand-induced complex stabilisation, similar to FLS2 and BAK1 receptor complex stabilisation by flagellin (Chinchilla et al., 2007). Because both the ABA receptors (14 members) and PP2Cs (nine members in clade A) are encoded by multigenes, combinatorial interactions between these two components could promulgate subtly distinct downstream

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messages. Although the picture is far from complete, the different receptors that have been tested display variable affinities to ABA isomers (Park et al., 2009; Szostkiewicz et al., 2010). Further, the selectivity observed between receptor and members of the clade A PP2C could constitute a second layer of control to fine-tune the ABA signal input. For example, PYL5/RCAR8 can bind HAB1, ABI1, ABI2, but not AHG3 (Santiago et al., 2009). PYL8/ RCAR3 was also shown to repress ABI1 and ABI2 in vitro and to stimulate ABA signalling in protoplasts (Szostkiewicz et al., 2010). The efficiency of ABA-mediated phosphatase inhibition was higher with ABI1 than ABI2, and higher with RCAR3 than RCAR1. To put this into perspective, halfmaximal inhibition of RCAR3/ABI1 was observed at 23 nM ABA, whereas RCAR1/ABI2 revealed a more than fourfold higher IC50 value of 95 nM ABA. This finding reflects major differences in the heteromeric receptor complexes with respect to ABA-mediated inhibition. Although these studies using different combinations of the PYR/PYL/ RCAR and PP2C hinted that these two components may function as coreceptor complexes (Szostkiewicz et al., 2010), structural studies (using models PYR1/RCAR11, PYL1/RCAR12, PYL2/RCAR13) revealed that ABA is actually bound deep inside an occluded protein cavity, rather than at the interface between receptors and the PP2Cs (Miyazono et al., 2009; Nishimura et al., 2009; Yin et al., 2009). This indicates that the PYR/PYL/ RCAR are direct ABA receptors and signal transduction partners. On the mechanistic level, ABA receptors therefore function more like the gibberellin receptor GID1, rather than like the auxin-linked TIR1 and AUX/IAA coreceptor complex in which both components cradle the hormone. PP2Cs are monomeric enzymes, and those implicated in ABA signalling interact with PYR/PYL/RCAR in equal molar ratio (Leube et al., 1998; Ma et al., 2009; Yin et al., 2009); nevertheless, the receptors themselves can form dimers through interaction between the so-called CL2 loop (or the proline gate) from each of the subunits, even in the absence of ABA (Nishimura et al., 2009; Yin et al., 2009). PYR1 in the presence of nonsaturating concentrations of ABA yield an ABA-bound and an ABA-free subunit, related by a
1708 rotation around a pseudo twofold axis. The PYR1 is thus an unusual symmetric homodimer. Moreover, a specific leucine residue (Miyazono et al., 2009; Nishimura et al., 2009; Yin et al., 2009) from the ABA-free subunit reaches across the PYR1 dimer to block the remaining cavity access, thus sequestering the ABA. In excess ABA, when both subunits are bound by the ligand, the dimer assumes an exact twofold symmetry, consisting of a flattened biconcave disc that has been described to resemble a red blood cell (Nishimura et al., 2009). The CL2 loop also forms the lid, to which PP2C binding apparently favours lid closure and decreases the ABA

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off rate. This could also explain the observation of tighter binding of ABA to PYR in the presence of a compatible PP2C (Ma et al., 2009; Nishimura et al., 2009; Yin et al., 2009). It has also been speculated that PP2C docking onto the CL2 loop of the receptor–ABA complex might require the prior dissociation of the dimer into monomers (Yin et al., 2009). These structural studies also clarified the nature of the dominant mutations abi1-1 and abi2-1 which were the first to establish the important role of these PP2C in ABA signalling (Koornneef et al., 1984; Leung et al., 1994, 1997; Meyer et al., 1994; Rodriguez et al., 1998). The corresponding mutant proteins are genetically defined as suppressors of ABA signalling, exactly the same role as the wild-type protein, suggesting that these dominant mutations might have rendered the phosphatases constitutively active (without them necessarily being hyperactive on a per enzyme basis). The interaction of PYL1/RCAR12 with ABI1 is mediated by the CL loops (especially the CL2 mentioned above and helix a2) through a network of van der Waals contacts, water-mediated contacts, and a few direct hydrogen bonds. Ser112 of PYL1/RCAR12 donates one hydrogen bond to Glu412 of ABI1 and accepts one from the amide of Gly180 which makes a hydrogen bond to the backbone carbonyl oxygen of Ser112. The mutation abi1-1, which converts Gly180 to Asp, or the equivalent Gly168 to Asp in abi2-1 would disrupt this amide bond with Ser112 of PYL, and the Asp substitution may cause additional steric hindrance into the interface between PYL1 and ABI1 (Miyazono et al., 2009; Yin et al., 2009). Hence, these dominant mutations impart immunity to these PP2C from recruitment by the ABA-receptor complex (Ma et al., 2009; Park et al., 2009). The Trp300 of ABI1 (presumably equivalent amino acids in the other close homologs) is accorded particular importance, as it is the only amino acid in simultaneous contact with a hydrophobic pocket of the receptor and the ABA molecule via a water molecule (Miyazono et al., 2009; Yin et al., 2009). B. ABA RECEPTOR IN THE CHLOROPLAST MEMBRANE

Another potential ABA receptor is ABAR, corresponding to the H subunit of the magnesium-chelatase (CHLH; Shen et al., 2006). The identity of this being a potential ABA receptor was based on homology with a 42-kDa protein (or a fragment of a larger protein) that has been affinity purified from the abaxial epidermis of Vicia leaves earlier by the same group (Zhang et al., 2002). Scatchard plot analysis of this protein showed an equilibrium dissociation constant of 21 nM and stereospecificity in that ()ABA and trans-ABA were incapable of displacing 3H-() ABA bound to the protein and () ABA was less effective than (þ) ABA in the competition. As a

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candidate ABA receptor, the Arabidopsis homolog ABAR is surprising in that it is a chloroplast membrane-localised protein of over 120 kDa, and moreover, it was identified previously as genome uncoupled (or gun)5, that disrupted a component of plastid-to-nucleus communication to coordinate expression of both nuclear- and chloroplast-localised genes encoding photosynthesis-related proteins (Susek et al., 1993). The wild-type protein GUN5 is one of three subunits of the Mg2þ-chelatase required for Mg2þ-protoporphyrin IX synthesis and has been postulated to be a monitor of porphyrin levels by binding excess proto and/or Mg2þProto, and sends a signal to the nucleus via a hypothetical downstream effector. This function is separable from that of ABA signalling. It has been proposed that, after direct ABA reception, GUN5/CHLH/ABAR binds via its C-terminal to at least three WRKY proteins that are negative regulators of ABA signalling in seed germination and postgermination growth (Shang et al., 2010; Fig. 1). Overexpression and RNA-interference transgenic Arabidopsis lines led to altered ABA responses in seed germination, postgermination growth, stomatal movement and expression of certain ABA-regulated genes (Shen et al., 2006). C. PLASMA MEMBRANE-LOCALISED ABA RECEPTORS: THE LINK TO G PROTEINS

Heterotrimeric G proteins are fundamental in transmembrane signalling by relaying a large variety of receptors to channels, enzymes and myriads of effector molecules (Wettschureck and Offermanns, 2005). In the mammalian genomes, multiple subforms of G proteins together with receptors, effectors and teams of regulatory proteins make up the components of a highly versatile signal transduction network (Wettschureck and Offermanns, 2005). G protein-mediated signalling is employed by virtually all cells in the organism and is centrally involved in diverse physiological functions, notably sensory perception, synaptic transmission, hormone release and cell contraction and mobility, just to name a few. The key members are the heterotrimeric G proteins—comprising the Ga, Gb, Gg subunits—as well as the G protein-coupled receptors (GCPRs). The Ga subunit, with both GTP-binding and GTPase activity, acts as a bimodal switch, typically a GDP-bound ‘‘off’’ state and a GTP-bound ‘‘on’’ state. The GPCRs classically act as guanine nucleotide exchange factors (GEF), and a change in GPCR conformation upon signal perception triggers an exchange of GDP for GTP at the Ga subunit. This triggers the dissociation of Ga from the Gbg dimer, both of which can interact with an array of downstream signalling elements. Note, however, plant genomes are notorious for their seemingly poor endowment of G proteins, at least based on the criterion of overt sequence homologies

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with their counterparts in other species. A. thaliana, for example, has a rather modest repertoire of one Regulator of G protein Signalling (RGS), one prototypical Ga (GPA1), one Gb (AGB1), two Gg (AGG1 and AGG2) subunits (Jones and Assmann, 2004). Disruption of GPA1 was previously shown to disturb normal ABA functions (Pandey and Assmann, 2004), a first hint suggesting the involvement of GCPRs. In silico analysis of the Arabidopsis has, however, recently revealed two plasma membrane-localised proteins with 45–68% sequence identity to the human protein GPR89, which was initially annotated as an orphan GCPR (Pandey et al., 2009), but functionally it is a pH-sensitive chloride channel resident in the Golgi (Maeda et al., 2008). These Arabidopsis homologs, which are conserved across phyla, are named GTG1 and GTG2 (GPCRtype G protein), and both have been shown to display GTPase activity in vitro. As compared to the wild type (accession Wassilewskija) or each of the single gtg mutants, the double mutant is hyposensitive to ABA at seed germination, seedling growth and stomatal closure. The two GTG proteins interact with GPA1, the sole canonical Ga subunit (see above), in the yeast split-ubiquitin two-hybrid system (SUS) designed to assess membrane proteins, and they coimmunoprecipitate from plant extracts. GPA1 binding stimulated GTP-binding activity of the GTGs but remarkably the latter’s GTPase activities are inhibited. The purified recombinant GTGs show saturable 3H-ABA binding with apparent Kd of between 30 and 40 nM. The dissociation constant could be even halved, considering that binding was completed by ()ABA, but not the biologically inactive ()ABA, and ABA binding was improved by GDP (Pandey et al., 2009). The stoichiometry of binding was low in these experiments, being
0.01 mol ABA/mol of GTG, perhaps due to the fact that the receptors require a membranous environment for optimal ABA binding (Pandey et al., 2009; Risk et al., 2009).

VI. THE SOLUBLE PYR SIGNALLING COMPLEX IS PART OF A SHORT PHOSPHO-RELAY CASCADE In the ABA signalling core complex, members of the protein phosphatases 2C belonging to clade A—as epitomised by the two closely homologous founding members ABI1 and ABI2 (Gosti et al., 1999; Merlot et al., 2001)—are negative regulators of ABA signalling. In an idealised environment devoid of stress (presumably no or low levels of ABA), these clade A PP2Cs would antagonise the pathway by inactivating specific downstream protein kinases, which function as positive regulators of the ABA signal transduction pathway (Umezawa et al., 2009; Vlad et al., 2009). Indeed,

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these PP2Cs can physically dock via a particular motif called domain II that is found in the noncatalytic C-termini of SnRK2.2, SnRK2.3 and OST1/ SnRK2.6/SRK2E (Fujita et al., 2009; Umezawa et al., 2009; Yoshida et al., 2006). The PP2Cs probably interact transiently with these SnRK2s because coimmunoprecipitation of the two proteins was found to be inefficient unless chemical cross-linkers were added (Vlad et al., 2009). These PP2Cs can efficiently dephosphorylate the multiple Ser/Thr residues in the activation loops of these kinases (Umezawa et al., 2009; Vlad et al., 2009). In response to stress, ABA binds to PYR/PYL/RCAR. As mentioned above, the change of the receptor protein conformation caused by the binding of ABA in turn leads to the creation of contact surfaces specific for at least some of the clade A PP2Cs, thereby restraining physically these negative regulators. In the absence of these free PP2Cs, the equilibrium of the SnRK2s is shifted towards their active states, permitting them to phosphorylate downstream targets. These core signalling steps have been successfully and elegantly recapitulated in vitro using a peptide derived from the ABA-responsive transcription factor ABF2 as the model target of phosphorylation (Fujii et al., 2009). The in vitro reconstruction of the signalling core is largely consistent with a number of in vivo observations. As alluded to above, the dominant nature of the mutation equivalent to abi1-1 in various PP2Cs owes this to the loss of a contact amino acid, allowing them to escape recruitment by the soluble receptors. In addition, the lower in vivo SnRK2 kinase activity in the dominant PP2C mutant genetic background, and inversely, their elevated activity in the PP2C knock-outs (Mustilli et al., 2002; Vlad et al., 2009) are in accordance with the direct binding and inactivation of SnRK2s by PP2Cs in vitro or in yeast (Umezawa et al., 2009; Vlad et al., 2009). The homologous SnRK2.2, SnRK2.3 and SnRK2.6/OST1/SRK2E are likely the major and direct targets of negative regulation by at least some of the clade A PP2Cs related to ABA signalling. Of these three kinase-encoding loci, SnRK2.6/OST1/SRK2E was independently identified in forward genetic screens for mutants that transpire excessively in conditions of low humidity (Merlot et al., 2002; Xie et al., 2006) and by reverse genetics in search of kinases activated by exogenous ABA (Yoshida et al., 2002). Earlier, the ortholog AAPK was purified from Vicia faba guard cells by virtue of its activation by ABA and was shown to be functionally important in mediating stomatal closure by ABA (Li et al., 2000). Thus, OST1 and AAPK seem to be the predominant SnRK2s in relaying the ABA signal in the guard cell. No forward genetic screen has identified SNRK2.2 and SNRK2.3, and one reason is that these two kinases may be functionally very similar (in those defined experimental conditions). This is supported by the fact that knock-out mutants for either SNRK2.2 or SNRK2.3 are phenotypically

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indistinguishable from the wild type, but the simultaneous loss of both loci causes altered root growth and seed germination behaviour on ABA (Fujii et al., 2007). Despite their apparent tissue-specific phenotypes that imply functional distinction, OST1 may still share a subset of common downstream targets with SNRK2.2 and SNRK2.3. In comparison to the double mutant, the triple knock-out snrk2.2 snrk2.3 snrk2.6/ost1/srk2e shows further reduction by several fold in ABA sensitivity, remains stunted in development, produces few seeds, and is prone to wilting when ambient humidity is not high (Fujii and Zhu, 2009; Umezawa et al., 2009). The severity of the phenotype throughout plant growth and a pronounced decrease in ABA sensitivity suggest that these three kinases act as key regulators of most of the elementary downstream ABA responses.

VII. ABA CONTROLS RAPID DROUGHT ADAPTIVE RESPONSES BY MODIFICATION OF SELECTIVE TRANSPORT ACROSS THE PLASMA MEMBRANE A. THE POTASSIUM CHANNELS

As an overview, most of the so-called rapid ABA responses are derived from studies using the guard cell as the model (Fig.1). These rapid responses are most likely conserved, at least at the qualitative level, as they have also been observed in cell cultures (Meimoun et al., 2010). One of the earliest detectable responses to drought onset is the reduced turgor pressure and volume of the pair of flanking guard cells leading to stomatal closure. The closing stimulus is mediated, at least in part, by the production of H2O2, and through increases in cytosolic [Ca2þ]cyt as well as enhancement of its sensitivity as a form of positive feedback (coined as ‘‘priming’’; Kim et al., 2010; Siegel et al., 2009). The Ca2þ signals prevent turgor increase by downregulating both the P-type Hþ-ATPases required for plasma membrane hyperpolarisation (Kinoshita et al., 1995) and channels for Kþ influx. In parallel, Ca2þ stimulates anion extrusion (Hedrich et al., 1990; Schroeder and Hagiwara, 1989), which contributes to the depolarisation of the plasma membrane. As well, in parallel to the Ca2þ signal, the associated cytoplasmic alkalinisation evoked by ABA promotes Kþ extrusion via efflux channels (Blatt and Armstrong, 1993). In plant cells, Kþ is the most abundant cation (which can make up to 10% of the dry weight if Kþ is unlimited in availability) and serves as a charge carrier, enzyme cofactor and an osmoticum, as in the case of the guard cell. There are six Shaker-type (named after the Drosophila founding member) Kþ

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inward-rectifying channels expressed in the guard cells, with the homologs KAT1 and KAT2 being responsible for majority of the Kþ influx activity that drives stomatal opening. Each of these two major Shaker channels, when expressed alone, can form homotetramers which constitute the functional unit of the Kþ channel. However, in plants, they preferentially form heterotetramers consisting of two subunits each of KAT1 and KAT2 (Lebaudy et al., 2010). KAT1–KAT2 heterotetramers generate synergistic enhancement in Kþ transport in that the current is much larger as compared to the equivalent amounts of either KAT1 or KAT2 homotetramers. In comparison to the large number of inward rectifiers, there is only one Kþ outward-rectifying channel, GORK, responsible for Kþ efflux in the guard cell requisite for stomatal closing. The voltage-gated Kþ channels presumably have evolved from a common Shaker-type ancestor because they share the characteristic framework of six TMDs, designated S1—S6, with the pore region between transmembrane segments S5 and S6 that form the major constriction and lining of the pore. Despite the sequence conservation among these Kþ transporters, they are impressive in their remarkable functional diversity. Previous work on the sole Kþ outward rectifier expressed in the stellar cells of the root, SKOR, has shown that its gating mechanism is achieved by certain amino acids deep in the S6 domain that opposes and contacts with the base of the helix pore (Johansson et al., 2006; Liu et al., 2006). However, the precise regulatory mechanism remains intriguing: it has been reported that the activity of SKOR is stimulated by internal [Kþ] (Liu et al., 2006), or alternatively, a more unusual mode that depends rather on external Kþ (Johansson et al., 2006) even though the function of SKOR is to extrude Kþ. It also appears that both intracellular and extracellular acidification inhibit SKOR (Lacombe et al., 2000). As described above, SKOR bears a ‘‘S6 gating domain’’, including the key residues D-M-I within the last transmembrane segment that opposes and interacts with the base of the pore helix, transmitting information about pore occupancy to the channel gate. Altering this interaction through residue exchange—either in the S6 gating domain or at the base of the pore helix— affects the Kþ sensitivity as well as the voltage-dependence of SKOR gating and, in the extreme, also renders the channel nonrectifying. These results raised the question of whether the S6 regions of Kin and Kout channels are key to understanding the divergence in their function. B. THE ANION CHANNELS

The long-sought after channel that is critical for slow sustained anion efflux, and the subsequent decrease in osmotic pressure, needed to drive stomatal closure was identified by independent genetic screens for mutants with

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altered sensitivity to ozone (Saji et al., 2008; Vahisalu et al., 2008) and to CO2 (Negi et al., 2008). The mutation slow anion channel-associated 1 (slac1, also variously known as rcd1, rcd3, or cdi3; Fig. 1) results in larger stomatal aperture, and the corresponding guard cells contain higher Kþ content, possible as a counter ion to the accumulation of malate and fumarate (Negi et al., 2008). As well, the mutant turns out to be reduced in sensitivity to a variety of other stomatal closing-inducing signals, but several details differ in the independent mutant characterisation in terms of its responses to H2O2, ABA and high CO2. For example, the mutant guard cells were said to be insensitive to ABA by two groups (Negi et al., 2008; Vahisalu et al., 2008) but were described as wild type by another (Saji et al., 2008). Both rapid transients and long-term O3-induced decreases in stomatal conductance were abolished in the mutant. Notably, only the S-type (but not the R-type) anion current was impaired by the mutation. This observation is important because it provides strong evidence that the S- and the R-type anion currents are mediated by distinct entities, rather than due to the same channel being modified differently, for example, by phosphorylation, as previously proposed. Whole-cell patch clamp techniques detected a permeability ratio for malate to chloride anions of 0.128 consistent with previous anion selectivity analyses of S-type anion channel currents. The corresponding protein (At1g12480) has low homology with the C4-dicarboxylate transporter/ malic acid transport protein domain defined from the E. coli TehA and Schizosaccharomyces pombe Mae 1 protein. C. THE P-TYPE PROTON PUMPS

Activated proton pump is required for hyperpolarisation of the plasma membrane that eventually drives stomatal opening. The pumping activity is substantially reduced in the phot1 phot2 mutant background, thus placing the Hþ-ATPases, such as OST2 (Fig. 1), in the phototropin-mediated pathway (Shimazaki et al., 2007). Plant proton pumps can functionally complement yeast mutants disrupted for the major Hþ-ATPase PMA1 (which is lethal for yeast), which has facilitated detailed studies into the structure–function relationship of these plant proton pumps. The activity of the proton pump is known to be influenced by protein phosphorylation and structural rearrangements. All P-type Hþ-ATPases have a cytosolic C-terminal domain of
100 amino acids which act as an autoinhibitory domain (Palmgren et al., 1991). The current model proposes that this domain suppresses proton pumping activity by folding back onto the rest of the protein. Note that the inactive form of the P-type Hþ-ATPase is dimeric and it is not clear how the individual folded proteins are arranged relative to each other. This presumed

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closed protein conformation, nonetheless, is consistent with the interaction between the C-terminus and the rest of the OST2 protein in trans by using the yeast split-ubiquitin (Merlot et al., 2007) and bimolecular fluorescence complementation (BiFC) assays in transfected tobacco epidermal cells (Sirichandra et al., 2009a). Recently, the N-terminal soluble portion of the Actuator domain has also been shown to possess autoinhibitory functions as well; in particular, an extrapolation from the available data suggests that deleting amino acids 4–10 in this domain of AHA2 led to enhanced yeast growth in acidic medium (Ekberg et al., 2010). This was further shown to correlate with an increased phosphorylation of a specific Thr947 and its subsequent ability to bind to 14-3-3 proteins in the C-terminus of the pump. Further deletions up to 20 amino acids negate this enhancement of yeast growth on acidic medium, and in a majority of the cases, without apparent effect on the stability these truncated AHA2 (Ekberg et al., 2010). There might be other subtle regulatory effects exerted by the N-terminal portion of the Actuator domain that are difficult to decipher by increasingly larger deletions which might disrupt the protein structure, as hinted by the existence of several point mutations further up to the first TMD (M1) capable of increasing the proton pumping activities as well (Merlot et al., 2007; Morsomme et al., 1996). Activation of the pump requires phosphorylation of the penultimate Thr, which then binds 14-3-3 proteins to assume the active conformation. This is also accompanied by the conversion from the dimeric to a hexameric form joint by six 14-3-3 proteins at the C-termini of the Hþ-ATPases (Kanczewska et al., 2005; Ottmann et al., 2007). At least for two of the most studied tobacco Hþ-ATPases, PMA2 and PMA4, do not heterodimerise. Moreover, the hexamer assumes a true sixfold symmetry rather than a threefold symmetry, consistent with a lack of heterodimeric forms between PMA2 and PMA4. This has also been interpreted that regions other than the C-termini might be engaged in the formation of this hexameric configuration (Ge´vaudant et al., 2007). Besides the bilateral autoinhibitory domains, the proton pumping activity is also reduced by phosphorylation by the calcium-dependent kinase PKS5 on a specific and conserved serine residue in the C-terminal domain that impedes subsequent 14-3-3 binding (Fuglsang et al., 2007). This might explain the sensitivity to Ca2þ inhibition of Hþ-ATPases as described above. PKS5 is itself inactivated by the chaperone J3, homologous to the E. coli DnaJ/hsp40 protein (Yang et al., 2010). The inactivation mechanisms are not clear; it could be that J3 decreases the affinity between Hþ-ATPase and PKS5, or J3 could directly inhibit the PKS5 kinase activity.

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Treatment with ABA is invariably associated with an increase in H2O2 production, as shown in Arabidopsis and in V. faba guard cells (Kwak et al., 2003; Pei et al., 2000; Zhang et al., 2001). In Arabidopsis guard cells, two of the 10 NADPH oxidases, AtrbohD and AtrbohF, are responsible for ABAinduced ROS production and subsequent stomatal closure. Until recently, it remained unknown how ABA activated these two NADPH oxidases. The ost1 mutant was observed to lack detectable H2O2 rise in the guard cells after ABA stimulation (Mustilli et al., 2002). This kinase has now been shown to activate H2O2 production by direct phosphorylation of AtrbohF (Ser13, Ser174; Sirichandra et al., 2009b; Fig. 1). Another H2O2 production pathway exists, independent of the core complex, but that requires phospholipase Da1 and phosphatidic acid (Zhang et al., 2008). Using Xenopus oocyte as the main functional assay system, OST1 has also been shown to be one of the kinases which directly phosphorylate (Thr303) and inactivate KAT1 (Sato et al., 2009; Fig. 1). However, the anion channel SLAC1 is activated by OST1 (Geiger et al., 2009; Lee et al., 2009) as well as the Ca2þ-dependent CPK21 (Geiger et al., 2010), while the channel’s inactivation is mediated by direct interaction with PP2CA (Lee et al., 2009), and indirectly by this same PP2C and ABI1 via interactions with OST1 (Geiger et al., 2009). Note that the physical interaction of these PP2Cs with their targets has not been directly shown to be dephosphorylation step, as this is only suggested by the coexpression of these components in Xenopus. H2O2 is an intriguing choice for a signalling molecule because it is induced by a variety of stresses and, being a reactive free-radical, can inflict severe cell damage if uncontrolled. The H2O2 signal must therefore be tamed if it were to serve as a signalling molecule in order to activate the proper physiological responses, especially the coreceptor ABA complex with ABI1/ABI2 (see below). Excess free radicals are, at least in part, removed by scavenger enzymes such as superoxide dismutase, catalase, peroxidases and enzymes involved in the ascorbate–glutathione cycle (Noctor and Foyer, 1998). It has been shown that glutathione peroxidase3 (ATGPX3) can interact with ABI2 and to a lesser extent, ABI1 (Miao et al., 2006). The redox state of both ATGPX3 and ABI2 was found to be regulated by H2O2. The in vitro phosphatase activity of ABI2 was reduced by fivefold in the presence of added ATGPX3. The reduced form of ABI2 was converted to the oxidised form by the addition of oxidised ATGPX3, suggesting that this latter protein plays a dual role in mediating ABA and oxidative signalling by relaying H2O2. Consistent with this is that T-DNA mutants of GLUTATHIONE

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PEROXIDASE3 show higher rate of water loss under drought, higher sensitivity to H2O2 treatment during seed germination and subsequent early development. The mutant also produces higher amount of H2O2 in the guard cells. In contrast, lines overexpressing AtGPX3 were less sensitive to drought stress than the wild type and displayed lesser transpirational water loss. The Atgpx3 mutation also disrupts ABA activation of Ca2þ channels and expression of ABA-/stress-responsive genes. Thus, besides being sequestered by PYR during ABA signalling, ABI2 (and perhaps ABI1 to a lesser extent) could also be inactivated by the oxidised ATGPX3. In fact, both ABI1 and ABI2 are sensitive to inactivation directly by H2O2 without ATGPX3, as demonstrated in vitro (Meinhard and Grill, 2001; Meinhard et al., 2002). So, both PP2Cs could also be direct targets of H2O2 in vivo.

VIII. TARGETS OF SnRK2s IN MEDIUM-TERM ABA RESPONSES-GENE EXPRESSION AND CHROMATIN MODELLING It has been estimated that about 5% of the plant transcriptome is under the influence of ABA. The direct class of downstream target genes is probably represented by those controlled by the characteristic ABA-responsive element (ABRE) motifs in the promoters (see below), which are binding sites of the b-ZIP class of transcription factors. In turn, some of these b-ZIP proteins are also in vivo substrates of SnRK2. A fragment corresponding to ABF2/ AREB1 (ABA Response Element Binding Factor) had been indeed used successfully in the reconstitution of a functional ABA core signalling complex in vitro (Fujii et al., 2009). In fact, a b-ZIP transcription factor (TaABF) was the first plant target identified for this kind of kinases in a yeast twohybrid screen by using the wheat homolog PKABA1 as the bait (Johnson et al., 2002). Further, PKABA1 was able to phosphorylate in vitro, among six test peptides derived from TaABF, one was described as relatively efficient (containing the sequence RMIKNRESAARSRARK). Independent work in rice cell cultures also showed that three representative members of the SnRK2 family, SAPK8, 9 and 10, were particularly effective in mediating expression of reporter genes containing the motif ABREs, which are recognition sites of b-ZIP proteins (Kobayashi et al., 2005). When SAPK10 was used as the model kinase, it was shown that it can indeed phosphorylate in vitro peptide fragments derived from the b-ZIP transcription factor TRAB1 and coimmunoprecipitated from rice cells transiently expressing the two proteins. Mapping the phosphorylation sites by MALDI-TOF mass spectrometry revealed two phosphoserines in a peptide with a sequence

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composition (RGQGSLTLPRTLSVKTVDEVW) that differs significantly from that for PKABA1 above. Detailed investigations in Arabidopsis using peptides derived from several shared domains in b-ZIP proteins (AREB1/ABF2, AREB2/ABF4 and ABF3) and test SnRK2s (SnRK2.2/SRK2D, SnRK2.3/SRK2I, SnRK2.6/OST1/ SRK2E, SnRK2.7/SRK2F and SnRK2.8/SRK2C). These studies allowed the deduction of phosphorylated (S/T) in a ‘‘consensus’’ motif R 3–X 2–X 1– (S/T) in the ABFs, which also first revealed the highly conserved arginine residue at the  3 position (Furihata et al., 2006). Phosphorylation at these particular Ser or Thr in the b-ZIP proteins was proven important for their function, as their replacement to Ala compromised severely their capacity to transactivate a reporter gene. Using SnRK2.8/SRK2C as a representative SnRK2 to phosphorylate in vitro
200 pools of semidegenerate peptides that represented 1012 unique sequences and then quantifying the influence by the neighbouring amino acids on phosphorylation of a fixed Ser or Thr, it was confirmed that Arg is indeed preferred at the  3 position, and further, this method also predicted that SnRK2s may have a general selectivity for hydrophobic amino acids (LIMVF) at the  5 position, in particularly Leu (Vlad et al., 2008). Indeed, there is a preponderance of Arg at  3 and Leu at  5 relatively to the phosphorylated serine in the peptides derived from AREBs/ ABFs that supported phosphorylation by the SnRK2s (Furihata et al., 2006). Promoter analyses carried out on a number of ABA-responsive genes led to the identification of a conserved motif, named the ABRE (with the consensus PyACGTGG/TC) enriched in their presumptive promoters. The core ACGTG also resembles the G-box (CACGTG) prevalent in promoters of genes whose expression is sensitive to light. As mentioned above, the ABRE is the binding site of b-ZIP transcription factors, some of which are targets of SnRK2s. Three of the b-ZIP transcription factors—AREB1, AREB2 and ABF3—together seem to assume a particular important status in the adaptive responses to high salinity, drought and exogenously applied ABA. As compared to the wild type, or to lower order mutants, the triple mutant disrupted in these genes was found to be highly resistant to inhibitory concentrations (up to
50 mM) of exogenous ABA in seed germination tests and root growth (Yoshida et al., 2010). More than 80% of the downregulated genes in the triple mutant contain two or more copies of ABRE in their promoters, consistent with the idea that these three AREBs control a major portion of the transcriptome triggered by water stress. It is striking though the transpiration remained nearly normal in this triple mutant, suggesting that there are as yet additional b-ZIP transcription factors (or other unrelated proteins) that regulate the guard cell responses to drought and ABA transcriptionally. When coexpressed in the same cell, these three b-ZIP

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transcription can form heterodimers and homodimers. Whether this might be the case as well in intact plants is not known, since whether all three proteins are simultaneously expressed in time and in space has not been investigated. Many of the downstream genes transcribed either directly, or perhaps indirectly, by these AREBs encode dehydrins, whose appearance has been known to coincide with water deficit. It should be noted that, besides being unstructured proteins, the diverse biochemical functions of these dehydrins in protecting the cells against water penury are still not fully elucidated.

IX. EPIGENETICS IN ABA REGULATION In addition to changes in specific genes brought about by interaction between b-ZIP transcription factors and promoters bearing target binding sites, it appears that ABA also mediate large-scale changes in gene expression through modification of chromatin changes. Epigenetics was coined by C. H. Waddington in 1942—prior to any knowledge concerning the physical nature of the gene and its role in heredity—as a conceptual model of how genes might interact with their surroundings to produce a phenotype. ‘‘Epi-’’ in epigenetics implies ‘‘above’’ genetics; thus epigenetic traits are addressing regulatory mechanisms that are more than those explainable by the traditional Mendelian basis of inheritance. The definition of epigenetics has evolved over time, and the modern usage now refers to heritable traits, stable over cell division and generations that do not entail changes in the DNA sequences. In molecular parlance, these epigenetic modifications are equivalent to chromosomal marks or imprinting, including cytosine 50 methylation, posttranslational modification of histones (generating the so-called histone code) and changes in compositions or positions of chromatin complexes (nonhistone proteins) along the DNA in response to some environmental or developmental signal. There are suggestions that modifying chromatin structure may serve to impose a sort of ‘‘stress memory’’ in plants (Chinnusamy and Zhu, 2009). A histone H1 variant induced by drought through an ABA-dependent pathway has been reported for tomato, as transgenic plants expressing an antisense directed at this histone variant resulted in higher stomatal conductance (Scippa et al., 2004). Some of the proteins with homologies to the polycomb group (PcG), whose role is to condense chromatin as a means to coordinately repress genes within a chromosomal region, are responsive to ABA in barley (Kapazoglou et al., 2010). As well, in Arabidopsis, the histone deacetylase HDA19 is associated with a chromatin complex that is required for suppressing stomatal conductance and ABA sensitivity (Song et al., 2005). ABA also represses the expression of the

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histone deacetylase gene AtHD2C (Sridha and Wu, 2006), the overexpression of which was shown to result in lower stomatal conductance. These results imply that chromatin modification underpins even the so-called rapid drought adaptive responses that include stomatal regulation. Further evidence for histone deacetylase in ABA and abiotic response emerged from the identification of the hos15 mutation (Zhu et al., 2008), whose affected protein shows similarities to the human WD-40 repeat TRANSDUCIN BETALIKE PROTEIN-1 (TBL1), which is a component of the chromatin repressor complex in histone deacetylation. HOS15 is induced not only by ABA, but also by cold and high salinity, and the protein is associated with histone H4. It might thus be possible that HOS15 modulates ABA and other abiotic stress responses by H4 deacetylation-dependent chromatin remodelling. The Arabidopsis PP2C, HAB1, besides functioning as a suppressor of SnRK2 activity to abrogate ABA transmission, may also directly regulate large-scale gene expression by modifying chromatin structure or nucleosome positioning. HAB1 interacts with SWI3B, an A. thaliana homolog of the yeast SWI3 subunit of SWI/SNF (SWITCH/SUGAR NONFERMENTING) chromatin-remodelling complexes (Saez et al., 2006). Based on BiFC and immunoprecipitation assays, this interaction is confirmed to take place in the nucleus of plant cells. This nuclear-confined action of HAB1 (representing less than 10% of the total HAB1) is reminiscent of another PP2C, ABI1, which has also been described to exert its negative regulatory effects in the nucleus, even for the socalled rapid plasma membrane transport events associated with stomatal closure, although the reason for this is not clear (Moes et al., 2008). In this respect, ABI1 (ABI2, PP2CA/AHG3) can also interact with SWI3B in plant cells (Saez et al., 2006) and suggests that ABA may reprogram gene expression by epigenetic modification. In the absence of ABA, HAB1 is enriched in the vicinity of ABRE and TATA elements upstream of classical ABA-inducible marker genes such as RAB18 and RD29B, but it is then evicted from these chromatin regions during ABA response. SWI3B also interacts with FCA (Sarnowski et al., 2005), suggesting a molecular link between stress, ABA and flowering time through the remodelling of an SWI/SNF complex.

X. MITOGEN-ACTIVATED PROTEIN KINASES IN ABA SIGNALLING Mitogen-activated protein kinases (MAPKs) have been described to be components in many signalling pathways mediating abiotic and biotic stress responses as well as during normal development such as cell division. Indeed, activities of MAPK associated with hormone actions, pathogen invasion, or

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harsh environmental treatments have often been reported. The most studied in Arabidopsis are MPK3, MPK4 and MPK6 which respond to myriads of stimuli. In particular, disruption of MPK6 also impaired downstream H2O2 production stimulated by ABA (Xing et al., 2008). Moreover, both ABA- and H2O2-induced stomatal closure was severely reduced in the presence of the MAPKK inhibitor PD98059 suggesting important functions for these kinases in reducing transpiration (Jammes et al., 2009). In silico examination of gene expression data revealed that the transcripts of MPK9 and MPK12 are highly enriched in guard cell protoplasts than those derived from mesophyll. The double, but not each of the single, TILLING mutants of these two MAPKs was impaired in stomatal closure induced by ABA, cold and H2O2 (Jammes et al., 2009). The activities of both kinases, as ascertained by immunoprecipitation and in vitro tests, were found to be enhanced by ABA and H2O2. Further, studies by electrophysiology found that in the double mutant, the anion channels are refractory to activation by either ABA or by Ca2þ. These two MAPK are likely functionally redundant, most likely downstream of the Ca2þ signal, as expression of MPK12 (fused to the epitopes, YFP and HA) alone was able to rescue the phenotypes of the double mutant. In animals, MAPKs can be deactivated by both dual-specificity and type 2C protein phosphatases. Although there is evidence that certain dual-specificity protein phosphatases may control ABA sensing (Monroe-Augustus et al., 2003; Quettier et al., 2006), the more solid evidence of MAPK inactivation in plant has come from the studies of PP2C. The plant PP2C shown to negatively regulate MAPK was the alfalfa MP2C, which directly interacts with the salt-stress-inducible SIMK and inactivates it by dephosphorylation of the Thr in the conserved pTEpY motif (Meskiene et al., 1998,2003). Similarly, the Arabidopsis AP2C1, which is the closest MP2C homolog, was also shown to interact with and dephosphorylate MPK4 and MPK6 (Schweighofer et al., 2007). These two MAPKs as well as MPK3 are also negatively regulated by a nuclear-localised phosphatase PP2C5, phylogenetically belonging to clade B (Brock et al., 2010). Depletion of PP2C5 and its closest homolog AP2C1 results in plants with increased stomatal aperture, partial ABA sensitivity during seed germination and reduced responsiveness of some ABA-inducible genes after ABA application (Brock et al., 2010).

XI. ROOT GROWTH IN RESPONSE TO ENVIRONMENT ‘‘Can science feed the world?’’ printed on the front cover of the leading journal Nature in the 29 July 2010 issue. Whether this is an open question or rather a desperate plea is hard to tell, but it is no doubt a blunt reminder of

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the momentous achievement by the Green Revolution between 1940 and 1970, and for which the founder member Norman Borlaug was recognised by the Nobel prize. One of the current efforts to increase yield without causing environmental damage is to enhance root development. Root delivers nutrients and water, two of the most essential and often limiting factors that a plant needs. Designer roots could thus be part of the future strategy to diminish fertiliser and/or water input while still maintaining optimal output. Obviously, this strategy, if successful, would also tender a plausible solution to avoid encroaching increasingly on more land to feed the growing world population. But we need to first get down to the basics with respect to the regulatory mechanisms of how a root normally develops, with its attendant complex tissue specialisation and how a root sense environmental stresses such as drought? Finally, how are the genetic programme and environmental variations integrated? Previous physiological studies have provided evidence that ABA might be required for root growth, even in conditions of the environmental constraints (Sharp et al., 2000; Spollen et al., 2000). There are other corroborating, but indirect, evidence such as high concentrations of ABA in the columella cells and the quiescent centre (Christmann et al., 2005). Root is also the active site of ABA biosynthesis and precursor conversion. The biosynthetic genes AtNCED2 and AtNCED3 are expressed in the pericycle at the site of lateral root initiation (Tan et al., 2003). The ABSCISIC ALDEHYDE OXIDASE (AAO)3 gene is highly expressed in root tips and vascular bundles and ABA2 is detectable in the branching points of lateral and mature roots (Cheng et al., 2002; Koiwai et al., 2004; Tan et al., 2003). Mutations affecting root patterning and development exist (e.g., cobra, werewolf, myb23, caprice, tryptychron), but exploiting them to understanding the link between development and environmental constraints has been limited. Roots, being underground, create more of a technical challenge for agricultural researchers to observe their growth patterns in their natural milieu. Their inherent growth flexibility could also confound the relative contributions from the tremendous genetic variations and the heterogeneity of the soil environment with a given phenotypic criterion or a physiological response. Nevertheless, when osmotic stress treatment goes on for extended period of time, notable changes in root architecture occurs. There had been a couple of reports describing bulbous and shortened root hairs in drought conditions that implicate the role of hormones, particularly auxin, GA and ABA (Schnall and Quatrano, 1992; Vartanian et al., 1994). Root hair development is also transiently inhibited by salt stress, but in time-course experiments, it was noted that development resumed after a few hours, suggesting some sort of physiological adaptation. This is correlated with fluctuation of

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certain hair-cell developmental marker genes and many of the repressed genes encode structural components of the cell wall, or in tricoblast differentiation (Dinneny et al., 2008; see below). It has also been observed that when plants were treated in mannitol (50–75 mM), lateral roots did not develop or delayed (Xiong et al., 2006). Lateral root growth is also very sensitive to inhibition by ABA. All ABAdeficient mutants have more lateral roots under normal conditions, but these same mutants are also less sensitive to the inhibition by mannitol. A forward mutant screen was thus conducted using inhibition of lateral root development as a typical response to mannitol. This screen has identified a mutant, dig3, that showed lateral root growth in experimental drought conditions, although the nature of the gene product is not yet known. The slight increase in lateral root primordia in slight decrease in osmotic potential in an artificial medium has also been exploited to identify another mutant, lateral root development(lrd)2, which displays a constitutive increased lateral root system in the repressive osmotic condition as well as in the absence of stress (Deak and Malamy, 2005). Again the nature of the LRD2 gene product is not yet known. In addition to the above mutant screens, recent whole-genome technologies have provided a glimpse of some of the molecular aspects of root adaptability to stress. A high-resolution spatial expression atlas of stressinduced genes in various Arabidopsis root tissues was constructed by using whole-genome RNA arrays (Dinneny et al., 2008). Radial patterns of gene expression maps were generated by GFP-reporters expressed in particular cell layers whereas longitudinal sections of the roots were used as a proxy of different developmental stages. These GFP-labelled cells were recovered by fluorescence activated cell-sorting and the RNAs were profiled by Affymetrix gene-chips. These whole-genome results revealed that, along the longitudinal axis, an increase in salt responsiveness was found in those cells at the elongation zone. This suggests that cells that are the most developmentally competent to respond to high salinity are those undergoing differentiation. In examining the radial pattern of gene expression in response to high salt, many of the induced genes are found to express in only one cell layer. This suggests that there is both developmental and tissue-specific competence (or constraints) in conferring salt responsiveness. In contrast, genes responding to ABA are not cell-type specific. In those genes that were induced by salt along the longitudinal zones, their corresponding promoters were enriched by many known cis-regulatory elements such as drought-responsive elements (DRE) and ABRE. These cis-elements were also found in genes that are classified as semiubiquitous based on the fact that they are expressed in at least three radial zones. Thus, although canonical stress-responsive pathways

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appear to be active in all cell layers, the authors argued that cell-type-specific responses are distinguishable at the promoter level and probably controlled by other cis-elements. ABA insensitive mutants are also partly resistant to salt (Achard et al., 2006), and the affected ABA-regulated genes are also saltresponsive in all cell layers of the root. This apparent widespread activity has been taken to mean that ABA might primarily mediate semiubiquitous transcriptional responses to salt. This picture is certainly more complex. In ABA-deficient mutants, salt-induced expression is diminished for many ABA-responsive cell-type-specific markers. This would also indicate that ABA regulates cell-type-specific responses to salt stress in a manner independent of characterised ABRE.

XII. ABA IS CONSERVED IN EVOLUTION AND HAS POTENTIAL TO IMPROVE HUMAN HEALTH Laying down the basic knowledge concerning the mechanisms of ABA action has obvious application in the field to enhance plant resilience to a variety of environmental constraints. For interested readers, there are many excellent reviews and primary publications that point in this direction, especially in view of climate change (Schroeder et al., 2001; Wang et al., 2005; Zhang et al., 2004). There are, however, tantalising and less-publicised advances in the medical field in using ABA as a experimental treatment for certain ailments. This comes in the wake of our increasing awareness of the general benefits of stress-protective compounds derived from plants for human health, a concept known as ‘‘xenohormesis’’ (Hooper et al., 2010). As we have said in the opening to this book chapter, biological sciences are inextricably tied to the theory of Evolution, which embodies the continuity in all life forms as its unifying concept. For the molecular biologists, this concept of continuity is inscribed by the modular nature of protein structures, and these composite functional domains can be found rethreaded together in myriads of different combinations across plant and animal kingdoms. Learning the potential function of an unknown gene from one organism by comparing its sequence against public data bases by ‘‘blast’’ is reaffirming faith in evolution. Despite all our open-mindedness, it still comes as a scientific cultural shock that this continuity actually goes beyond genes, but encompasses ABA, which also implies that large portions of pathways of biosynthesis and signal transduction must be somehow conserved or reinvented by convergent evolution. ABA was first isolated from plants in the 1960s as a substance that can induce bud dormancy and fruit abscission, and thus has logically inherited

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the designation of phytohormone. However, this tacit acceptance has been jolted by its rediscovery in marine sponges, fresh-water hydra, parasites, and even in humans (Wasilewska et al., 2008). Although the pathways by which ABA is synthesised in organisms other than plants is not known, its discovery in animals has naturally sparked interests in the possible parallels between its role in plant and animal stress response systems. In sea sponges, ABA acts as a trigger to increase water filtration to cool the body in response to high sea temperature (Zocchi et al., 2001). This requires cyclic ADP-ribose as the second messenger, and thus the stimulus is thought to pass through Ca2þ release by means of ryanodine receptors in the sarcoplasmic reticulum. The ABA–cyclic ADP-ribose–Ca2þ connection reappears in Toxoplasma gondii (Nagamune et al., 2007), a worldwide parasitic protozoan (found in faeces in domestic animals such as cats) that causes congenital retinitis and brain damage. T. gondii can remain dormant in the cystic form throughout the host’s lifespan, but can be reactivated to lytic growth (egress) in immunocompromised hosts, such as patients suffering from AIDS. A series of elegant experiments conducted by Sibley and colleagues (Nagamune et al., 2007) have helped to track down the origin of the ABA signal. Stress causes ABA synthesis in the apicoplast, a remnant organelle of an algal endosymbiont. The presence of ABA was confirmed by high-performance liquid and gas chromatography, conjugated to mass spectrometry. In a cell culture model, the addition of the classical ABA synthesis inhibitor fluoridone can block specifically egress of the pathogen, but not its replication and host invasion. Moreover, exogenous reapplication of ABA rescues egress, by Ca2þ release through cADP ribose to promote secretion of the marker parasite adhesion MIC2 (microneme protein2). Importantly, in whole animal studies, fluridone inhibition of the ABA-dependent lytic parasite cycle protected mice against toxoplasmosis. In humans, ABA was first detected in the brain (Le Page-Degivry et al., 1986), then rediscovered in granulocytes (Bruzzone et al., 2007), in pancreatic islets (Bruzzone et al., 2008), in monocytes (Magnone et al., 2009) and in the plasma of mice fed with an AIN-93-G-based rodent diet (Bassaganya-Riera et al., 2010). The biosynthetic origins of ABA in mammals have still to be worked out, but it has hormone-like effects as nanomolar concentrations are sufficient to modulate the immune system (Magnone et al., 2009). The value of the public Arabidopsis resources for these new medical investigations are unquestionable, as they provided leads on over 1000 human orthologs that are related to those implicated in ABA signalling (Bassaganya-Riera et al., 2010). By predicting interactions among all proteins by informatics treatment, combined with available data on tissue-specific expression of these proteins, four human homologs of plant ABA-related genes that would anchor in a peroxisome proliferator–activator receptor g (PPARg) network were

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identified. PPARg is the target of the popular though controversial insulinsensitising drugs, thiazolidinediones (TZDs) for the treatment of type II diabetes. Based on studies using cell lines (3T3-L1 preadipocytes), ABA was shown to activate the expression of PPARg and that ABA can prevent the onset of type II diabetes in obese mice (Guri et al., 2007). Importantly, the antidiabetic effect of ABA, based on glucose homeostasis and macrophage infiltration, was reduced in mice deleted for PPARg, thereby providing in vivo genetic data that PPARg is a key player in the mechanisms of ABA antidiabetic action. There is no evidence that PPARg is the direct-binding site for ABA, however. Binding of ABA to the cell surface was observed in granulocytes, monocytes and aortic smooth muscle cells (Magnone et al., 2009; Sturla et al., 2009). In human granulocytes, ABA-induced responses including Ca2þ rise and ROS production were observed to require LANCL2, a plasma membrane-localised protein through N-terminal myristoylation with homology to the prokaryotic lanthionine synthetase (Sturla et al., 2009). Moreover, transfection of a transgene encoding LANCL2 into HeLa cells created ABA-binding sites (Sturla et al., 2009). It should be emphasised that, in contrast, a previous report for a cell-surface ABA receptor with homology to lanthionine synthetase (named GCR2) in Arabidopsis has remained controversial (Gao et al., 2007; Illingworth et al., 2008), and there is no evidence for direct ABA binding to LANCL2. In human monocytes, ABA induces nuclear translocation of NF-kB which regulates the expression of several inflammatory proteins (Magnone et al., 2009). Remarkably, ABA acts as a chemoattractant for granulocytes (Sturla et al., 2009), as well as monocyte or aortic smooth muscle cell migration and positively stimulate MCP-1 (believed to be the primary chemoattractant for monocytes to the antherosclerotic plaque) secretion from monocyte to constitute a positive feedback loop; these cellular behaviour forms part of the important repertoire for the development of antherosclerotic lesions. Both this study with human monocytes and those using the mouse model (Guri et al., 2007) converge on the potency of ABA as an immune modulator.

XIII. CONCLUDING REMARKS Transpiration and photosynthesis are intrinsically linked in gas-exchange processes. Biomass accumulation requires light interception by leaves and stomatal opening (Tardieu, 2003). Light, especially in the blue spectral range, stimulates stomatal opening by hyperpolarisation of the plasma membrane. In contrast, drought and high CO2 stimulate stomatal closing. As the stomatal pore is the only channel allowing communications between plant and

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environment, the trade-off of these two processes is critical for biomass, with important implications for agricultural productivity in view of the climate change. Within the past 2 years, one of the core complexes in ABA signalling has been solved. It consists of three essential components: the soluble receptor, a PP2C and an SnRK2. The binding of ABA promotes sequestration of the PP2C by the receptor, thereby releasing the SnRK2 to phosphorylate downstream targets. Because each of the above components are members of rather large families, their combinatorial actions could fine-tune downstream signalling intensity, although it remains unclear how this is related to the positive role played by low concentrations of ABA in plant development. This is also the second core signal complex that begins with the unusual receptor of
120 kDa resident in the chloroplast membranes, ABAR (GUN5), which was first identified as an essential component in retrograde signalling between the nucleus and chloroplast. It turned out that once bound to ABA, ABAR then becomes competent to interact with several nuclear repressors of the WRKY family, presumably to relieve transcriptional repression of target genes. Although the PYR and the ABAR pathways affect some common reporter genes, it will be exciting to see how they are coordinated (cross-talk) to control the repertoire of ABA responses. Electrophysiological and cell biological approaches have also indicated the presence of cell-surface ABA receptors, and two of these (GTG1 and GTG2) with homology to chloride channels in mammalian cells have been proposed as candidates. However, their affinity to ABA has been questioned (Risk et al., 2009). The genome of Arabidopsis has 129 annotated ABC transporters (Verrier et al., 2008). At least two have been assigned functions in ABA export (AtABCG25) and import (AtABCG40). The genetic and physiological evidence available suggests that they modulate the cellular content of ABA, and hence, perhaps the intensity of the ABA response (much like the combination of soluble PYR receptor and the different PP2Cs). With the discovery of ABAR, one could expect ABA transporters in the chloroplast envelope as well. AtABCB14 and SLAC1 are an interesting combination, because they have counter directions in malate transport, as would be expected, for example, when stomatas are exposed transiently to high atmospheric CO2. The long-distance transport of ABA, and whether this also involves active mechanisms, is unclear. As some of the ABA biosynthetic genes are strongly expressed in cells juxtaposing the vascular tissues these cells might represent sites of maturation and delivery of ABA to the rest of the plant. The SLAC1 and the potassium inward-rectifying channel KAT1 are direct targets of OST1. This same kinase is able to phosphorylate members of the b-ZIP transcriptional activators which control a substantial portion of the ABA

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transcriptome. Both lines of evidence indicate that OST1 (or the AAPK homolog of Vicia) is a key element in this core signalling pathway. Three b-ZIP transcription factors in Arabidopsis seem to control up to 80% of the ABA-inducible transcriptome. These proteins can form heterodimers, at least if they happen to be coexpressed in the same cell, but whether this is accompanied by changes in binding affinity to target promoters as another level of fine-tuning regulatory mechanism is not known. Another key regulator in the core ABA complex is the PP2C (represented by members ABI1, ABI2, HAB1, PP2CA, etc.) that acts as a coreceptor of ABA along with PYR, and the phosphatase is also the upstream suppressor of the OST1 kinase. ABI2 and, to a lesser extent, ABI1 are also rapidly inactivated by H2O2, which can be produced by OST1 phosphorylation of AtrbohF, which altogether, suggests a mechanism of mutual regulation between these two proteins. Moreover, HAB1 has a role in chromatin modification by binding to SWI3 that predicts alteration of the ABA transcriptome by epigenetics, besides those genes directly regulated by ABRE-binding transcriptional activators. The mRNA levels of ABI1, ABI2 and PYR/PYL/RCAR are also either induced or repressed by ABA treatments, but the current available data are somewhat contradictory, probably of the cryptic influence of the different experimental conditions (Shang et al., 2010; Szostkiewicz et al., 2010). It will be fascinating to see the extension of the nascent ABAR signalling pathway, as it implicates completely unexpected elements so far. Even the recognition and binding to ABA by ABAR seem to involve distinct mechanisms than those of the PYR. For example, the active group of ABA that is buried deeply within PYR was actually used to chemically couple ABA to an affinity column used to purify the 42-kDa protein from Vicia and ABAR from Arabidopsis. This predicts that ABAR will recognise a different part of the ABA molecule. The mechanisms of delivering WRKY from the nucleus to ABAR in the chloroplast are not known. Another obvious question, no less fascinating, will be potential points of cross-talk between ABAR and PYR to coordinate downstream events. One of the connecting point to ABAR could be somehow through the APETELA-related nuclear transcription factor ABI4, which has been shown to work downstream of the chloroplast protein GUN1 in retrograde signalling between the chloroplast and the nucleus (Koussevitzky et al., 2007). Root response to drought (and high salinity) remains a difficult subject for forward genetic approach. Roots alter their growth patterns when exposed to prolonged dehydration. Although there are several mutants with altered root development, how the activities of their corresponding genes might be linked to environmental constraints are not yet clear. The high-resolution transcriptomic atlas that recorded gene activities along the longitudinal and radial

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axes, indicate very complex and intertwined gene networks that are influenced by salt, ABA, developmental and tissue-specificity signals. In general, genes responding to exogenous ABA are not confined to any cell layers, nor to particular developmental stages, but are widespread. The most astounding findings in the last years is the rediscovery of ABA in a wide range of nonplant organisms that span several kingdoms. Thus, the existence of ABA is probably universal. The ability of fluridone to inhibit egress of T. gondii may open up development of novel antiparasitic compounds with minimal side effects on mammalian hosts. Extending this logic, if the entire pathway is worked out, from ABA perception to secretion of the microneme proteins, a wider range of targets will be available to combat this type of parasitic infections. However, results in studies using human monocytes (Magnone et al., 2009) contradict somewhat the interpretation of ABA as an anti-inflammatory agent in stromal vascular cells extracted from murine white adipose tissue, in which MCP-1 expression was inhibited instead (Guri et al., 2008). In both of these cases, the results converge on the fact that ABA is produced in mammals, and acts a powerful modulator of the immune system. Not surprisingly, some of the cellular components that are important for ABA signal transduction in plant have homologs in humans, but it is too early to pronounce whether they assume the equivalent functional roles.

ACKNOWLEDGEMENTS A. J. S. is supported by a postdoctoral fellowship from the French Agence Nationale de la Recherche ANR-08-BLAN-0123-01. JL and CV are grateful for the support from the Centre National de la Recherche Scientifique. JL thanks Dr. Philip Hooper (MD) at the University of Colorado, USA for the inspiring exchanges, reprints, preprints, and the philosophical waxing on xenohormesis.

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