An Update on Abscisic Acid Signaling in Plants and More …

Molecular Plant

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Volume 1

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Number 2

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Pages 198–217

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March 2008

REVIEW ARTICLE

An Update on Abscisic Acid Signaling in Plants and More . . . Aleksandra Wasilewskaa, Florina Vlada, Caroline Sirichandr aa, Yulia Redkob, Fabien Jammesc, _ Frei dit Frey _ a and Jeffrey Leunga,1_ Christiane Valona, Nicolas

ABSTRACT The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a ß-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H1-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.

INTRODUCTION Abscisic acid (ABA) was first discovered in the 1960s, initially under the names of either dormin or abscissin in young cotton fruits and sycamore leaves. Since then, this hormone has been found in a great number of plant species and appears to be of universal occurrence among vascular plants and mosses. The most documented role of ABA is in seed maturation processes, acquisition of desiccation tolerance and dormancy. All of these traits in seed development have important applications for

crop species due to the widespread problem of pre-harvest sprouting (the germination of the physiologically mature 1 To whom correspondence should be addressed. E-mail Leung@isv. cnrs-gif.fr, fax 01 69 82 36 95. These authors contributed equally to this work.

ª The Author 2008. Published by Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssm022, Advance Access publication 14 January 2008

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a Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, 1 Avenue de la Terrasse, Baˆt. 23, 91190 Gif-sur-Yvette, France b CNRS UPR9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France c Laboratoire Signalisation et Re´gulation Coordonne´e du Me´tabolisme Carbone´ et Azote´, Institut de Biotechnologie des Plantes (UMR8618), Universite´ ParisSud, F-91405 Orsay Cedex, France

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Newer findings now suggest the possible existence of a novel ABA-signaling pathway regulating lateral root development— a role that has been traditionally attributed to auxin. The most remarkable discovery, however, is that ABA has been detected unequivocally in several metazoans, representing on the evolution scale the most primitive to the most advanced (i.e. sponges and humans, respectively). Surprisingly, ABA has hormonal properties in animals as well, and there is a close parallel between the ABA signaling mechanism in plants and in human granulocytes. These cross-kingdom comparisons have brought to light that the mechanisms of ABA signaling may have an ancient origin.

A SHORT PRIMER ON ABA BIOSYNTHESIS, RELEASE, AND CIRCULATION IN PLANTS The biosynthetic pathway of ABA in higher plants has been largely delineated (Nambara and Marion-Poll, 2005; MarionPoll and Leung, 2006). Arabidopsis as a genetically tractable plant model has contributed much to the clarification and identification of the main enzymes of the catabolic pathway. Localizing the tissue expression of the corresponding genes should soon provide a precise description of the dynamics of ABA synthesis, transport of the intermediates, and the sites of its action during the entire lifecycle of the plant. Briefly, the early precursor of ABA—isopentenyl diphosphate—is synthesized primarily in plastids from glyceraldehyde 3-phosphate and pyruvate via the methylerythritol phosphate pathway. This leads to the successive production of phytoene and lycopene as the intermediates, the latter of which is cyclized and hydroxylated to form zeaxanthin, the first oxygenated carotenoid (Figure 1). The subsequent conversion of zeaxanthin to either violaxanthin or neoxanthin also occurs in the plastid. The enzymatic step that catalyzes the all trans-violaxanthin to the all trans-neoxanthin is the latest to be solved by positional cloning of the ABA4 gene from Arabidopsis (North et al., 2007). The predicted neoxanthin synthase—with an estimated molecular weight of 17 KDa and highly conserved among plant species—had indeed been identified previously in the proteome of the chloroplast envelope membrane (Ferro et al., 2003), which is the same as the subcellular compartment of its substrate violaxanthin and product neoxanthin. As the in vitro catalytic activity has not been successfully demonstrated with the recombinant form of ABA4 expressed in E. coli, the authors have suggested that the protein may bind the substrate rather than carrying out the catalytic conversion of violaxanthin to neoxanthin (North et al., 2007). Alternatively, the active form of this enzyme may need posttranslational modifications or regulatory subunits absent in the recombinant protein. The protein may be important for the de novo ABA synthesis specifically during dehydration, because the ABA level is low in water-stressed aba4 mutant plants while it is similar to that in wild-type plants in the absence of stress (North et al., 2007). In Arabidopsis, both

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grain on the parent plant prior to harvest) that occurs in many regions of the world. Also, during vegetative growth, ABA is the key hormone that confers tolerance to environmental stresses, most notably drought and high salinity, thereby permitting plants to colonize ecological niches where water availability is limited or sporadic. With water shortage predicted as the most severe environmental problem for the twenty-first century, modifying the biosynthesis and perception of ABA has also attracted attention as potential means to enhance drought resistance in crops (Schroeder et al., 2001; Wang et al., 2005; Shinozaki and Yamaguchi-Shinozaki, 2007). Even though ABA has mostly been studied in the context of stress signaling during vegetative growth, it has also been reported to control certain developmental or physiological functions in non-stress situations. First, plants deficient in ABA continue to display phenotypic abnormalities, even in well watered conditions (Barrero et al., 2005). Second, there have also been reports of ABA being involved in heterophyllous induction in the aquatic fern Marsilea quadrifolia (Lin et al., 2005), sex determination in Canabis sativa (Mohan Ram and Juiswal, 1972), pollination (Kovaleva and Zakharova, 2003), and senescence (Hunter et al., 2004). Lastly, ABA restrains the production of ethylene (Sharp and LeNoble, 2002; Benschop et al., 2007), while ethylene has been reported to induce ABA synthesis (Grossmann and Hansen, 2001; Chiwocha et al., 2005), suggesting a self-maintained negative regulatory loop. Several recent reviews that cover either the biosynthesis (Nambara and Marion-Poll, 2005; Marion-Poll and Leung, 2006) or the physiological and molecular aspects of ABA signal transduction (Finkelstein et al., 2002; Finkelstein and Rock, 2002; Himmelbach et al., 2003; Riera et al., 2005; Roelfsema and Hedrich, 2005; Israelsson et al., 2006; Marion-Poll and Leung, 2006; Shinozaki and Yamaguchi-Shinozaki, 2007) are available for more background details. A perusal of the current literature easily turns up over 100 genes, identified by forward and reverse genetics screens, that are implicated in ABA signaling and new ones are continually being reported at a regular pace. The functions assumed by these predicted proteins are of such vast diversity as to defy a comprehensive concept to explain the molecular basis of ABA signaling. Perhaps such a universal model does not exist because the different cell types in which ABA is active manifest distinct physiological outputs; therefore, an identical signaling circuit common to all plant cells is unlikely. For heuristic purposes, all current models are thus simplistic by necessity. In most cases, the subcellular contexts in which these elements are proposed to interact have not been experimentally verified. This short update will focus on discoveries made largely within the last 3–4 years. As mentioned above, ABA has been up to now associated mostly with adaptive responses to abiotic stress, but recent studies have revealed that the hormone is also active in biotic stress response in a pathosystem-dependent manner. In the context of development, the role of ABA in seed maturation processes has been relatively well charted (Finkelstein et al., 2002; Feurtado and Kermode, 2007) and will not be dealt with here.

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violaxanthin and neoxanthin are alternative in vivo substrates of nine cis-epoxycarotenoid dioxygenase (NCED) to produce xanthoxin—the first cytoplasmic precursor for the catalytic conversion to ABA. In addition to de novo synthesis, ABA can be circulated throughout the plant as an inactive glucose ester conjugate and then released into an active form by apoplastic and endoplasmic reticulum b-glucosidases. This implies that the intensity of the ABA signal at the site of action is not necessarily correlated with the absolute amount of the hormone per se and is governed by the efficiency of enzymatic modification and release. The chemical properties of ABA glucose ester are well suited for its long-distance translocation in the xylem because of the low permeability of biomembranes for this conjugate (Jang and Hartung, 2007). Since the glucose ester form

can be activated by cleavage, this may represent a fast response mechanism that is always poised as a first-line defense against a fluctuating environment. Consistent with this idea is that an Arabidopsis mutant deficient in b-glucosidases, containing lower ABA levels in leaves, was found to display stress-sensitive phenotypes (Lee et al., 2006b). ABA glucose ester is stored in vacuoles or apoplastic space (Dietz et al., 2000), but it is transported by an unknown mechanism to the endoplasmic reticulum in response to dehydration, where it is then cleaved to release active ABA (Lee et al., 2006b). Moreover, biochemical studies showed that water deficit triggered a rapid polymerization of b-glucosidases in microsomes (presumably including membranes from the endoplasmic reticulum) of wild-type Arabidopsis leaves yielding four times higher activity than in unstressed controls. ABA glucose ester

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Figure 1. The Biosynthetic Pathway of ABA. ABA is formed by cleavage of carotenoids, which are derived from five-carbon precursors, isopentenyl diphosphate. Phytoene is the common precursor for all plant carotenoids. The backbone molecule is formed by phytoene synthase after condensation of two molecules of geranylgeranyl diphosphate. Desaturation and isomerization reactions convert this into the all-trans-lycopene, which is then cyclized into b, b-carotene. Further hydroxylation reactions by the b, b-carotene hydroxylase yield zeaxanthin. This figure emphasizes the details on the synthesis from zeaxanthin to ABA. All of the catalytic steps, with the exception of the last two, occur in the plastids. The enzymes and their corresponding plant species in which studies have been carried out are shown next to the catalytic steps. The Arabidopsis ABA1 and the tobacco ortholog encode an epoxidase. The NCED is a dioxygenase that catalyzes the cleavage of cis-xanthophyll and it is the first committed step for ABA synthesis. The Arabidopsis ABA2 gene encodes a short chain dehydrogenase/reductase, and AAO3 encodes an aldehyde oxidase that requires molydenum cofactor for its activity. ABA3 encodes the molydenum cofactor sulfurase that adds the sulfur atom to the molydenum center. The identity of the cis-isomerase(s) is/are presently unknown.

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ABA RECEPTION SITES IN THE NUCLEUS AND CHLOROPLAST ABA reception sites have been detected over a decade ago by physiological means, for instance, by measuring stomatal responses after releasing caged ABA into different cellular compartments (MacRobbie, 1995; Leung and Giraudat, 1998). We learned from these early studies of the existence of perception sites located on the ‘inside’ as well as the ‘outside’ of cells; yet, to this day, the hunt for the corresponding proteins is still very much open. Besides physiological means, ABA analogues provide alternative tools for molecular biologists to probe the nature of the perception sites. These analogues are synthetic chemicals that can act either as agonists or antagonists of the natural ABA [(+)-S-ABA]. An example is (+)-8#-acetylene ABA, which is an irreversible inhibitor of the catabolic enzyme 8#-hydroxylase (Cutler et al., 2000). It is reasoned that since this analogue is more stable, it might be useful to identify genes induced only weakly by natural ABA (Huang et al., 2007). Moreover, the differential sensitivity to analogues has been used as a premise to screen mutants that are altered in their capability to distinguish between ABA isomers (Nambara et al., 2002). No mutations affecting bona fide receptors were identified from such screens, however, even though these ABA analogues presumably exert their physiological effects initially by binding to a receptor. It is noteworthy that all targeted genetic screens based on the classical criterion of altered sensitivity to exogenous ABA that have revealed a plethora of signaling intermediates have so far failed to identify a receptor. There are several possible reasons why such classical genetic screens have failed. Obviously, the omnipresent problem of functionally redundant genes could be one compromising factor, because recessive mutations could likely have been masked. The opposite problem with dominant (and viable) alleles of mutant receptors is that they might be rare. It is equally possible that the ABA receptors have other unexpected functions such that their mutations engender other notable phenotypes besides altering ABA sensitivity. A quick succession of papers have unveiled two candidate receptors based on their ability to bind ABA. FCA—a nuclear RNA-binding protein—has actually long been renowned for its role in the so-called autonomous pathway controlling flowering time (Macknight et al., 1977). The hint that it might be an ABA receptor came from some earlier work by the same group on ABAP1—a 52-kDa plasma membrane-localized protein with a WW-domain from barley (Razem et al., 2004). ABAP1 binds ABA, and a search for homologs in Arabidopsis by BLAST turned up FCA as the closest (our independent observations). The overall homology between ABAP1 and FCA is about 40% (our own comparisons), with most of the conservation limited to the WW domain (reviewed by Finkelstein 2006). ABA binds to an unspecified amino acid segment near the C-terminus of FCA, which disrupts its subsequent interaction, via its WW domain, with FY. The latter

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can therefore be cleaved with much higher rates to release the active ABA from the microsomes to the cytosol. Further release to the apoplast would be facilitated by the pH gradients across the mesophyll plasma membrane to generate an intensified ABA signal at the apoplastic space. The dynamic distribution of ABA in whole plants has been tracked by using two ABA-sensitive promoters (pAtHB6 and pRD29B) fused to reporter genes consisting of either b-glucuronidase or luciferase. In the absence of stress, reporter signal is barely detectable throughout the transgenic Arabidopsis. Nonetheless, above this background, low concentrations of ABA are present in the shoot apical meristem, veins, and hydathode of the cotyledons and in guard cells. In the root, clear reporter activity was restricted to the columella cells and the quiescent center. These localized pools of ABA, which are likely to be biologically active, suggest that the hormone has many subtle developmental roles, independent of stress, that are yet to be discovered (Christmann et al., 2004). Water deprivation at the roots induces reporter expression in guard cells and in the vasculature of leaves prior to detectable signals in the vascular tissues in the roots. The dynamics in the re-distribution of ABA would indicate a leaf-derived source of ABA. Indeed, the expression profiles of several known ABA biosynthetic genes confirm that leaves can synthesize ABA, with the sites of synthesis primarily near or at vascular bundles. However, it is also known that 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; Tan et al., 2003; Koiwai et al., 2004). Expression of these other genes indicates that certain catalytic conversion steps occur in the roots. It is unclear at present whether the promoter–reporter constructs are not sensitive enough to detect ABA in these cells, or the intermediates of these particular conversion steps are not capable of activating the ABA-sensitive promoters used in these studies. However, exogenous ABA application can activate these reporters, indicating that all cells are competent to respond to the hormone (Christmann et al., 2004). The dynamics in the tissue distribution of the reporter activity reveal two intriguing results. First, the current model argues for a translocation of the root-derived ABA to the shoot during soil drying (Jang and Hartung, 2007), which is spatio-temporally opposite to the above observations based on the sequential appearance of reporter activities. It is not clear if the difference is due to the unique characteristics of the plants used in the respective studies, the methods of measurements, or whether the universality of the current model needs to be re-examined. A second and perhaps more tantalizing possibility raised by the studies using ABA-sensitive promoters is that water stress perceived by the roots relays this signal to shoots by an unknown messenger to stimulate ABA accumulation in the leaves.

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by transcriptome analysis (Marquardt et al., 2006). Lastly, there is still the cell-surface receptor to account for the typical physiological responses elicited by externally applied ABA on guard cells (Anderson et al., 1994) and on cell cultures (Jeannette et al., 1999). Although candidates have been proposed, none of these proteins possesses all of the characteristics expected of an authentic receptor.

POST-TRANSCRIPTIONAL REGULATION IN ABA RESPONSES ABA inhibits flowering (Levy and Dean, 1998). At the molecular level, this observation has been explained by a dramatic increase in the level of the FLOWERING LOCUS (FL)C mRNA (Razem et al., 2006; Schroeder and Kuhn, 2006). The gene encodes a MADS-box-type transcription factor that is the central repressor of flowering in Arabidopsis (Michaels and Amasino, 1999; Sheldon et al., 2000; Ba¨urle and Dean, 2006). The fact that FCA can bind ABA has, at least in part, fuelled the motivation for a deeper exploration of the relationship between ABA and flowering. The ABH1 gene, which encodes the large subunit of a heterodimeric nuclear cap-binding protein, has been identified as a mutant with enhanced sensitivity to ABA based on seed germination (Hugouvieux et al., 2001). The abh1 mutant displays early flowering under both shortand long-day regimes (Kuhn et al., 2007), and, moreover, it has also been independently isolated as a suppressor of the flowering mutant frigida (FRI) (Bezerra et al., 2004), suggesting that ABH1 may regulate an important process in flowering time. Examination of the RNA profile in the abh1-7 mutant revealed that of the 19 flowering-time regulators, CONSTANS (CO), FLC and FLM showed significant changes in either their transcript abundance or relative ratios of their splicing intermediates when compared to the wild type. For FLC, the correct splicing of the first intron is disturbed in abh1-7, leading to about an 80% decrease in the wild-type transcript. The decrease in the level of this transcript may explain the early flowering phenotype despite the fact that abh1 is hypersensitive to ABA. There is also evidence based on RT-PCR assays that the FLM (a MADS-transcription factor that is an inhibitor of flowering) is prematurely terminated or degraded in the abh background as well (Kuhn et al., 2007). In addition to ABH1, the disruption of two other genes encoding mRNA cap-binding proteins—CBP80 and CBP20—also leads to altered sensitivity of plants to ABA and stress (Xiong et al., 2001; Papp et al., 2004). It is difficult to reconcile at present how lesions in these general RNA metabolic proteins bring about such precise stress-related phenotypes. Based on our current state of knowledge, de-capped nuclear RNAs are rapidly degraded, and the resulting phenotypes would be expected to be rather pleiotropic, even lethal. This paradox extends to other ‘house-keeping’ RNA metabolic proteins as well. A specific nuclease, encoded by ABA HYPERSENSITIVE GERMINATION (AHG)2, is hypothesized to be necessary for the processing of the poly(A) tail of mRNA (Nishimura et al.,

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is also an RNA-binding protein with homology to the yeast polyadenylation and 3#-end processing factor PsF2p (Simpson et al., 2003). The FCA–FY interaction is required to control the correct splicing of the FCA transcript in a negative feedback loop. The disruption of this interaction by ABA may provide one explanation on how this hormone delays flowering (Levy and Dean, 1998). The mutant fca, however, shows no substantial alteration in most of the classical ABA responses (such as seed germination or transpiration), except at the level of the lateral roots, which are reduced in sensitivity to the inhibitory effect of applied ABA (Razem et al., 2006). It has been speculated that potential phenotypes in these other tissues may have been cloaked by a distantly related FCA homolog in the Arabidopsis genome (reviewed by Schroeder and Kuhn, 2006). Another candidate ABA receptor is the H subunit of the larger trimeric Mg2+-chelatase complex, and this H subunit has been previously shown to bind Mg2+-protoporphyrin in the chloroplast. The CHLH has a homolog in Vicia faba which had been biochemically characterized by the same group as an ABA-binding protein (Zhang et al., 2002). The Arabidopsis counterpart is encoded by the ABAR gene which turned out to be identical to GENOME UNCOUPLED5 (GUN5), discovered earlier as being essential for plastid–nucleus communication, and, more specifically, in the control of chlorophyll synthesis by either the metabolism or sensing of the tetrapyrrole signal Mg2+-protoporphyrin-IX. This GUN5/CHLH subunit has therefore dual functions: binding to Mg2+-protoporphyrin in chlorophyll synthesis and binding to ABA. The physiological consequences of signaling through ABA seem independent of those in chlorophyll synthesis. Treatment with exogenous ABA significantly decreases the amounts of chlorophyll and protoporphyrin but increases Mg2+-chelatase activity and Mg2+-protoporphyrin content, and stimulated ABAR expression. Conversely, knock-down abar mutants have normal content of chlorophyll; the ABA-inducible genes are downregulated, while two homologous negative regulators of ABA signaling—ABA-INSENSITIVE1 (ABI1) and ABI2—are upregulated. Phenotypically, the knock-down abar mutants display reduced sensitivity to ABA in all of the elementary responses including germination, dormany and transpiration; an over-expression of the ABAR gene causes the complementary set of phenotypes. Finally, null mutations at the GUN5/ ABAR locus cause lethality at early embryonic stage due to deficiency in lipid and mature protein bodies, similar to the phenotypic defects observed in abi3. This latter gene encodes a B3domain transcriptional activator that has been extremely well characterized with respect to its role in seed maturation (Parcy et al., 1994). ABAR is thus essential throughout the plant lifecycle and it is expressed in all vegetative tissues of the plant. Besides the FCA and CHLH proteins, there are almost certainly additional intracellular receptors. As we have alluded to above, the fca mutant displays no ABA-related phenotypes in tissues other than lateral roots. This is also correlated with the lack of altered expression in ABA-regulated genes revealed

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the stress-activated promoter derived from rd29A as a reporter have also turned out a wealth of mutants affected in ABA responses as well (Ishitani et al., 1997; Lu and Fedoroff, 2000). Noteworthy is the mutant fiery (fry)2, which is slightly resistant to ABA and salt but exhibits increased sensitivity to cold, is mutated in a protein domain with similarity to RNA polymerase II C-Terminal Domain phosphatase and to DSRM—the prototype double-stranded RNA-binding motif (Xiong et al., 2002). Another double-stranded RNA-binding protein—HYPONASTIC LEAVES (HYL)1, which affects microRNA metabolism—has been implicated in some aspects of ABA signaling as well as other pleiotropic phenotypes (Lu and Fedoroff, 2000; Han et al., 2004; Vazquez et al., 2004). In the next section, we will describe the characterization of the RNA-binding protein AKIP from Vicia faba. With the exception of miRNA159 and AKIP, the mechanistic link between RNA metabolism and ABA signaling remains to be explored.

REVERSIBLE PROTEIN PHOSPHORYLATION: TRANSCRIPTION, TRANSPORTERS, AND THE Ca2+ CONNECTION Pharmacological studies coupled with genetic screens of mutants have converged on reversible protein phosphorylation as an early and central event in ABA signal transduction, at least in the guard cell (Schmidt et al., 1995; Leung et al., 1997; Himmelbach et al., 2003; Sokolovski et al., 2005). This is not too surprising, given the known versatility of phosphorylation as a regulatory mechanism, which is well suited to control the dynamic movement of guard cells in rapid response to a large spectrum of environmental and hormonal stimuli including light, CO2, low humidity, ABA, and jamonic acid (JA). The first kinase likely to be pivotal to ABA signal transduction in guard cells was purified from Vicia faba. This kinase is activated by ABA, and hence the name AAPK for ABAActivated Protein Kinase (Li and Assmann, 1996; Li et al., 2000). Reverse genetics showed that a mutation created in the P-loop of the AAPK, which leads to a loss of enzymatic activity, can still block ABA response in transformed guard cells (Li et al., 2000). AAPK can directly phosphorylate in vitro an RNA-binding protein AKIP (AAPK Interacting Protein), which in turn binds to a mRNA encoding a dehydrin, as detected by RT-PCR using specific primers. Whether there might be other drought-associated mRNAs co-regulated post-transcriptionally by AKIP remains an interesting question to be explored. AKIP is constitutively nuclear-localized, but becomes reorganized in ‘nuclear speckles’ subsequent to ABA activation (Li et al., 2002). The orthologous kinase from Arabidopsis, variously known as OPEN STOMATA (OST)1/Srk2e/SnRK2.6, was discovered independently and by different experimental approaches aimed at identifying key regulatory elements in osmotic stress adaptation (Merlot et al., 2002; Mustilli et al., 2002; Yoshida et al., 2002; Boudsocq et al., 2004; Xie et al., 2006). The ost1 mutations impede stomatal response to

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2005). Likewise, the gene EARLY RESPONSIVE to DEHYDRATION (ERD)15 encodes a small acidic protein that is postulated to interact with the C-terminal domain of poly(A)-binding proteins. Both have been implicated in ABA signaling as defined by genetic and reverse genetic studies (Wang and Grumet, 2004; Kariola et al., 2006). The ahg2 mutant has other abnormalities, including 50% higher ABA content and hypersensitivity to salicylic acid (Nishimura et al., 2005). STABILIZED1 (STA1) —a stress-up-regulated protein with a significant overall homology to the human U5 snRNP-associated 102-kD protein and yeast pre-mRNA splicing factors—might be required for splicing under cold stress conditions (Lee et al., 2006a). This is consistent with the observation that the cold-induced transcript COLD RESPONSIVE (COR)15a remains unspliced in the mutant (Lee et al., 2006a). Besides COR15a, there are approximately 70 other transcripts whose steadystate levels are also affected under normal conditions in sta1 (Lee et al., 2006a). The sta1 mutant—similar to abh1—has serrate leaves, hypersensitivity to ABA, and displays early flowering. To explain the well defined phenotypes from mutations affecting RNA-binding proteins that perform seemingly house-keeping functions will require identifying their direct RNA ligands. Towards this goal, our laboratory is developing a method aimed at capturing RNA still bound to its receptor protein in live cells (J. Redko, N. Frei dit Frey and C. Valon, unpublished data). It is conceivable that these RNA-binding proteins influence the decay rates of certain mRNAs encoding key regulators of ABA sensitivity. For example, the long 5#untranslated leader of ABI3 impedes efficient expression, which can be dramatically improved by its removal (Ng et al., 2004). The other possible explanation is that the ‘specific’ phenotypes are due to incomplete characterization of the mutant. In any case, efficient capping of nuclear mRNAs seems to be less critical in plants than other model organisms such as yeast and mammals. In addition to splicing, modification of the cap, and poly(A) structures, much excitement in recent times has come from the realization that the stability of mRNA or its translatability can be negatively regulated by pairing of complementary microRNAs with mode sizes of 21–24 bp. Several microRNAs whose expression is associated with stress have been reported (JonesRhoades and Bartels, 2004; Sunkar and Zhu, 2004). However, some of these miRNA are also expressed in the absence of stress, suggesting that they may perform other developmental functions (Sunkar and Zhu, 2004). The level of the microRNA miR159 is up-regulated by drought, and, among several hormones, by ABA. In this latter case, the accumulation of miR159 is contingent on the wild-type function of the B3 domain transcriptional activator ABI3 (Reyes and Chua, 2007). The microRNA mediates cleavage of the transcripts encoding MYB101 and MYB33—two positive regulators of seed germination. The degradation of these two transcripts would thus suggest a homeostatic mechanism to desensitize ABA signaling during seedling stress responses. Several other genetic screens in the past based on seed germination or the use of

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Figure 2. The Canonical Architecture of a b-ZIP Transcription Activator. The four highly conserved C subdomains and the b-ZIP domain are highlighted. The b-ZIP domain is required for dimerization and binding to the target promoter. The C1 and C2 domains are strongly phosphorylated by four SnRK2 in vitro (noted by the stars). The C3 contains a weak phosphorylation site. The serines and threonines residues, which are the targets of phosphorylation, are shown in red and the Arg residues at the –3 positions are shown in bold. The C2–C3 domains of ABF4, which contains the target Ser110 of phosphorylation by AtCPK32 (Choi et al., 2005), are also highly homologous to the equivalent regions in the b-ZIP transcription activators shown in the figure. Whether the C4 domain can be phosphorylated in vitro has not been tested by Furihata et al. (2006), but it contains the characteristics of a SnRK2 phosphorylation target, and, in the case of AREB2, it is also highly homologous to one of the 14-3-3 binding sites identified by Schoonheim et al. (2007b).

A proteomics approach was also used to identify targets of SnRK2.8 in planta (Shin et al., 2007) by comparing the profiles of the phosphoproteins in transgenic plants ectopically expressing SnRK2.8 to those of a corresponding insertion mutant. These experiments concentrated on novel phosphoproteins migrating between pI values of 3.9–5.1 and pI 4.7–5.9 because the authors estimated that ;70% of the total phosphoproteins fall within these defined ranges of isoelectric points. Nine candidate proteins were chosen from a roster based on their repeated occurrence in the different experiments. Seven of these were confirmed in vitro to be phosphorylated by SnRK2.8 produced in E. coli. Among the seven candidates, three were 14-3-3 isoforms, and, by comparing their sites of phosphorylation, the deduced motifs were DYRPSKVE on 14-3-3j, DYRPSKIE on 14-3-3v (Shin et al., 2007). Note that SnRK2.8 was the test kinase common to both of the above independent studies (Furihata et al., 2006; Shin et al., 2007), and yet no conservation in the motifs was observed in the 14-3-3 proteins and those in the b-ZIP transcription factors. Perhaps the sample sizes are still too small to draw any conclusion concerning a ‘consensus’ phosphorylation site of SnRK2, and, moreover, we cannot exclude the possibility that the two different experimental approaches may have hidden technical biases. Some b-ZIP transcription factors may also be the targets of calcium-dependent protein kinases (CPKs), at least as

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ABA, leading to hypersensitivity to mild drought conditions (Mustilli et al., 2002; Yoshida et al., 2002). Like the AAPK in Vicia faba, OST1 is also activated by ABA, and by hyperosmotic stress independently of ABA (Mustilli et al., 2002; Yoshida et al., 2002; Boudsocq et al., 2004). The closest AKIP homologs in Arabidopsis are the so-called UBA2a and UBA2b proteins (for poly(U)-Binding Associated due to their interaction with another poly(U)-binding protein), each of which is characterized by two RNA-Recognition Motifs (RRM), typically found in RNA-binding proteins with affinity to single-stranded RNA (Lambermon et al., 2002). In vitro, UBA2a has an apparent affinity for poly(U) over other monoribonucleotide chains. Similar to AKIP, UBA2a is also translocated to subnuclear structures resembling ‘speckles’ in response to exogenous ABA. Despite the high degree of conservation of the individual proteins, however, OST1 phosphorylates neither UBA2a nor UBA2b in vitro (Riera et al., 2006). Thus, the functional relationships of the kinase to the RNA-binding protein in ABA signaling seem to have diverged between Vicia and Arabidopsis. OST1 is one of the ten members belonging to the Sucrose Non-Fermenting Related Kinase2 (SnRK2) family (for nomenclatures of the different families of kinases, consult Hrabak et al., 2003). These kinases have often been described as plant-specific, but, as their name implies, they are structurally related to the Sucrose Non-Fermenting kinase1 (SNF1) in yeast or the mammalian counterparts, the AMP-activated kinases. SNF1 and AMP-activated kinases have been primarily studied as a metabolic regulator that is activated in response to energy deprivation (Kahn et al., 2005). Although there is ample information on the biochemical characteristics of these kinases, data on their in vivo function in a physiological context are still rather limited to only a few examples. Besides OST1, two other members in the same clade—SnRK2.2 and SnRK2.3—are also highly inducible by exogenous ABA (Boudsocq et al., 2004). A parallel situation exists in rice. Three of the ten kinases (here known as Stress-Activated Protein Kinases or SAPK) are also inducible by ABA (Kobayashi et al., 2004). The Arabidopsis OST1, SnRK2.2, SnRK2.3, as well as SnRK2.8 (which responds to hyperosmotic stress independently of ABA), all phosphorylate in vitro a motif in the so-called Constant (C) subdomains found among basic-leucine zipper (b-ZIP) transcription factors, including ABA Responsive Element Binding protein (AREB)1, AREB2, and ABI5 (Furihata et al., 2006) (Figure 2). The phosphorylated residues in the motifs are either Ser or Thr. These motifs in the C domains are characterized by a highly conserved Arg at position –3 relative to the Ser or Thr that is the target residue for phosphorylation (Furihata et al., 2006), which is arbitrarily designated as position zero (the symbol ‘–’ designates the residues upstream from ‘0’). In rice, SAPK also phosphorylates a b-ZIP transcription factor in vitro (Kobayashi et al., 2005) at two Ser residues, both of which are similarly preceded by an Arg at –3. Thus, the kinases of Arabidopsis and rice are highly conserved not only in their protein architecture and mode of activation by ABA, but also with respect to their downstream targets.

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onstrated negative regulators of ABA, the observations on the physiological impact caused by over-expression of the FsPP2C gene support the hypothesis that this PP2C might be a positive regulator. An endogenous PP2C that positively regulates ABA sensitivity has not been identified in Arabidopsis, however. RecentstudiesonABI1suggestthatitmightbindtophosphatic acid (PA) generated by cleavage of the plasma membrane lipids by phospholipase Da1 (PLDa1) (Zhang et al., 2004a). In vitro, PA binds to ABI1 and the mutation of Arg73 in the N-terminal domain abolishes this binding. The extrapolation to an in planta scenario is that PA sequesters the PP2C to the plasma membrane, thereby effectively preventing its negative regulation of ABA-mediated stomatal closure (Mishra et al., 2006). Expression of a mutant transgene bearing Arg73 in Arabidopsis disrupted in its endogenous copy induced ABA insensitivity, presumably by escaping PA titration (Mishra et al., 2006). If PA does have a major role in vivo in the regulation of ABI1 by titration, a testable hypothesis is that the dominant phenotypes of abi1-1 (in which Gly180 is mutated to Asp) should be relieved by exogenous application of PA (Zhang et al., 2004a). ABI1, but not its closest homolog ABI2, interacts with a small stretch of amino acids in the regulatory C-terminal domain of OST1 in the yeast two-hybrid assay (Yoshida et al., 2006). It is not known whether ABI1 negatively regulates the OST1 kinase activity, or works as a presumptive scaffold protein in analogy to ABI2/Salt Overly Sensitive (SOS)2 kinase interaction (Ohta et al., 2003). The upstream kinases that activate OST1 are presently unknown, but OST1 seems to already exist as a phosphoprotein even in the absence of the stress stimulus (Boudsocq et al., 2007). Thus, the stress signal may stimulate changes in the pattern of phosphoamino acids in OST1 such that some are dephosphorylated (for example, by ABI1) while others are de novo phosphorylated. Besides PP2Cs, mutations that disrupt PP2As (Kwak et al., 2002; Pernas et al., 2007) and at least two putative dual-specificity protein phosphatases, when mutated, lead to altered ABA sensitivity (MonroeAugustus et al., 2003; Quettier et al., 2006). The further observation that a PP2A scaffold subunit can interact with the C-terminal autoinhibitory domain of the H+-ATPase AHA2 in the yeast two-hybrid system (Fuglsang et al., 2006) suggests a novel phosphorylation-based pathway involving PP2A and SnRK3 (one of which is PSK5) that link ABA signals to membrane transport (see section below on H+-ATPases). ABA regulates repetitive cytosolic free Ca2+ ([Ca2+]cyt) elevations in guard cells (Mori and Muto, 1997). Experimentally imposing [Ca2+]cyt transients reveals two distinguishable Ca2+dependent stomatal closing responses: a rapid ‘Ca2+-reactive’ stomatal closing response, and a long-lasting ‘Ca2+programmed’ stomatal response, which prevails even after Ca2+ transients have been terminated (Israelsson et al., 2006). The long-lasting Ca2+-programmed response, but not the rapid Ca2+-reactive stomatal closing response, depends on the Ca2+ transient pattern. In guard cells, [Ca2+]cyt elevation activates S-type anion channels via reversible protein

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demonstrated in heterologous experimental systems. These kinases contain an intrinsic Ca2+-activation domain with four EF-hand Ca2+-binding sites. Using the C2–C3 domains of the ABA-Responsive Element Binding Factor (ABF)4 as the bait, AtCPK32 was identified as a partner using a two-hybrid screen (Choi et al., 2005). Ser110 of ABF4 is phosphorylated by AtCPK32 in vitro, which is important for the interaction of the two partner proteins, and for the transcriptional activity of ABF4. This Ser110 is also preceded at –3 by an Arg and shows other conserved residues with the SnRK phosphorylation motifs (Figure 2). AtCDK10 and AtCDK30, which belong to the so-called subgroup III of CDPK, also interact with ABF4. Conversely, AtCDK32 can also interact with two other transcription factors—ABF2 and ABF3 (Choi et al., 2005). Thus, there may be combinatorial regulations between multiple b-ZIP transcription factors and CDPKs or SnRK2. However, in planta, it is not known whether interactions might be less promiscuous than those demonstrated here in heterologous systems, especially if only certain proteins are co-compartmentalized in the plant cells. These b-ZIP transcription factors bind to the consensus motif ABRE (for ABA-Responsive Element) characterized by the core motif ACGTGGC found in many promoters that are responsive to ABA and calcium (Kaplan et al., 2006). Details concerning transcription networks that are regulated by ABA have been the subject of recent reviews (Shinozaki and YamaguchiShinozaki, 2007). The major group of protein phosphatases (PP) that have been identified in ABA signaling are the PP2Cs. Of the 69 to 75 predicted PP2C genes in the Arabidopsis genome, at least four of them (ABI1, ABI2, AtPP2CA, HAB1; Schweighofer et al., 2004) are genetically defined as negative regulators of ABA. Loss-of-function mutations in each of these genes, isolated either as T-DNA insertion or second site intragenic revertants of dominant mutations, all led to hypersensitivity to ABA. Furthermore, the physiological characterization of hab1-1 and two new T-DNA insertion alleles of abi1 (abi1-2, abi1-3) showed that double mutants are more sensitive to ABA relative to each of the single mutants alone, which would suggest that HAB1 and ABI1 may control parallel ABA signaling pathways, rather than being redundant regulators in the same pathway (Saez et al., 2006). One of the direct targets of ABI1-mediated negative regulation is a putative homeodomain transcription factor encoded by the AtHB6 gene (Himmelbach et al., 2002) whose promoter was used as one of the ABA sensors described above. However, not all PP2Cs are negative regulators of ABA sensitivity. An ABA-inducible gene encoding a PP2C (FsPP2C) had been isolated previously from Fagus sylvatica (beech). Because transgenic work is not possible in beech, this gene was overexpressed under the control of the cauliflower mosaic virus 35S RNA promoter in transgenic Arabidopsis to produce enhanced ABA sensitivity in seeds and vegetative tissues, as well as dwarf phenotype and delayed flowering (Reyes et al., 2006). These effects, moreover, were reverted by application of GA3. Thus, in marked contrast to the endogenous Arabidopsis PP2C, which are dem-

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ABA-MEDIATED STOMATAL CLOSURE: ACTION AT THE PLASMA MEMBRANE ABA signaling events at the membrane level, involving many channels and transporters, have also been extensively studied in the guard cell for two decades because of the clear role of ABA in stomatal closing to limit water loss through transpiration. The control of transpiration (Caemmerer and Baker, 2007), especially from the viewpoint of genetics (Nilson and Assmann, 2007), has been recently reviewed. Light induces stomatal opening, while ABA promotes closing. During the course of the day, the plant must therefore continually adjust the conflicting need for CO2 uptake destined for photosynthesis, while minimizing water loss through transpiration. Light activates H+-ATPases to hyperpolarize the plasma membrane, which drives potassium uptake and the bowing of the guard cells to open the stomatal pore. Apart from a number of P-type ATPases that transport Ca2+ or heavy metals (Axelsen and Palmgren, 2001), the H+-ATPase is the only primary active transporter in the plasma membrane, making it the probable central component in driving stomatal opening. In contrast, any role of these proton pumps in stomatal closing mediated by ABA had been rather contentious. ABA activates R- (rapid) and S- (slow) ion channels for [Cl–] efflux. In parallel, the hormone stimulates [K+] outward- and inhibits [K+] inward-rectifying channels to promote stomatal closure. In the current model, it is assumed that the activation of the anion channels by ABA is necessary and sufficient to over-ride the proton pump to depolarize the membrane potential (Schroeder and Keller, 1992; Schwartz et al., 1995; Ward et al., 1995; Levchenko et al., 2005). The recent characterization of the ost2 mutations (Merlot et al., 2002, 2007) revealed that they constitutively activate an H+-ATPase named AHA1. Importantly, both mutant ost2 dominant alleles block completely the guard cell response to ABA, but not to two other stomatal closing signals, such as high CO2 and darkness. These observations suggest that ABA activation of the anion channels alone is not sufficient to depolarize the plasma membrane, but it must be preceded by the attenuation of specific H+-ATPases, like AHA1 (Merlot et al., 2007). The mechanisms by which the AHA1 activity might be selectively suppressed by the ABA signaling pathway are not known. An intriguing hint, however, has come from our preliminary tests indicating that the inward-rectifying potassium channel KAT1 can physically interact with AHA1 in the yeast two-hybrid system based on split ubiquitin (C. Valon, unpublished results). This suggests that AHA1 might be a component in the KAT1 pathway which is subjected to ABA inhibition of K+ influx during stomatal closure. H+-ATPase activity is in part regulated by autoinhibition. This is mediated by the binding of the Cterminal regulatory domain with other parts of the protein (Palmgren, 2001; Lefebvre et al., 2003). These H+-ATPases are also inactivated by phosphorylation on a specific Ser residue in a highly conserved region in the C-terminal regulatory domain (Fuglsang et al., 2007). In the case of AHA2—the closest

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phosphorylation events (Schmidt et al., 1995), implying that Ca2+-regulated kinases may be involved in decoding the Ca2+ signal. Taking advantage of enriched expression and inducibility in guard cells as a criterion (Leonhardt et al., 2004), insertion mutants of two calcium-dependent protein kinases, cpk3 and cpk6, were selected and tested as candidates. Indeed, disruption of either one or both kinases impaired the up-regulation of anion currents by ABA (Mori et al., 2006), confirming that these kinases are two of the key transducers of the Ca2+ signal in the guard cells. When guard cell protoplasts were incubated in low extracellular Ca2+, the S-type anion channels only respond moderately to 2 mM Ca2+ in the pipet. This response can be enhanced in the wild-type, but, importantly, not in the mutant cpk protoplasts, by the addition of ABA. The authors’ interpretation is that ABA, as a stomatal closing signal, mediates ‘priming’ of guard cell Ca2+ sensors (that is, CDPK) such that they can respond to an elevated Ca2+ signal. The molecular nature of this ‘priming’ of CDPK is worth further experimental pursuit. These two CDPKs, furthermore, may have additional roles in the ABA regulation of Ca2+-permeable (ICa) channels (Israelsson et al., 2006; Mori et al., 2006). It is not known whether these CDPKs phosphorylate b-ZIP factors. There are likely to be other sensors that process the Ca2+ signals or signature, as strongly suggested by the suppression of the CBL9 gene, encoding a calcium-binding protein, resulting in hypersensitivity to low K+ and ABA (Cheong et al., 2003; Pandey et al., 2004; Xu et al., 2006). CBL9, as well as another calcium-binding protein CBL1, which may perform ABAindependent functions, can alternatively bind to CIPK23 (SnRK3.23) to regulate and phosphorylate the K+ transporter AKT1 (Li et al., 2006a; Xu et al., 2006). The same two CBL also alternatively bind to CIPK1, probably directing the kinase to the plasma membrane. The loss of function of cipk1, similar to cbl9, leads to ABA hypersensitivity (D’Angelo et al., 2006). The SOS2 kinase (CIPK24 or SnRK3.11) and the SOS3 calciumbinding unit Calcineurin B-Like protein4 (CBL4), which are the founding members of the CIPK/CBL class of plant-specific kinases, regulate the Na+/H+ antiporter SOS1 to bring about ion homeostasis under salt stress. The available evidence thus suggests that CBL–CIPK interactions, in a network-like combination, may regulate the phosphorylation state of various ion transporters. The CIPK members are characterized by a 21-amino acid FISL motif as part of the C-terminal autoinhibitory domain. The C-terminal domain is also characterized by a 33-amino acid PPI motif, and, in the case of SOS2, constitutes the binding site of ABI2 (Ohta et al., 2003). This phosphatase interaction scaffold motif is conserved from yeast to humans (Sa´nchez-Barrena et al., 2007). The precise role of the ABI2 phosphatase in the SOS2/SOS3 complex is not known, although it seems unlikely that ABI2 is a direct negative regulator of SOS2 kinase activity (Ohta et al., 2003). As suggested by studies on a homolog of ABI2, AtPP2CA, these protein phosphatases 2C could also directly regulate the phosphorylation status of K+ channels (Che´rel et al., 2002).

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ABA SIGNALING VIA 14-3-3 PROTEINS We have alluded to the binding of a 14-3-3 protein to the penultimate phosphothreonine residue on the H+-ATPase as part of the activating mechanism of the proton pump. 14-3-3 are ;30-KDa acidic proteins that form homo- or heterodimers in a clamp-shaped structure that can either interact with one target protein at two different positions, or with two different target proteins, as is the case with H+-ATPases (Roberts, 2003). Our work on the ost2 mutations described above provides indirect evidence of 14-3-3 proteins in ABA signaling (Merlot et al., 2007). In Vicia faba guard cells as well, ABA has been reported to antagonize blue-light-dependent H+-ATPase activity and cause the release of the 14-3-3 protein (Zhang et al., 2004b). The hormone can also suppress K+ channels activated by a 14-3-3 protein in barley roots (van den Wijngaard et al., 2005), and the maize ABA signaling effector, VIVIPEROUS (VP)1, interacts with 14-3-3 in the yeast two-hybrid system (Schultz et al., 1998). Besides the few examples mentioned above, direct evidence of specific 14-3-3 isoforms in ABA signaling had been rather sparce. Now, the de Boer group has provided much stronger evidence that ABA affects both the expression and protein levels of a subset of the five 14-3-3 isoforms in embryonic barley roots (Schoonheim et al., 2007b). In particular, the presence of Hv14-3-3D and Hv14-3-3E is prolonged by ABA treatment. RNAi-mediated silencing of each one of the five 14-3-3 genes impairs ABA induction of a reporter, suggesting that all 14-3-3

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proteins somehow intervene in ABA signal transduction. Using a two-hybrid screen, three ABA-responsive element binding factors (ABF, ABRE or b-ZIP transcription factors) were identified among a collection of 132 interacting proteins. Closer examination of one of these—HVABI5 (named after the homolog ABA-INSENSITIVE5 of Arabidopsis)—allowed the authors to highlight two putative 14-3-3 binding motifs— REIPT160AP and RRTLT350GP, which are also important for the transactivating function of this protein in a transient expression assay consisting of a promoter-gene reporter. These results suggest that transcription of the 14-3-3 genes and at least some of their corresponding proteins are under tight control by ABA. The 14-3-3 proteins, in turn, also control ABA action by providing a platform to secure specific signaling proteins.

THE EMERGING ROLE OF ABA IN PATHOGEN RESPONSE Salicylic acid (SA), JA, and ethylene are the three major hormones in triggering pathogen resistance responses (Grant and Lamb, 2006). SA, furthermore, is predominantly associated with the establishment of systemic acquired resistance. Although ABA has been linked to pathogen susceptibility in the past, the extent of its involvement and its relationship to these other hormones have attracted even more attention recently (Anderson et al., 2004). Interested readers can refer to a didactic review, particularly the positive role of ABA in callose deposition (Mauch-Mani and Mauch, 2005). The advent of large-scale experiments has allowed the surveillance of gene activities across the whole genome. A significant (;42%) overlap exists in the gene activities influenced by ABA and type III virulence factors, such as those from the biotroph Pseudomonas syringae DC3000 (de Torres-Zabala et al., 2007). Exogenous application of ABA promotes colonization by both virulent (DC3000) and non-virulent (hrpA–) P. syringae. Collectively these results suggest that ABA negatively regulates post-invasion pathogen immunity. In line with this argument is that reducing the synthesis and sensitivity of the plant to ABA, either by mutations (such as abi1-1 and abi2-1) or by over-expression of relevant genes (for example, 35S::HAB1), leads to a 20–80-fold increase in pathogen resistance as compared to that of wild-type plants. Conversely, mutants that are hypersensitive to ABA support, on average, 30-fold more P. syringae DC3000 multiplication. However, the role of ABA appears to be more complex, and may vary among different pathosystems. For example, mutants deficient in ABA are more sensitive to infection by the fungal pathogens Pythium irregulare (Adie et al., 2007) and Leptosphaeria maculans (Kaliff et al., 2007). In these two cases, ABA thus appears to have a positive role in activating the pathogen defense system. This situation becomes even more complicated when pathogens are tested on ABA signaling mutants, such as abi4, which displays opposite resistance responses towards these two fungi. Along the same line of

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homolog of AHA1—the calcium-dependent kinase PSK5 has been implicated as the negative regulating kinase (Fuglsang et al., 2007). The phosphorylation on Ser931 of AHA2 by PSK5 interferes with the binding of a 14-3-3 protein to the penultimate phosphothreonine (amino acid 947) of the H+-ATPase, which is necessary to maintain the proton pump in the activated conformation. The AHA2 gene alone is capable of complementing the growth defect of a yeast mutant disrupted in its endogenous H+-ATPase. However, yeast cells co-transformed with the PKS5 and AHA2 genes, or the SCaBP1 and AHA2 genes grew very poorly, consistent with the notion that the kinase and its interacting calcium-binding protein conjointly downregulate AHA2.New phosphorylatedaminoacids on H+-ATPases are being discovered (Nu¨hse et al., 2003); thus, there might still be more regulatory sites. Dominant mutations in the protein phosphatases ABI1 and ABI2 also block suppression of global H+-ATPase activities by ABA (Roelfsema et al., 1998), although it is not known whether these two phosphatases can directly dephosphorylate and inactivate H+-ATPases. We have also mentioned above that AHA2 can interact with a PP2A scaffold (Fuglsang et al., 2006), hinting that PP2As might also be regulators. An inactive form of an H+-ATPase is dimeric, while the active form is hexameric; however, the mechanism by which it passes from the inactive to active configuration, and vice versa, has not been entirely elucidated (Kanczewska et al., 2005; Ottmann et al., 2007).

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not passive ports of bacterial entry, but are part of the innate immunity defense involving the OST1 kinase that acts as a barrier against bacterial infection.

LATERAL ROOT DEVELOPMENT A substantial body of evidence indicates that normal development of the lateral root system is controlled primarily by auxin. However, the root architecture is notoriously flexible, as the intrinsic developmental program (depth of root growth, mass, rate of root elongation, lateral root number) is modified by many external factors, of which the most important is the availability of water and nutrients in the soil. Our knowledge on how the root architecture is controlled and adjusted in accordance to environmental variations remains rudimentary. ABA has been implicated, at least in part, in altering the developmental program of lateral roots in adaptation to environmental stresses (De Smet et al., 2006). Model plants in the Brassica genus, including Arabidopsis, respond to prolonged drought stress by developing the so-called ‘short roots’, which are specialized lateral roots characterized by their stubby tuberized structures (Vartanian et al., 1994). Once initiated, these short roots then switch to a dormant mode, if the condition of sub-lethal drought persists. Upon rehydration, however, these dormant short roots resume growth and eventually replace the dehydrated old lateral roots (Vartanian et al., 1994). This remarkable morphological and physiological adaptive capacity is severely compromised in mutants that are deficient in ABA. In certain mutants insensitive to ABA—like abi1-1—lateral root buds are significantly lower in number. This suggests that ABA, and some signaling elements that control ABA sensitivity, are important factors in the initiation of the short-root developmental program. Note that this process is also interrupted by the auxin-resistant (axr)1 mutation (Vartanian et al., 1994) which confers auxin resistance. Additional evidence of ABA-auxin interaction in modulating lateral root development has come from the work on the maize transcription factor VP1 (also described above in the interaction with 14-3-3 proteins) (Suzuki et al., 2001) and more recently on its Arabidopsis ortholog ABI3 (Brady et al., 2003). For brevity, we will only describe the more recent work on ABI3. The function of ABI3 has been relatively well documented in seed development, but it is also expressed in vegetative tissues, although its precise functions in non-seed contexts have attracted comparatively less attention (Rohde et al., 2000). ABI3 contains a DNA-binding domain, named B3, that is conserved in plant-specific transcription factors, including Auxin Response Factors (ARF), which regulate the expression of auxin-responsive genes. Comparison of the lateral roots of the mutant abi3 plants (specifically the strong allele abi3-6) and those from the wild type revealed that the mutant has a higher requirement for auxin (indole-3-acetic acid or IAA) to initiate lateral roots (Brady et al., 2003). The promoter of the ABI3 gene is also responsive to auxin. In contrast, mutations in ENHANCED RESPONSE TO ABA1 (specifically the era1-3

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observation, the mutations abi1-1 and abi2-1 actually foster differential resistance responses against the same pathogen, L. maculans (Kaliff et al., 2007). Transcriptome and metaanalysis of expression profiles altered by infection with the necrotroph Pythium irregulare identified many JA-induced genes but also highlighted the importance of ABA as a regulator, as the ABA responsive element (ABRE) appears in the promoters of many of the defense genes (Adie et al., 2007), consistent with the interpretation that ABA is a positive regulator from parallel mutant studies. It is very likely that the final response output to a particular pathogen may be influenced by mutually synergistic or antagonistic interactions with other hormones (Anderson et al., 2004). Another potential complicating factor is that these studies do not always take into account that exogenously applied ABA (involving variables such as hormone concentrations and the duration of treatment) and endogenous sources of ABA may not exert the same physiological effects (Christmann et al., 2004). There are many other possible connections between pathogen resistance and ABA signaling, not the least of which is that both pathogen attack and ABA trigger the formation of the secondary messenger hydrogen peroxide which is dependent on the NADPH oxidases RbohD and RbohF (Kwak et al., 2003; Torres and Dangl, 2005). This second messenger participates in the regulation of the stomatal pore through several input pathways, including that of ABA. In fact, recent studies suggest that the pore is a critical component of the innate immunity defense against pathogen invasion (Melotto et al., 2006). P. syringae mutants defective in producing coronatine—a structural octadecoanoid mimic of JA and its precursor 12-oxo-phytodienoic acid—fail to cause disease when deposited on the leaf surface, but remain virulent when delivered directly into the leaves by injection (Melotto et al., 2006). Melotto and colleagues report that virulent P. syringae moves towards open stomata when inoculated at the leaf surface, and although the deposition of the bacteria provokes rapid closure of the stomata, they re-open within 3 h. Mutant bacteria lacking coronatine fail to colonize the leaf interior, which correlates with the failure to re-open stomatal apertures. Initial rapid stomatal closing thus seems to protect plant leaves from bacterial entry which is countered by coronatine. Mutant plants deficient in FLS2—the receptor to the flg22 peptide derived from the most conserved region of bacterial flagellins—are more susceptible to infection by P. syringae. The flg22 peptide alone can also trigger stomatal closure in wildtype Arabidopsis, suggesting that its corresponding receptor is implicated in the stomatal response to pathogen invasion. The stomata of ost1 fail to close upon treatment with flg22 and these mutants also support the multiplication of P. syringae, regardless of whether it does or does not produce coronatine (Melotto et al., 2006). Thus, the ABA signaling pathway in stomatal closure regulated by OST1, which acts upstream of hydrogen peroxide production (Mustilli et al., 2002), and the immune response pathway dependent on FLS2 are interconnected. These observations argue that the stomata are

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ABA SIGNALING IN METAZOANS The concept of ABA as a strict ‘phytohormone’ has been hammered into our collective minds ever since our first botany course. Thus, a report more than 20 years ago claiming traces of ABA and its ester glucose have been found in the brains of rats and pigs—and judging from the silence from the plant community—must have seemed a heresy (Le Page-Degivry et al., 1986). Surely, we could have better explained away these observations by supposing that the trace of ABA was dietary in origin, rather than an endogenous hormone of mammals. That long silence has now been breached again by the detection of ABA in several metazoans. More surprisingly, ABA, when served as a dietary supplement, can help to counter diabetes

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or obesity-related inflammation at least in mice (Guri et al., 2007). ABA even exists in the oldest metazoan—the sea sponge—in which it acts as an early trigger in a hightemperature signaling pathway to stimulate water filtration (Zocchi et al., 2001). This ABA-mediated adaptive response may have a significant impact in shaping the survival and evolution of marine organisms as ocean temperatures continue to rise. The hormone is also found in marine hydroids, which are evolutionarily slightly more advanced than sponges. In the hydroids, ABA plays an indispensable role in their extraordinary capacity to regenerate damaged body parts (Puce et al., 2004). In humans—the pinnacle of evolution—ABA acts as a pro-inflammatory cytokine in granulocytes (Bruzzone et al., 2007). The circulating granulocytes can produce reactive oxygen species (ROS) and nitric oxide (NO), which result in the destruction of pathogens. Note that ROS and NO are also intermediates in ABA signaling in plant defense. The immunity response in granulocytes triggered by ABA requires a pertussissensitive receptor/G protein complex, cyclic ADP ribose and [Ca2+]i (Bruzzone et al., 2007), again reminiscent of some aspects of ABA perception and signaling in plants (Liu et al., 2007). Biotin-labelled ABA has detected binding sites on the surface of the plasma membrane from granulocytes, with approximately the same Kd of 11 nM as that described for plants (Yamazaki et al., 2003; Bruzzone et al., 2007). Thus, it seems that ABA is not only produced naturally in metazoans, but several intermediates in the signal transduction chain are also conserved between animals and plants. This close parallel in ABA signaling across two kingdoms raises the possibility that the origin of the hormone predates the great divide of metazoan and metaphyta.

CONCLUDING REMARKS The ABA biosynthetic pathway is largely delineated. The conversion of all trans-violaxanthin to the all trans-neoxanthin requires a neoxanthin synthase, which is now known to be encoded by the ABA4 gene (North et al., 2007). The precise conversion mechanism remains to be elucidated as the catalytic activity of the enzyme has not yet been reconstituted in vitro. Also, since ABA4 does not convert trans-violaxanthin to its cis isomer, an unknown isomerase is still one of the missing links to the biosynthetic pathway (North et al., 2007) (Figure 1). In the future, detailed characterization of the expression profiles of all of these biosynthetic genes should soon provide a ‘tissue map’ through which precursors and intermediates are shuttled, finally ending with ABA deposited mainly in few cell types (Christmann et al., 2004). In Arabidopsis, drought appears to cause remobilization of ABA from leaves to roots (Christmann et al., 2004). This observation is surprising because it contradicts the widespread notion of a root-derived source of ABA being the long-distance messenger of dehydration. One tantalizing possibility is that the messenger may not be ABA, but a novel substance that communicates the hydraulic signal. These long-distance

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allele), the genetically defined upstream regulator of ABI3, augment the number of lateral roots. Like ABA, high nitrate in the medium also inhibits root growth in wild-type Arabidopsis plants and this inhibition is less severe in ABA-deficient mutants and certain signaling mutants, such as abi4 and abi5 (Zhang et al., 2007). Note, however, that recent progress hints at the possibility that the pathways promoting lateral root dormancy could be controlled by novel genes that are distinct from those in seed dormancy (Zhang et al., 2007). The lateral roots show about a 10-fold higher sensitivity to exogenous ABA relative to, for example, seed germination (De Smet et al., 2003, 2006). The fact that FCA is expressed in lateral root tips, and that the fca mutation reduces ABA sensitivity in the lateral roots (Razem et al., 2006), have been cited as supporting evidence for this speculation (De Smet et al., 2006). Moreover, the formation of lateral root primordia in response to only a slight decrease in osmotic potential in an artificial medium has been observed (Deak and Malamy, 2005). It is tempting to draw the parallel between the lateral root primordia and the ‘short roots’ induced by progressive drought described above. In this experimental system, mutants deficient in ABA showed an overall larger root system, which led the authors to propose that ABA functions as a suppressing hormonal signal (but necessary to initiate lateral root primordia). This system has been used to carry out a genetic screen, and allowed the identification of a mutant called lateral root development2 (lrd2) which has an increased lateral root system in both repressive osmotic conditions and in the absence of stress (Deak and Malamy, 2005). The mutant is hypersensitive to ABA signal, which correlates with the increased number of lateral root primordia. In contrast, lrd2 as well as two ABA-deficient mutants, aba2 and aba3, produce more lateral roots in the presence of 2,3,5-triiodobenzoic acid (TIBA) or N-1-naphthylphtalamic acid (NPA)—two inhibitors of polar auxin transport. The lrd2 is mapped to a 762-kb region containing 205 ORFs on chromosome 1, but the gene has not yet been identified (Deak and Malamy, 2005). Nonetheless, it seems again to confirm an antagonistic relationship between ABA and auxin signaling in controlling lateral root development.

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transcriptional platform to enlist other regulatory proteins on target promoters containing the ABRE motif (Figure 3). Would these ‘other regulatory proteins’ be found among the interactors of the 14-3-3 protein (Schoonheim et al., 2007a)? If the working model is correct, then one can start the search for the corresponding in vivo components that are expressed at the same time in the same subcellular compartments. The metabolism of RNA appears to be an important regulatory component in ABA signaling. The most direct evidence is the binding of ABA to FCA (Razem et al., 2006). Moreover, how mutations in cap-binding proteins, poly(A) processing enzymes and double-stranded RNA-binding proteins lead to altered ABA sensitivity remain an important paradox to be resolved. One possibility is that some classes of molecules relevant to ABA signaling are exquisitely sensitive to perturbed RNA metabolism. Identifying direct physiological RNA targets that are processed or transported by each of these binding proteins will be necessary but the classical method of in vitro immunoprecipitation may not be always reliable (Mili and Steitz, 2004). Most RNA-binding proteins bind sequences that are rich in U or AU. Such featureless sequence motifs do not lend an easy explanation to the functional specificity of these proteins. Clearly, more creative approaches, including highresolution predictions of sequence motifs or secondary structures in RNA, will be needed. Based on the fact that ABA can modify the levels of certain microRNA, flowering time, and some components of gene silencing like HYL1, their potential influence on chromatin structure (loosely known as epigenetics) should also be explored. The guard cell has long been the preferred model among physiologists and geneticists working on ABA signaling. Theoreticians have now joined the field (Li et al., 2006b). Simulating gene disruptions and pharmacological interventions have shown that the network might be surprisingly robust against a significant fraction of the perturbations. Some disruptions, however, such as the loss of membrane depolarizability, predicted clear vulnerability irrespective of initial conditions or of relative timing that would lead to insensitivity to ABA. This is indeed experimentally supported by identifying the ost2 mutations which render the AHA1 H+-ATPase constitutively activated (Merlot et al., 2007). We note, however, a few striking differences between the mathematical model and a previous one founded on experimental considerations (Leonhardt et al., 2004). Incongruences in the models could arise from different theoretical assumptions, for example, on allelic strength of a mutation, or the molecular basis of its dominance (dominant-negative, or a gene dosage phenomenon referred to by geneticists as haplo-insufficiency, or, inversely, hypermorphs). Also, a mutation may cause a loss of enzyme activity but increase the stability of the protein relative to its wild-type counterpart, leading to a more pleiotropic phenotype. Lastly, the species origin and the physiological state of the biological material are also hidden parameters in weighing the ‘importance’ of a signaling intermediate

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messengers might be analogous to the graft transmissible carotenoid-derived signaling molecules in Arabidopsis (Booker et al., 2004; Norman et al., 2004) and in peas (Sorefan et al., 2003) that affect leaf development or branching. One simple experiment that might resolve the question on the origin of the ABA signal is to bisect a plant into shoot and root prior to submitting them to dehydration stress. ABA content is expected to rise in the source tissue. The mechanism by which ABA glucose ester is transported from the vacuole or apoplastic space to endoplasmic reticulum for the cleavage release step is also an open question. There are hundreds of putative transporters in the Arabidopsis genome but only a few have been characterized, especially with respect to ABA signaling (for examples, see Klein et al., 2003, 2004). The pursuit of the elusive ABA receptor for over a decade has culminated in the identification of two candidates (FCA and CHLH) that might represent the intracellular receptors. The will undoubtedly be others, but genes encoding ABA receptors have been surprisingly refractory to classical genetic screens. This situation is reminiscent of riboregulators. It is now known that these non-coding RNAs are pervasive in many developmental pathways, but they have rarely been identified by genetic screens. Could there be, if one dares to stretch the imagination beyond the narrow confines of the protein world, intracellular receptors of ABA that are riboswitches—a particular type of regulatory RNAs that bind to metabolites (Sudarsan et al., 2003)? Mutations affecting the few bases within the RNA that contact specifically with the ABA molecule would therefore represent extremely rare targets for the mutagens. Also, the intensive hunt for the first extracellular receptor is still open. Although a high-profile candidate ‘surface receptor’ has recently been proposed (Liu et al., 2007), the protein is likely to be the plant homolog of the bacterial lanthionine sythetase (Gao et al., 2007). What are the respective downstream elements of these two intracellular receptors? Is there any cross-talk among the two different pathways, and how? These are all intriguing and exciting questions that we are anxious to answer. The implications of 14-3-3 proteins in ABA signaling have been reinforced (Schoonheim et al., 2007a). 14-3-3 proteins interact with the penultimate phospho-Thr of H+-ATPases such as AHA1 in Arabidopsis, and b-ZIP transcription factors in barley. Such binding may stabilize and thus prolong the activities of their partner proteins. Several b-ZIP transcription factors in Arabidopsis are also the direct targets of protein phosphorylation by SnRK2 kinases, among which three are activated by ABA (Furihata et al., 2006). Note that one of the 14-3-3 binding sites (RRTLT350GP) identified in HVABI5 is highly conserved in the C4 domain of some of the Arabidopsis b-ZIP transcription activators (Figure 2). It is possible that phosphorylation of the C4 domain by an SnRK2 creates a binding site for a 14-3-3 protein. Because 14-3-3 proteins function as dimers, their interaction with the C4 domains explains readily why the active form of b-ZIP transcription factor is dimeric (Katagiri et al., 1992). This complex of b-ZIP and 14-3-3 dimers may form the basic

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Results from independent approaches suggest that SnRK2 can interact physically (based on two-hybrids) and phosphorylate b-ZIP transcriptional activators using peptides or protein fragments containing motifs that are found in the C domains. A single study (Shin et al., 2007) based on comparisons between transgenic plants and mutants suggests that some 14-3-3 proteins may also be targets of phosphorylation by SnRK2.8. Also, b-ZIP transcriptional activators may also be phosphorylated by certain calcium-dependent CPKs, which recognize similar or even identical C-domain motifs as the SnRK2s. The phosphorylated 14-3-3 protein dimers, which have been described as resembling a vice, can grip onto amphipathic helices (structure typically recognized by 14-3-3 proteins) from the dimers of b-ZIP transcriptional activators to form the stable platform. Recruitment of other regulators will lead to the final competent transcriptional complex. The downstream target genes are statistically enriched in ABRE sequences in their promoters, which respond to ABA in gene reporter assays and are the binding motifs of b-ZIP transcription factors. The OST1/SnRK2.6 interacts with the ABI1 protein phosphatase based on the two-hybrid system but the impact on ABA signaling is not known. Because most of these results have been obtained by in vitro and heterologous expression systems, the true identities of the components await confirmation by in planta experiments. This next stage will be more challenging, as all of these components are encoded by multigene families.

(Marten et al., 2007). Such discrepancies, however, can be useful in providing novel insight not intuitively obvious, which can then be submitted to experimental testing. How much of the signaling network in the guard cell constitutes the core will need comparison to ABA signaling in other cell types. In this respect, the recent revelation that ABA has a role in lateral root development is a welcome addition as a model. The most astonishing discovery, however, is the existence of ABA in metazoans, providing signaling models from another kingdom. The close parallel between the signaling intermedates found in plants and in the human granulocytes during pathogens defence would undoubtedly invite closer cross-kingdom comparisons to learn more about the versatile and surprising roles of this ancient hormone.

ACKNOWLEDGMENTS We thank S. Merlot and the anonymous referees for their helpful comments on the manuscript. Ge´noplante Post-Doctoral Fellowships (AFF2001073 and GEN026 to JL, FJ); Centre National de la Recherche Scientifique Post-Doctoral Fellowship (YR); European Union Marie-Curie Early Stage Training Fellowships MEST-CT2005-020232-2 ADONIS (AW, FV); Ministe`re de la Recherche Doctoral Fellowship (CS). No conflict of interest declared.

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Figure 3. A Model Depicting a Possible Branch of ABA Signaling Pathway that Leads to Transcriptional Activation of a Gene Network.

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