Review

The genetics of adaptive responses to drought stress: abscisic acid-

dependent and abscisic acid-independent signalling components

Marta Riera, Christiane Valon, Francesca Fenzi, Jerome Giraudat and Jeffrey Leung*

Institut des Sciences du Vegetal, Centre National de la Recherche Scientifique UPR2355 Bat. 23 Avenue de la Terrasse, 91198 Gif-sur-Yvette. France*Corresponding author, e-mail: leung@isv.cnrs-gif.fr

Received 30 June 2004; revised 22 October 2004

Drought stress is the major limitation to crop productivity.

However, crops are genetically complex with many loci

contributing quantitatively to a given physiological trait.Nonetheless, significant in-roads into the molecular mechan-

isms of drought-adaptive responses have been made from the

use of Arabidopsis thaliana. In this special review, we will

discuss results gleaned from reverse and forward geneticstudies that revealed the involvement of both ABA-dependent

and ABA-independent components. In particular, mutant

analyses have highlighted the surprising prevalence of RNAmetabolism in many key steps. We will also discuss our recent

use of infrared thermography to visualize stomatal closure in

response to dehydration as a means to identify novel regula-

tory genes. This has allowed us to recover mutations belonging

to at least eight complementation groups. Analysis of six of

these loci revealed that all of their corresponding mutationsaffect either abscisic acid (ABA) biosynthesis or perception.

Hence, in contrast to molecular studies on gene networks

which pointed to the clear existence of multiple ABA-independ-

ent pathways in the control of dehydration tolerance, ourresults reinforce ABA-based signalling pathways as the

predominant factor in primary or rapid responses. Finally,

we will provide some details learned from the molecular ana-lysis of OPEN STOMATA1 (OST1), a gene that encodes an

ABA-activated kinase issued from this targeted genetic

approach.

Introduction

Fresh water scarcity will be one of the major global pro-blems in the new century. It is thus obvious that under-standing the molecular and physiological mechanisms bywhich land plants adapt to water deficit is vital towardsrational improvements in agricultural practices. Droughtcauses severe disruption in cropping programmes. How-ever, the consequences can be more dire than simple loss inrevenue. It also reduces livestock, threatens human health,engenders widespread ecological degradation andincreases potential for international conflicts (Poster et al.1996). Plants currently account for around 65% of globalfresh water use, but unlike other important commoditiessuch as oil or metals, fresh water has no substitute inagricultural use. It is also not cost effective to transportthe large quantities of water needed in agriculture andindustry more than several hundred kilometres. Diverting

and desalting sea water is also unfeasible because of theevident signs of deterioration in the aquatic environment,broad decimation of fish populations and the extinction ofnumerous beneficial species. It is clear that in the longterm, the only viable and environmental-friendly solutionis to vastly improve prevailing farming practices, whichwill certainly include creating plant varieties through trans-genic and traditional means that are more drought resis-tant (Kates and Parris 2003).

Drought adaptation in plants, however, is a physiolog-ically complex phenomenon. Depending on the intensityor the duration of the stress stimulus, modifications canrange from rapid changes in ion fluxes to optimize cel-lular osmotic pressure, stomatal closing to reduce waterloss through transpiration (. 90%), production of var-ious osmo-protectants to stabilize cellular structures(sugars, proline and proteins). Prolonged drought can

PHYSIOLOGIA PLANTARUM 123: 111–119. 2005 doi: 10.1111/j.1399-3054.2005.00469.x

Printed in Denmark – all rights reserved Copyright # Physiologia Plantarum 2005

Abbreviations – DRE, dehydration-responsive; PP2C, protein phosphatase 2C.

Physiol. Plant. 123, 2005 111

also lead to dramatic alteration in plant growth patterns.For example, in Brassica species which include Arabidop-sis, specialized short roots characterized by their tuber-ized structure and by a lack of hairs are formed whichmay serve to enhance recovery in the aftermath of severedehydration (Vartanian et al. 1994). Despite the complexnature in these responses, it has been noted that anincrease in abscisic acid (ABA) cellular content is largelyassociated with the induction of these adaptive processes.Altered sensitivity to exogenous ABA, particularly at theseed germination stage (Finkelstein et al. 2002), hastherefore been widely used as the phenotypic criterionby which many mutants affected in drought tolerancewere identified in Arabidopsis. These mutants furnishedthe entry points to isolate key regulatory genes and as asource of material to understand the mechanistic aspectsthat underpin the diverse adaptation processes. Morerelevantly, many of these genes in Arabidopsis that relateABA signals to dehydration have orthologues or homo-logues in other species, including economically importantcrops (rice, wheat, barley, maize, etc.), suggesting thatArabidopsis is an adequate surrogate for acceleratingknowledge acquisition concerning many fundamentalregulatory processes in drought physiology.

This brief update is not meant to be an exhaustivereview in the ABA or in the stress physiology field, butan emphasis on the most current advances in addressingthe genetic approach of dissecting adaptive responses todrought. Drought stress activates many ABA-dependentand ABA-independent events and much effort has beendevoted to identifying the components in the major genenetworks in order to understand how the stress informa-tion may be processed and integrated (‘cross-talk’) (Shi-nozaki and Yamaguchi-Shinozaki 2000). Our laboratoryhas recently developed a forward genetic screen to iden-tify mutations that ‘specifically’ disrupt adaptiveresponses to low humidity. Some of the new perspectiveson drought molecular physiology illuminated by theserecent studies are discussed below.

A search for the molecular components – ABA–

dependent and ABA–independent events

Some of the pioneering work in identifying cellular com-ponents in Arabidopsis in osmotic stress regulation hasbeen based on comparative analyses with other modelorganisms. The molecular events in osmo-protection arerelatively well-established in Saccharomyces cerevisiae, inwhich two transmembrane proteins, SHO and SLN1,work as sensors. Mutations affecting SLN1 (sln1D orsln-ts) which encodes a two-component histidine kinase,are lethal because of constitutive activation of a specificmitogen-activated kinase cascade (HOG1) (Maeda et al.1994). The lethal phenotype engendered by sln1D can berescued by the Arabidopsis gene ATHK1, which encodesa structurally similar two-component histidine kinase(Urao et al. 1999). The ATHK1 gene is highly expressedin the roots, particularly under both low- and high-

osmolarity conditions, and therefore, has beenspeculated as the osmosensor in Arabidopsis. Eventsdownstream from the osmosensor are much less clear.The genome of Arabidopsis harbours at least 20 MAPK,10 MAPKK and . 80 MAPKKK (Jonak et al. 2002).The few MAP kinases that have been studied are in factactivated by multiple stress conditions including drought(Mizoguchi et al. 1996, Ichimura et al. 2000). TheMEKK1, MKK2 and two downstream MAP kinasesMPK4 and MPK6 have been proposed to be compo-nents of a MAP kinase module involved in salt andcold stress responses (Teige et al. 2004). However, inother studies, MPK4 has been shown to be weaklyinduced by salt, and only in a narrow range of concen-trations using detached leaves (Ichimura et al. 2000).Moreover, a null mutant for this kinase is not alteredin salt sensitivity (Petersen et al. 2000). There is as yet noevidence to suggest a direct connection between theMAPK module with ATHK1 and ABA signalling.

Owing to the experimental strategy chosen (Ishitaniet al. 1997, Shinozaki and Yamaguchi-Shinozaki 2000),most of the recent emphasis has been placed on dissect-ing the mechanisms of the major transcriptional systemin controlling responses to dehydration. One generalconclusion that can be distilled from these numerousstudies is that many of these drought-induced genes arealso responsive to high salinity and cold (Seki et al.2002), suggesting cross-talk regulation of these environ-mental stresses (Fig. 1). One level of signal cross-talk andintegration of cold and drought stimuli in fact occursdirectly at the promoters of the various downstreamtarget genes due to the striking similarity between thecis-acting dehydration-responsive (DRE), and the cold-responsive (C repeat) elements which share the coremotif CCGAC (Yamaguchi-Shinozaki and Shinozaki1994, Haake et al. 2002). Transcription factors, knowncollectively either as DREB or CBF, that bind specific-ally to the DRE/C repeat have been isolated. While thetranscripts of the first three CBF factors identified wereinduced by cold, the fourth known as CBF4 was distinc-tive in its responsiveness to both ABA and dehydration(Haake et al. 2002) (Fig. 1). Despite the distinctiveness ofthe input stimuli that activate these transcription factors,over-expression of CBF4 alone is sufficient to induce corgenes that are implicated in cold tolerance (for example,cor15a and cor78a). Furthermore, over-expression ofCBF4 in transgenic Arabidopsis was found to conferboth freezing tolerance and to increase the capacity ofthe plant to recover after dehydration stress withoutprior acclimation or drought treatment (Haake et al.2002).

Many drought-, cold stress-, as well as high salinity-inducible genes are also responsive to ABA (Shinozakiand Yamaguchi-Shinozaki 2000) because the promotersof these genes contain another motif known as the ABA-Responsive Element or ABRE (PyACGTGGC). TheseABRE motifs are known target binding sites for theb-zip transcription factors (Shinozaki and Yamaguchi-Shinozaki 2000) (Fig. 1). The rd29B gene of Arabidopsis

112 Physiol. Plant. 123, 2005

is induced by drought as well as high salinity, and itsdrought responsiveness requires endogenous ABA as thisis severely inhibited in mutants that are defective ineither ABA biosynthesis or signalling. The promoterregion of this gene, however, does not possess a DRE/C repeat element, but two copies of the ABRE andtherefore corroborates perfectly with its ABA-inducibil-ity. In contrast, the rd29A gene contains both the DRE/C repeat and the ABRE elements and can thereforeintegrate input stimuli from cold-, drought, high-saltand ABA signalling pathways (Fig. 1). In fact becausethis promoter integrates many stress signals, it has pro-vided a very powerful tool as a general reporter in iden-tifying mutations affecting osmotic and cold-stress signaltransduction (Ishitani et al. 1997, Xiong et al. 1999, seebelow).

A second ABA-dependent pathway was revealed fromthe analysis of rd22, another drought-responsive gene(Yamaguchi-Shinozaki and Shinozaki 1993) (Fig. 1).Motifs that are MYC and MYB recognition sequencesare essential for induction of expresson of this gene byABA and drought; furthermore, ABA-inducible MYCand MYB transcription factors may function co-opera-tively in the ABA-dependent expression of rd22 (Uraoet al. 1993, Abe et al. 1997, 2003). This different timingof induction of these transcription factors coupled withthe diverse regulatory motifs in the promoters of their

target genes have provided a simple heuristic model inexplaining the duration and integration of networks instress adaptation (Shinozaki and Yamaguchi-Shinozaki2000).

Direct identification of regulatory genes in drought

response

We are particularly interested in dissecting drought stressresponses by identifying relevant mutations that disruptkey regulatory genes. Even a small genome such as thatof Arabidopsis is, however, replete with gene families.Nonetheless, we believe that a well-designed forwardgenetic approach can often surmount the problem ofgenetic redundancy as attested by the numerous muta-tions that have already been uncovered affectinghormone signalling that involve gene families. As men-tioned above, previous screens for mutants affected indrought response relied on altered ABA-sensitivity inseed germination, and more recently, based on the activ-ity of a rd29A-Luciferase reporter whose expression isaltered by multiple abiotic stresses (Ishitani et al. 1997).This latter genetic screen, exploited with unprecedentedsuccess by the Zhu laboratory, has produced someremarkable insights into the cast of molecular compon-ents of the salt/cold/drought/ABA signal transductionnetwork. Among these mutants (including some in

Fig. 1. Cross-talk among several ABA-independent and ABA-dependent stress responsive pathways (modifed from Shinozaki and Yamaguchi-Shinozaki 2000; Haake et al. 2002). Different abiotic stresses, such as low temperature or drought, can activate distinct transcription factors.These pathways may interact at multiple levels, one of which takes place directly at promoters of downstream target genes. Hence, for example,rd29A can integrate signal inputs from cold as well as ABA-dependent and ABA-independent pathways owing to the coexistence of elementsthat are binding sites for specific transcription factors. Some of the transcription factors of the b-zip class seem to have specific affinity for theABRE elements found in ABA-responsive promoters. Certain b-zip proteins may be phosphorylated (Uno et al. 2000). The Figure is modifiedfrom Haake et al. (2002) and Shinozaki and Yamaguchi-Shinozaki (2000).

Physiol. Plant. 123, 2005 113

ABA biosynthesis, Xiong et al. 2001a, 2002a), fiery1(fry1) was shown to be impaired in a bifunctional enzymewith inositol polyphosphate 1-phosphatase activities,providing evidence that phosphoinositides function assecond messenger in stress and ABA signalling (Xionget al. 2001b). Other components resulted from thisscreens clearly underline mRNA metabolism as a sur-prisingly recurring theme in the stress signalling network.fry2, which causes a slight resistance to ABA and to salt,but increase sensitivity to cold, is mutated in a geneencoding a novel protein with domains similar to RNApolymerase II C-Terminal Domain phosphatase and toDSRM (the prototype double-stranded RNA-bindingmotifs) (Xiong et al. 2002b). The DSRM motifs arecritical for the function of the protein, as a particulargenetic lesion (fry2-1) removing these motifs behavessimilar to null alleles (Xiong et al. 2002b). This hints atthe prospect of some structured RNA as potential inter-mediates in stress and ABA signalling. The LOS4 locusencodes a DEAD-box RNA helicase which positivelyregulates cold acclimation and cold signalling throughat least CBF3-mediated regulons (Gong et al. 2002). Thecorresponding mutant, however, also shows a slighthypersensitivity to ABA based on the higher expressionof the luciferase reporter gene as compared to that in thewild type. At present, it is unclear how RNA metabolismmediated by LOS4 leads to the proper mounting ofprotective mechanisms against chilling stress. In fact,with only one exception (Dalmay et al. 2001), the phy-siological functions of the 50 or so helicases in the Ara-bidopsis genome are not known. It could be that LOS4may be required to unwind cold-stabilized secondarystructures in RNAs (for example, CBF mRNA), a situ-ation that has precedence in cyanobacteria during chillingresponse (Yu and Owttrim 2000). The SAD1 gene – iso-lated through a mutant that is defective in the positivefeedback in drought-induced ABA biosynthesis –encodes a polypeptide similar to the multifunctionalSm-like U6 small nuclear ribonucleoproteins (snRNP)in animals and yeast that are required for mRNA meta-bolism and implicated in regulating nuclear receptoractivities (Xiong et al. 2001c). Gene profiling of thesad1 mutant indicated that the lesion affected surpris-ingly few genes, including those encoding protein phos-phatases 2C (PP2Cs) such as ABI1. Since these PP2Cscan act as negative regulator of ABA and salt signalling,this could partly explain the hypersensitivity of themutant to these stress stimuli (Xiong et al. 2001c). Infact, the basal transcript level of another PP2C is affectedin the ABA hypersensitive mutant abh1 reduced in anuclear mRNA cap-binding protein (Hugouvieux et al.2001). Moreover, it appears that disruption of CBP80gene encoding another putative nuclear mRNA cap-binding protein renders the plant less sensitive to ABA(discussed in Xiong et al. 2001c). How such seeminglygeneral machinery in mRNA metabolism can generatehighly biased phenotypes is intriguing. It is possible thatthese latter RNA-binding proteins may regulate thedecay rate of mRNAs of components that are involved

in the early steps of ABA signalling. In addition to FRY2mentioned above, another double-stranded RNA-bind-ing protein HYL1, which affects small RNA metabolism,has been implicated in some aspects of ABA signalling,among other pleiotrophic effects (Lu and Fedoroff 2000,Han et al. 2004, Vazquez et al. 2004). This class of RNA-binding proteins may constitute critical regulators ofstress responses since these adverse conditions can alsoinduce the expression of many micro or small interferingRNAs that can form transient double-stranded regionswith the target transcripts (Sunkar and Zhu 2004). How-ever, most of these small RNAs show a high basal level ofexpression (Sunkar and Zhu 2004), suggesting that theymay have an additional role in plant growth processesthat is not related to stress.

To increase the coverage of mutations that mightaffect ‘specifically’ drought-responsive pathways, wetook advantage of stomatal movements as a natural,rapid and robust cellular reaction to low humidity.Because this is a microscopic response, the technicalchallenge is how best to quantify this appropriately fora large-scale genetic screen in which specific mutationsare expected to occur at relatively low frequencies incomparison with the high background level. Manyyears ago, an ingenious idea of observing stomatalresponse to ABA in barley by using remote infraredthermography was proposed (Raskin and Ladyman1988). The underlying principle is that transpirationleads to cooling of leaf temperatures due to latent heatloss. In response to reduced humidity (or ABA treat-ment), the stomatal pores close within minutes to reducetranspiration, and consequently, increases the averagefoliar temperature. We have adapted this imaging tech-nology to screening mutagenized populations of Arabi-dopsis (Merlot et al. 2002) for mutants that might bealtered in stress adaptation (Fig. 2). The progressivedrought conditions in our experimental set-up areclose to those encountered in natural field conditions(Vartanian et al. 1994): after a period of « normal »growth, the plants are deprived of water for 3 to 4 daysand the foliar temperatures are visualized with the aid ofan infrared camera. Wild-type plants invariably closetheir stomata in response to the mild water deficit,whereas mutants that fail to respond correctly wouldcontinue to transpire and are betrayed by their coolerleaf surface temperature. We have isolated a diversity ofmutations that displayed enhanced sensitivity todrought, which belong to at least eight genetic comple-mentation groups. Four of these affect ABA biosynthesis(aba1, aba2, aba3 and possibly a new allele of aao3;Merlot et al. 2002; M. Riera, C. Valon and J. Leung,unpublished data) with the remaining ones blockingABA responses (abi1-1, ost1, ost2 and possibly a novelmutation on chromosome 5) (Merlot et al. 2002;M.Riera, C. Valon and J. Leung, unpublished data).Thus, although our genetic screen is based directly onprogressive drought stress and stomatal closing as thediagnostic physiological response, all of the mutationsrecovered nonetheless affect biosynthesis of, or sensitivity

114 Physiol. Plant. 123, 2005

to, ABA as part of their phenotypic consequences.These results are somewhat unanticipated becausenumerous studies based on gene expression profileshad proposed concerted action of both ABA-dependentand -independent pathways. Our genetic screen ratherreaffirms ABA as the predominant factor, at least inactivating immediate or rapid physiological responsesto low humidity. We note, however, many of theseresponsive mutants, with the exception of ost1 (seebelow), are actually pleiotropic in phenotypes (F.Fenzi and J. Leung, unpublished observations),indicating that the genetic screen can capture generaldrought-sensitive mutations and not just those affectingstomatal movements.

We initially focused on OST1 as the target for ourmolecular studies because the mutational effects seemedconfined to guard cells, as we could not detect anyobvious altered ABA sensitivity in the roots nor in theseeds (Mustilli et al. 2002). This clearly hints at theadvantage of using the OST1 gene to eventually engineerplants with enhanced tolerance to drought because itwould modify the behaviour of only a single cell type,and would not be expected to have dramatic impact onthe architecture of the plants which may have commer-cial value. It is also noteworthy that even though the ost1guard cells are insensitive to ABA, they responded nor-mally to two other environmental signals tested, CO2

and light, which are also known to stimulate stomatalmovements. Again, in the context of plant engineering,

modifying OST1 expression would not expect to com-promise other important signal transduction pathwaysexcept drought/ABA responses.

We isolated the OST1 gene by combining positionalcloning and gene candidate approaches (Mustilli et al.2002) (Fig. 3); but it was also cloned simultaneously andindependently in K. Shinozaki’s group by using a reversegenetics strategy (Yoshida et al. 2002). Whether OST1/snRK2e corresponds to the ABA-activated kinase thatcan phosphorylate the two ABA-Responsive ElementBinding proteins (AREB1 and AREB2) is an intriguingpossibility (Uno et al. 2000, Yoshida et al. 2002). Thepredicted gene product of OST1 is composed of an N-terminal domain that is homologous to the SNF1(Sucrose Non-Fermenting) family of serine–threoninekinases, and a unique C-terminal segment that suggestsregulatory functions (Fig. 3A). The OST1 protein sharesbetween 68 and 80% sequence homologies with nineother homologues in the Arabidopsis genome (Fig. 3B).The SNF1 kinases are highly conserved in many organ-isms as well, including yeast and mammals, and theaction of this class of kinases is generally attributed toenergy balance. The tissue-specific expression of OST1as ascertained by promoter–reporter gene fusion isrestricted to, not surprisingly, guard cells, and to rootvascular tissues (Mustilli et al. 2002). The precisefunction of this kinase in root vascular tissues is not clearas no obvious abnormality has been discerned fromvisual and limited physiological analyses in the ost1mutant (F. Fenzi; data not shown). It is possible thatsome of the other OST1-related kinases could beexpressed in the root tissues, thereby providing morefunctional redundancy in this tissue in comparison withguard cells. Another possibility is that OST1 may regu-late other pathways in the root which are unrelated toresponses to dehydration or ABA. Under our experimen-tal conditions of progressive drought or brief ABA treat-ments, the level of the OST1 transcript does not changeappreciably (Mustilli et al. 2002), but it seems to bestimulated by about two-fold in guard cell protoplastsisolated from plants 4 h after spraying with 100mM ABA(Leonhardt et al. 2004). The physiological significant ofthis transcript up-regulation in the context of stomatalclosure is not clear. In contrast, while the endogenousOST1 kinase activity is barely detectable in plants grownunder non-stressed conditions, it is up-regulated by lowhumidity (Yoshida et al. 2002) or by exogenous ABAtreatments (Mustilli et al. 2002, Yoshida et al. 2002).These results clearly indicate post-translational control ofOST1 as critical for its physiological functions in rapidresponses, but the possibility that the gene may also betranscriptionally up-regulated in prolonged ABA/stressconditions can not be excluded (Leonhardt et al. 2004).Taken together, the data suggest that OST1 could beregulated by several distinct mechanisms.

The advantage offered by Arabidopsis is that since it isa genetically tractable organism, it is often possible toplace the putative physiological role of a gene productwithin a signalling cascade by tests of epistasis. The

Fig. 2. The principle of the genetic screen for Arabidopsis mutantsaltered in adaptive responses to progressive drought stress usingstomatal closure as a diagnostic phenotype. Remote infraredthermography was used as an indirect means to measure the rateof transpiration. Wild-type plants respond to mild dehydration byclosing their stomata to limit water loss through transpiration andthereby display a warmer leaf temperature (approximately 22.5�C inthis example). Three mutants altered in this natural response areshown as examples. The mutants ost1 and ost2 are altered instomatal response to ABA and to low humidity as they continue totranspire excessively (approximately 21.8�C). Similarly, the aba1mutant also fails to mount a proper response to dehydration due toinsufficient increase in ABA biosynthesis. The leaf temperatures arenot absolute but relative values read by the camera using ambienttemperature (24�C) as the reference. These temperatures are thenconverted into false colours using imagery programs publiclyavailable (NIH, version 4) and are displayed on the scale to theright.

Physiol. Plant. 123, 2005 115

mutation, abi1-1, affecting a specific protein phosphatase2C has been shown to cause a disruption in ABA-inducedproduction of reactive oxygen species (ROS or H2O2) andCa21 increase (Murata et al. 2001). That the OST1 couldfunction in the ABI1 pathway was suggested by the factthat the ost1 mutation also severely reduces ABA-inducedROS production (Mustilli et al. 2002). Indeed, applicationof exogenous H2O2, or another downstream signallingintermediate Ca21, restores stomatal closure and ABAinhibition of stomatal opening in the ost1 mutant (Mustilliet al. 2002). Moreover, ABA-inducible OST1 activity isalso blocked by abi1-1 (Mustilli et al. 2002), but interest-ingly not by abi2-1, a mutation affecting the closest PP2Chomologue (Fig. 4). Despite the highly similar architectureof these two protein phosphatases, and that both abi1-1and abi2-1 affect the same amino acid substitutions in therespective phosphatase domains, they seem to have differ-ent impact on some of the responses to exogenous ABA orwater stress (Gilmore and Thomashow 1991, Vartanianet al. 1994, Gosti et al. 1995, de Bruxelles et al. 1996,Soderman et al. 1996, Pei et al. 1997, Savoure et al. 1997,Guo et al. 2002). This further supports the suggestion thatABI1 and OST1 indeed function in the same pathway.Altogether, these physiological tests coupled to genetictests have allowed us to establish the rough sequence ofevents (Fig. 5) leading to stomatal response to ABA/drought. The levels of two of the ABA-inducible tran-scripts, rd22 and rd29B, are also reduced in the ost1 null

mutant (Yoshida et al. 2002), consistent with the hypoth-esis that the kinase may have transcription factors such asb-ZIP and MYC/MYB as downstream targets (Shinozakiand Yamaguchi-Shinozaki 2000) (Fig. 5).

The closest relative to OST1 in the sequence data baseis the ABA-activated protein kinase AAPK of Vicia faba

Fig. 3. A, Gene structure ofOST1. The shaded boxesrepresent the exons and thesize of the gene is denoted bythe number of nucleotides. Thedifferent alleles, consisting ofthree base changes and theT-DNA insertion (snRK2e),are shown above the gene. Theost1-2 is an amino acidsubstitution in the ATP-binding site, while both ost1-1and ost1-3 affect spliceacceptor sites. The predictedprotein is 362 amino acid(41kDa is theoretical molecularweight) with homology to SNF1kinases in its N-terminaldomain, while the C-terminalpart is divergent in sequence ascompared to other members inthis family, thereby suggestingthat it may be regulatory infunction. B, Relation tree ofOST1 and related proteins fromArabidopsis and other plantspecies (AAPK, Vicia faba;PKABA, barley).

Fig. 4. In planta kinase activity of OST1. Root extracts from 2-week old plants treated with 10 mM ABA (1) or solvent (–) werefractionated by electrophoresis in polyacrylamide gels containinghistone as a substrate for the kinase. Extracts from guard cellprotoplasts (Mustilli et al. 2002) and root tissues gave similarresults. Roots have been frequently used because they can beconserved easily and they also allow larger scale experiments to beperformed. Ler correspond to the wild-type Landsberg erecta. Themutations abi1-1 and abi2-1 affect two homologous proteinphosphatases 2C which have been implicated in ABA anddrought signal transduction. The predicted 41 kDa kinase activityin Ler is activated by ABA, whereas this activity is suppressed bythe mutations ost1-2 and abi1-1, but not by abi2-1. The 41 kDakinase activity is OST1 as demonstrated in an independent study byimmunoprecipitation with specific anti-OST1 antibodies (Yoshidaet al. 2002). Our results also indicate that ost1 is epistatic to abi1-1,suggesting these two elements may belong to the same signallingpathway.

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(Fig. 3B), a guard cell protein kinase initially identifiedby in-gel kinase assays (Li et al. 2000). The AAPKcDNA was cloned based on peptide sequence obtainedby de novo mass spectrometric sequence analysis. OST1is 79% identical to AAPK. In ost1 mutant Arabidopsisguard cells and in V. faba guard cells expressing a dom-inant negative form of AAPK, both exhibit a failure toeffect stomatal closure in response to ABA. Nonetheless,one apparent major difference between the two kinases isthat ost1 also causes ABA-insensitivity in stomatal open-ing, whereas this is not the case with AAPK. OST1 isalso homologous to the barley PKABA1 (Fig. 3B),another kinase more distantly related to OST1, whichfunctions in seed maturation (Gomez-Cadenas et al.1999).

In a series of elegant experiments, the laboratory ofS.Assmann has identified a downstream target of AAPKphosphorylation. This target is a specific RNA-bindingprotein named AKIP1, which when activated by phosphor-ylation, can bind to dehydrin mRNA and becomes concen-trated in nuclear speckles (Li et al. 2002). Since expression ofdehydrin mRNA is unlikely to be restricted to guard cells,the functional significance of its association with AKIP isthus not clear and may reflect a more general response toABA/drought. The closest Arabidopsis RNA-binding pro-tein homologues to AKIP1 is the UBA2a protein (56%identity) (Lambermon et al. 2002). If the OST1/AAPKpathway is functionally conserved, this would suggest thatUBA2a may be a target of OST1 phosphorylation. WithArabidopsis as a more accessible genetic model, the variousroles of these proteins in the physiological context of ABAor drought signalling can now be better defined.

Conclusions

Osmotic stress activates the expression of many plantgenes through both ABA-dependent as well as ABA-inde-

pendent pathways. In identifying major transcriptionnetworks, the promoters from target genes induced byosmotic shock have been employed to dissect cis-actingsequences and to isolate corresponding transcriptionfactors. Pioneering work on rd29A and rd29B has led toidentification of both ABA- and drought-responsivemotifs that are essential for their activation by specificstresses. Although many of their upstream transcriptionfactors, such as DREB1 (cold) and DREB2 (osmoticchange), are induced by distinct environmental assaults,target promoters (for example, rd29A) are often promis-cuous in that they contain a combination of motifs thatwould permit signal cross-talk directly at the level oftarget gene transcription (Fig. 1). This is also consistentwith recent mRNA profiling which demonstrated thatmany of the same genes are indeed receptive to multiplestress input (Cheong et al. 2002, Seki et al. 2002).

We have established an original experimental condi-tion to impose progressive drought and the visualizationof stomatal movement as the phenotypic criterion toidentify mutations specifically affecting adaptiveresponses. Isolation of the OST1 gene has provided apotentially useful tool for engineering drought-tolerantplants because the mutation seems to alter only thestomatal response. Moreover, its kinase activity itself isup-regulated by either ABA or low humidity suggestingthat in normal growth conditions, the gene should notcompromise plant growth by, for example, constitutiveexpression. In collaboration with Marise Borja of theFundacion Promiva, we have introduced the genomicclone encoding OST1 into a male-sterile variety of Petunia(unpublished results), because this popular garden planthas a major drawback of being a heavy consumer of freshwater. This test will allow us to determine whether OST1could function as a quantitative sensor of dehydration inanother plant species, and if so, it would also stronglysuggest that many of the other components in the Arabi-dopsis OST1 pathway would be also conserved in Petuniaas well as other plants. Similar work in the past hasconfirmed the utility of using Arabidopsis genes, includingtranscription factors or stress-activated MAP kinasecomponents, not only as models of investigation into thefundamental nature of their regulation, but also as pro-mising tools to improve stress tolerance in other plantspecies (for some examples, see Jaglo-Ottosen et al. 1998,Kasuga et al. 1999, Shinozaki and Yamaguchi-Shinozaki2000, Thomashow 2001, Teige et al. 2004)

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