aba signallingh

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Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998. 49:199222

Copyright c 1998 by Annual Reviews. All rights reserved

ABSCISIC ACID SIGNAL

TRANSDUCTION

Jeffrey Leung and J er ome GiraudatInstitut des Sciences Vegetales, Unite Propre de Recherche 40, Centre National de la

Recherche Scientifique, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette, France;e-mail: [email protected]

KEY WORDS: guard cell, mutants, second messenger, seed development, stress tolerance

ABSTRACT

The plant hormone abscisic acid (ABA) plays a major role in seed maturation

and germination, as well as in adaptation to abiotic environmental stresses. ABA

promotes stomatal closure by rapidly altering ion fluxes in guard cells. Other

ABA actions involve modifications of gene expression, and the analysis of ABA-

responsive promoters has revealed a diversity of potential cis-acting regulatory

elements. The nature of the ABA receptor(s) remains unknown. In contrast,

combined biophysical, genetic, and molecular approaches have led to consid-

erable progress in the characterization of more downstream signaling elements.

In particular, substantial evidence points to the importance of reversible pro-

tein phosphorylation and modifications of cytosolic calcium levels and pH as

intermediates in ABA signal transduction. Exciting advances are being made in

reassembling individual components into minimal ABA signaling cascades at the

single-cell level.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

ABA SIGNAL TRANSDUCTION IN SEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Seed Dormancy and Germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200Reserve Accumulation and Acquisition of Desiccation Tolerance . . . . . . . . . . . . . . . . . . . 202

ABA SIGNAL TRANSDUCTION IN STRESS RESPONSE . . . . . . . . . . . . . . . . . . . . . . . . . . 206

Regulation of Gene Expression in Response to Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Regulation of Stomatal Aperture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

CONCLUSION AND PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

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1040-2519/98/0601-0199$08.00


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200 LEUNG & GIRAUDAT

INTRODUCTION

The scientific origins of abscisic acid (ABA) have been traced to several in-

dependent investigations in the late 1940s, but it was only in the 1960s thatABA was isolated and identified (1). Mutants affected in ABA biosynthesis

are known in a variety of plant species (39, 146). The characterization of these

mutants, together with physicochemical studies, has enabled the pathway of

ABA biosynthesis to be elucidated in higher plants (134, 135, 146, 166). Two

ABA biosynthetic genes have been recently cloned (88, 135) and should provide

insights into the regulation and the sites of ABA biosynthesis.

A large body of evidence indicates that ABA plays a major role in adap-

tation to abiotic environmental stresses, seed development, and germination.

The present review focuses on our current knowledge concerning how the ABAsignal could be faithfully transduced to mediate these well-characterized phys-

iological and developmental processes. Physicochemical and molecular ge-

netic approaches have already provided fundamental insights into the diversity

of ABA perception sites and other downstream components that could couple

ABA stimuli to particular responses. It should be emphasized that many con-

cepts about the relative importance of these components in the ABA signaling

network are far from firmly established. Nonetheless, it is clear that the present

development of single-cell systems should allow some of these components to

be assembled into minimal signaling pathways within a cellular context.

ABA SIGNAL TRANSDUCTION IN SEEDS

Endogenous ABA content peaks during roughly the last two thirds of seed de-

velopment before returning to lower levels in the dry seed (121). ABA is thus

thought to regulate several essential processes occurring during the develop-

mental stages that follow pattern formation of the embryo. These processes

include the induction of seed dormancy, the accumulation of nutritive reserves,

and the acquisition of desiccation tolerance.

Seed Dormancy and Germination

Exogenous ABA can inhibit the precocious germination of immature embryos

in culture (121). Embryos of the ABA-biosynthetic mutants from maize exhibit

precocious germination while still attached to the mother plant or vivipary

(91). Seeds of the ABA biosynthetic mutants fromArabidopsis thaliana (71, 77)

andNicotiana plumbaginifolia (88) fail to become dormant. These observations

support that endogenous ABA inhibits precocious germination and promotes

seed dormancy.Thus far, our knowledge on the signaling elements that mediate the regulation

of seed dormancy and germination by ABA is primarily derived from genetic

analysis. As summarized in Table 1, mutations that alter the sensitivity to ABA


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ABA SIGNALING 201

Table 1 Main characteristics of the various mutations known to affect ABA sensitivity

Species Mutation Dominancea Phenotype Gene productb Ref.

Hordeum cool ABA insensitivity 118

vulgare in guard cells

Zea mays vp1 R ABA insensitivity Seed-specific 92, 1 20

in seeds transcription

factor

rea R ABA insensitivity 144

in seeds

Craterostigma cdt-1 D Constitutive ABA Regulatory 31

plantagineum response in callus RNA or short

polypeptide

Arabidopsis abi1 SD ABA insensitivity Protein phos- 72, 78, 9 7thaliana phatase 2C

abi2 SD ABA insensitivity Protein phos- 72, 7 9

phatase 2C

abi3 R ABA insensitivity Seed-specific 38, 7 2,

in seeds transcription 102, 108

factor

abi4/5 R ABA insensitivity 27

in seeds

era1 R ABA hypersensitivityc subunit 24

of farnesyl

transferaseera2/3 R ABA hypersensitivityc 24

gca1/8 ABA insensitivityd 10

axr2 D Resistance to auxin, 163

ethylene, and ABAd

jar1 R Resistance to MeJa and 143

hypersensitivity to ABAc

jin4 R Resistance to MeJa 11

and hyper-sensitivity

to ABAc

bri1 R Resistance to brass- 22

inosteroids and hyper-sensitivity to ABAd

sax R Hypersensitivity to auxin f

and ABAe

Nicotiana plum- iba1 R Resistance to auxin, Molybdenum 13, 8 0

baginifolia (aba1)g cytokinin, and ABAc cofactor

biosynthesis

aDominance of the mutant alleles over wild-type; D, dominant; SD, semidominant; R, recessive.bMolecular function of the product of the wild-type gene.cSensitivity of seed germination to exogenous ABA.dSensitivity of seedling (root) growth to exogenous ABA.eSensitivities of root growth and stomatal closing to exogenous ABA.fM. Fellner and G. Ephritikhine, personal communication.gThe IBA1 locus was renamed ABA1 when it was found that iba1 and other alleles are in fact ABA-deficient, as

a result of a defect in the biosynthesis of a molybdenum cofactor that is required for multiple enzymatic activitiesincluding ABA aldehyde oxidase (the last step in ABA biosynthesis).


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in seeds have been described in maize and in A. thaliana, and several of the

corresponding genes have been cloned.

The maize vp1 (viviparous1) (119) and rea (red embryonic axis) (144) muta-

tions lead, like ABA biosynthetic mutations in this species, to vivipary. How-ever, vp1 (120) and rea (144) embryos do not have reduced ABA content but

rather exhibit a reduced sensitivity to germination inhibition by exogenous

ABA in culture. As is discussed below, the VP1 gene encodes a seed-specific

transcription factor (92).

The A. thaliana ABA-insensitive ABI1 to ABI5 loci were all identified by

selecting for seeds capable of germinating in the presence of ABA concen-

trations (310 M) that are inhibitory to the wild type (27, 72). The abi1

(72), abi2 (72), and abi3 (72, 101, 108) mutants also exhibit, like A. thaliana

ABA-deficient mutants, a marked reduction in seed dormancy. These threeloci thus seem to mediate the inhibitory effects of endogenous ABA on seed

germination. The ABI1 (78, 97) and ABI2 (79) genes encode homologous pro-

tein serine/threonine phosphatase 2C (PP2C) (12, 79). The ABI3 gene is the

ortholog of the maize VP1 gene mentioned above (38). It is presently unclear

whether the ABI1, ABI2, and ABI3 loci act in the same or in partially distinct

ABA signaling cascade in seeds (28, 110).

Mutations in the ERA1 to ERA3 (Enhanced Response to ABA) loci of A.

thaliana were identified by a lack of seed germination in the presence of low

concentrations of ABA (0.3 M) that are not inhibitory to the wild type (24).

The era1 mutations also markedly increase seed dormancy. The ERA1 gene

encodes the subunit of a farnesyl transferase, which may possibly function as a

negative regulator of ABA signaling by modifying signal transduction proteins

for membrane localization (24). The exact relationships between ERA1 and the

above-mentioned ABIloci are unknown.

Reserve Accumulation and Acquisition

of Desiccation Tolerance

The accumulation of nutritive reserves and the acquisition of desiccation tol-

erance are associated with the expression of specific sets of mRNAs (60, 121).

Transcripts encoding either storage proteins or late-embryogenesis-abundant

(LEA) proteins thought to participate in desiccation tolerance can be preco-

ciously induced by exogenous ABA in cultured embryos (60, 121). The char-

acterization of ABA-deficient mutants in A. thaliana (70, 77, 96, 101, 112) and

in maize (91, 109) supports a contribution of endogenous ABA in the develop-

mental expression of these genes in seeds.

The functional dissection of such ABA-responsive promoters, conducted pri-marily in transient expression systems, has identified several cis-acting elements

involved in ABA-induced gene expression.


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ABA SIGNALING 203

Table 2 cis-acting promoter elements involved in the activation of gene

expression by ABA

Gene (Species) Element Sequencea Ref.

Rab16 Motif I GTACGTGGC 141

(Oryza sativa) Motif III GCCGCGTGGC 107

Em Em1a ACACGTGGC 44

(Triticum aestivum) Em1b ACACGTGCC 44

HVA22 ABRE3 GCCACGTACA 138

(Hordeum vulgare) CE1 TGCCACCGG 138

HVA1 ABRE2 CCTACGTGGC 139

(Hordeum vulgare) CE3 ACGCGTGTCCTC 139

C1 Sph CGTGTCGTCCATGCAT 65(Zea mays)

CDeT27-45 AAGCCCAAATTTCACA- 104

(Craterostigma GCCCGATAACCG

plantagineum)

aThe ACGT core in G-box-type ABREs is underlined.

CIS-ACTING PROMOTER ELEMENTS A first category of elements is exemplified

by the related motif I of the Rab16LEA gene from rice (107, 141) and Em1a

motif of the wheat Em LEA gene from wheat (44). These sequences share

a G-box ACGT core motif (Table 2) and have been designated ABREs, for

ABA Response Elements. ABRE-related sequence motifs are present in many

other ABA-inducible genes, although their function in ABA signaling often

remains speculative in the absence of experimental tests. For instance, only

a subset of the multiple ABRE-like motifs present in certain promoters are

indeed required for ABA regulation (20, 48, 117, 138, 139). A second type of

cis-acting element has been identified in the maize C1 gene, a regulator of

anthocyanin biosynthesis in seeds. The ABRE-like motifs present in C1 are

not major determinants of the ABA responsiveness of this gene, whereas the

distinct sequence motif designated as Sph element (Table 2) is essential for

ABA induction of the C1 promoter (65).

Multimerized copies of ABREs (107, 141, 155) or of the Sph element (65)

can confer ABA responsivity to minimal promoters. Single copies of these

elements were, however, not sufficient for ABA response, suggesting that mul-

timerization substituted for other aspects of the native promoter contexts of

these elements. In fact, the smallest promoter units designated ABRCs shown

to be both necessary and sufficient for ABA induction of gene expression appearto consist of (at least) two essential cis-elements. ABRCs can be composed of

two G-boxes as in the case of the wheatEm promoter, where both the Em1a and


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Em1b G-box motifs (Table 2) contribute to the activity of the 75-bp Complex

I (44, 155). In contrast, several other ABRCs consist of an ABRE and of a

cis-element not related to G-boxes. The G-box-type motif I and the distinct

motif III are essential for the ABA responsiveness of a 40-bp fragment of thericeRab16B promoter (107). In the barleyHVA22 promoter, ABRC1 is a 49-bp

fragment that comprises ABRE3 and the Coupling Element CE1 located 20-bp

downstream (138). In the barley HVA1 promoter, the 22-bp long ABRC3 con-

sists of the coupling element CE3 directly upstream of ABRE2 (139). These

results indicate that a diversity of ABRCs participate in ABA signaling in

seeds.

TRANS-ACTING FACTORS The nature of the transcription factors that mediate

ABA regulation of seed genes via the various above-mentioned cis-elementsremains largely unknown. Most proteins that bind DNA sequences with ACGT

core motifs contain a basic region adjacent to a leucine-zipper motif (bZIP-

proteins). Several plant bZIP-proteins that bind to ABREs in vitro and/or are in-

ducible (at the mRNA level) by ABA have been cloned (44, 68, 74, 100, 103, 105).

However, few of these genes are known to be expressed in seeds, and thus far

none has been unambiguously shown to be involved in ABA signaling in vivo

(83).

In contrast, genetic and molecular evidence supports that the maize VP1

and A. thaliana ABI3 proteins are homologous transcription factors essential

for ABA action in seeds. The vp1 (91, 109) and abi3 (28, 101, 108, 112) mu-

tants are similarly altered in sensitivity to ABA, and in various other aspects

of seed maturation including the developmental expression of storage proteins

and LEA genes. The maize VP1 (92) and A. thaliana ABI3 (38) genes, as

well as the rice OsVP1 (47) and Phaseolus vulgaris PvALF(19) genes, encode

homologous proteins (Figure 1). These genes are all exclusively expressed

in seeds (19, 47, 92, 112). However, when ectopically expressed in transgenic

A. thaliana plants, ABI3 rendered vegetative tissues hypersensitive to ABA and

Figure 1 Schematic diagram of the architecture of the VP1/ABI3-like proteins. The maize VP1

(92), A. thaliana ABI3 (38), rice OsVP1 (47), and Phaseolus vulgaris PvALF (19) proteins display

a similar and novel structural organization. They contain, in particular, four domains of high aminoacid sequence identity: the A domain located in the large acidic () N-terminal region and three

basic domains designated as B1, B2, and B3 in order from the N terminus. The sizes of these

proteins range from 691 (VP1) to 752 (PvALF) amino acids.


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ABA SIGNALING 205

activated expression of several otherwise seed-specific genes in leaves when ex-

ogenous ABA was supplied (110, 112). Similarly, in transient expression stud-

ies, VP1 (65, 92, 155), OsVP1 (47, 48), and PvALF (18, 19) could trans-activate

various seed-specific promoters. When investigated, these trans-activationswere found to require the acidic N-terminal domain of the relevant VP1/ABI3-

like protein (48, 92), and domain-swapping experiments showed that the acidic

N-terminal regions of VP1 (92) and PvALF (19) are indeed functional transcrip-

tional activation domains. Thus, taken together, the above-mentioned results

indicate that VP1/ABI3-like proteins act as transcription factors.

The maize vp1 (56, 91, 109) and A. thaliana abi3 (70, 77, 101, 108, 112) mu-

tants exhibit a larger spectrum of seed phenotypes than the ABA-deficient

mutants in the corresponding species. A possible explanation would be that the

physiological roles of VP1 and ABI3 are not strictly confined to ABA signal-ing and rather that these proteins integrate ABA and other seed developmental

factors. This model is also consistent with the observation that VP1/ABI3-

like proteins can substantially trans-activate seed promoters in the absence

of (added) ABA (19, 48, 92, 110), and that, within a given promoter, the cis-

elements involved in VP1 responsiveness are partially separable from those

involved in ABA responsiveness (65, 155).

In the wheat Em promoter, the ABRE Em1a (Table 2) that is essential for

ABA induction also mediates transactivation by VP1 and synergism between

ABA and VP1 (155). The conserved domain B2 of VP1 is required for Em

transactivation (54), but its exact role in this process remains unclear (54, 131).

Furthermore, no specific binding of VP1 to G-box-like ABREs has been ob-

served thus far (54, 92, 145), suggesting that VP1 is a coactivator protein that

physically interacts with G-box-binding proteins (91, 155). Unlike for Em,

the combined response of the maize C1 promoter to ABA and VP1 is less

than additive , and transactivation of the C1 promoter by VP1 is mediated ex-

clusively by the sequence CGTCCATGCAT located within the Sph promoter

element (Table 2) (65). The conserved basic domain B3 of VP1 is essential

for C1 transactivation, and the isolated B3 domain binds specifically to the

above-mentioned sequence element in vitro (145). VP1 is, however, likely to

interact with other DNA-binding proteins involved in ABA regulation of C1,

because the entire Sph element is required for responsiveness of C1 to both

ABA and VP1 (65). The VP1/ABI3-like proteins thus appear to be multifunc-

tional transcription factors that integrate ABA and other regulatory signals of

seed maturation, most likely by interacting with distinct trans-acting factors

that remain to be identified.

UPSTREAM SIGNALING ELEMENTS Genetic studies in A. thaliana have iden-

tified a few other intermediates in the ABA regulation of gene expression in

seeds. The already mentioned abi4 and abi5 mutants share with abi3 mutants


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a decreased ABA sensitivity in germinating seeds, as well as a reduced devel-

opmental expression of certain LEA genes. These three loci have thus been

proposed to act in a common regulatory pathway of seed maturation (27). In

addition, the recent analysis of abi3 fus3 and abi3 lec1 double mutants in-dicates that ABI3 acts synergistically with the FUSCA3 (FUS3) and LEAFY

COTYLEDON1 (LEC1) genes to control multiple elementary processes dur-

ing seed maturation, including sensitivity to ABA and accumulation of storage

protein mRNAs (111). The ABI4, ABI5, FUS3, and LEC1 gene products are

currently unknown.

Studies on barley aleurone protoplasts indicate that reversible protein phos-

phorylation is likely to be implicated in the regulation of gene expression by

ABA in seeds. ABA rapidly stimulated the activity of a MAP kinase, and

this stimulation appeared to be correlated with the induction of the Rab16

mRNA (69). Okadaic acid (an inhibitor of the PP1 and PP2A classes of ser-

ine/threonine protein phosphatases) inhibited the induction of the HVA1 (73)

and Rab16 (51) mRNAs by ABA, and phenylarsine oxide (an inhibitor of ty-

rosine protein phosphatases) blocked the induction ofRab16(51). The protein

kinase and phosphatases revealed by these studies remain to be isolated.

Finally, suggestive evidence indicates that modifications of intracellular cy-

tosolic free Ca2+ levels ([Ca]i) and cytoplasmic pH (pHi) may act as intracellular

second messengers in the ABA regulation of gene expression in seeds. In barley

aleurone protoplasts, ABA triggers an increase in pHi (153) and a decrease in

[Ca]i (35, 158). However, the exact contribution of these alterations in [Ca]i and

pHi to the regulation of gene expression by ABA could not be clearly established

(152, 153).

ABA SIGNAL TRANSDUCTION IN STRESS RESPONSE

During vegetative growth, endogenous ABA levels increase upon conditions

of water stress, and ABA is an essential mediator in triggering the plant re-

sponses to these adverse environmental stimuli (166). As is discussed below,

substantial evidence supports that the increased ABA levels limit water loss

through transpiration by reducing stomatal aperture. ABA is also involved

in other aspects of stress adaptation. For instance, ABA-deficient mutants of

A. thaliana are impaired in cold acclimation (87) and in a root morphogenetic

response to drought (drought rhizogenesis) (154). The role of ABA in signaling

stress conditions has also been extensively documented by molecular studies

showing that ABA-deficient mutants are affected in the regulation of numer-

ous genes by drought, salt, or cold (39, 60, 140). It should, however, be notedthat the adaptation to these adverse environmental conditions also involves

ABA-independent pathways (140). Finally, ABA is involved in the induction

of gene expression by mechanical damage (116).


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ABA SIGNALING 207

Regulation of Gene Expression in Response to Stress

PROMOTER STUDIES The regulation of gene expression by ABA in vegetative

tissues appears to involve several signaling pathways. The ABA induction of

distinct genes exhibit differential requirements for protein synthesis (164). In

addition, several types ofcis-acting elements are involved in ABA-induced gene

expression in vegetative tissues. Many of the LEA genes that are abundantly

expressed in desiccating seeds are also responsive to drought stress and ABA in

vegetative tissues (60, 121). For several of these genes, the G-box-type ABREs

already described (Table 2) are required for ABA induction both in seeds and

in vegetative tissues (20, 44, 48, 107, 117).

In contrast, ABRE-like motifs are not involved in the ABA regulation of other

stress-inducible genes such as theA. thaliana RD22 (63) and the Craterostigmaplantagineum CDeT27-45 genes (104). The distinct sequence motif shown in

Table 2 is essential for ABA responsiveness of CDeT27-45 (104). In this

case, as for ABREs (see section on ABA Signal Transduction in Seeds), the

corresponding physiological trans-acting factors are presently unknown. Genes

that are induced by ABA and encode other types of potential transcription

factors include the A. thaliana homeobox gene ATHB-7(142) and several myb

homologues from A. thaliana (151) and Craterostigma (62). The role of these

genes in ABA signaling has, however, not been tested experimentally.

POTENTIAL INTERMEDIATES Studies performed on various model systems

have identified several potential signaling intermediates in the ABA activation

of gene expression in response to stress.

The resurrection plant Craterostigma plantagineum can tolerate extreme de-

hydration. However, in vitro propagated callus derived from this plant has a

strict requirement for exogenously applied ABA in order to survive a severe

dehydration (31). This property has been exploited for isolation of dominant

mutants by activation tagging, in which high expression of resident genes acti-

vated by insertion of a foreign promoter would confer desiccation tolerance to

the transformed cells without prior ABA treatments. One gene was identified

(CDT-1), whose high expression did confer the expected phenotypes in calli

and led to constitutive expression of several ABA- and dehydration-inducible

genes (31). The function of the CDT-1 gene is not immediately obvious, be-

cause it encodes a transcript with no large open reading frame. It is possible

that the biologically active product of CDT-1 is a regulatory RNA or a short

polypeptide (31).

The ABA regulation of gene expression in vegetative tissues is likely to

involve reversible protein phosphorylation events. Several stress- and ABA-inducible mRNAs that encode protein kinases have been identified (5759). In

epidermal peels ofPisum sativum, the ABA-induced accumulation of dehydrin

mRNA was reduced by K-252a (an inhibitor of serine/threonine protein kinases)


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and also by okadaic acid and cyclosporin A (an inhibitor of the PP2B class of

serine/threonine protein phosphatases) (53).

The involvement of particular protein phosphatases 2C was revealed by the

cloning of the ABI1 (78, 97) and ABI2 (79) genes ofA. thaliana. Their corre-sponding mutants show decreased sensitivities to the ABA inhibition of seedling

growth, and defects in various morphological and molecular responses to ap-

plied ABA in vegetative tissues (28, 41, 72). Despite the highly similar archi-

tecture of the ABI1 and ABI2 proteins (79), their functions are unlikely to

be completely redundant for all ABA actions. The abi1-1 and abi2-1 muta-

tions lead to identical amino acid substitutions at equivalent positions in the

ABI1 and ABI2 proteins, respectively (79), but have (at least quantitatively)

different effects on some of the responses to exogenous ABA or water stress

(25, 33, 41, 124, 142, 154).The identification of stress- and ABA-inducible mRNAs that code for a Ca2+-

binding membrane protein in rice (30) and for a phosphatidylinositol-specific

phospholipase C in A. thaliana (55), respectively, provides circumstantial ev-

idence for the possible involvement of Ca2+ in ABA signaling in vegetative

tissues. In addition, ABA has been shown to induce increases in [Ca]i and pHiin cells of corn coleoptiles and of parsley hypocotyls and roots (32).

SINGLE-CELL SYSTEMS Ambitious attempts are being made to reconstruct

minimal stress and ABA signaling pathways by reassembling candidate com-

ponents in single-cell systems.

In maize leaf protoplasts, the barley HVA1 promoter fused to reporter genes

could be activated by applied ABA or by various stress conditions (136). In the

absence of these stimuli, expression of the reporter gene could be induced by

treating the protoplasts with 1 mM Ca2+ plus Ca2+ ionophores, or by overex-

pressing constitutively active forms of the normally Ca2+-dependent ATCDPK1

and ATCDPK1a protein kinases fromA. thaliana. Overexpressing the catalytic

domain of the wild-type A. thaliana ABI1 PP2C inhibited the activation of

HVA1 by ABA or by ATCDPK1 (136).

Microinjection experiments were conducted in hypocotyl cells of the tomato

aurea mutant to investigate the signaling cascade that mediates ABA activa-

tion of the A. thaliana rd29A (165) and kin2/cor6.6 (157) promoters fused to

the GUS reporter gene. This work supports the existence of a minimal linear

cascade in which (a) ABA triggers a transient accumulation of cyclic ADP-

ribose (cADPR) (for information on cADPR, see 3), (b) cADPR induces a

release of Ca2+ from internal stores, and (c) a K-252a-sensitive kinase acting

downstream of Ca

2+

is required for activation of rd29A and kin2 (Y Wu &N-H Chua, personal communication). Microinjection of the mutant abi1-1

protein inhibited ABA-, cADPR-, and Ca2+-induced gene expression, and these


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ABA SIGNALING 209

effects could be reversed by an excess of wild-type ABI1 protein (Y Wu, A

Himmelbach, E Grill & N-H Chua, personal communication).

These two sets of results illustrate that combining genetics with single-cell

analyses represents a promising approach for deciphering possible epistaticrelationships between candidate components in ABA signaling cascades.

Regulation of Stomatal Aperture

The stomatal pore is defined by a pair of surrounding guard cells. The closing

and opening of the pore result from osmotic shrinking and swelling of these

guard cells, respectively. In conditions of water stress, the increase in cellular

ABA (45) or in apoplastic ABA at the surface of guard cells (161) is thought

to provoke a reduction in turgor pressure of the guard cells, which in turn

leads to stomatal closure to limit transpirational water loss. Applied ABA

inhibits the opening and promotes the closure of stomatal pores (8, 16, 159). The

enhanced transpiration of ABA-biosynthetic mutants (71, 77, 88) and transgenic

plants expressing an anti-ABA antibody (7) provides evidence for such a role

of endogenous ABA.

Unlike most other cells in higher-plant tissues, guard cells at maturity lack

plasmodesmata (162). This property has afforded a single-cell system, directly

accessible in planta, to investigate how ABA is perceived and transduced to

trigger integrated physiological responses.

ABA PERCEPTION Available evidence suggests that guard cells possess at least

two sites of ABA perception involved in the regulation of stomatal aperture,

one located at the plasma membrane and a second located intracellularly.

Stomatal closure can be triggered by extracellular application of ABA. How-

ever, the protonated form of the weak acid ABA (ABAH) readily permeates

the lipid bilayer of the cell membrane (64). Guard cells ofCommelina commu-

nis appear to have, in addition, a significant carrier-mediated uptake of ABA

(86, 133). The requirement for an extracellular ABA receptor is indicated by

the failure of ABA to inhibit the stomatal opening when microinjected directly

into the cytosol of Commelina guard cells (5). In this species, extracellular

ABA was, however, less effective in regulating stomatal aperture at high than

at low external pH (that favors passive uptake of ABAH), providing indirect

evidence for an intracellular ABA receptor (5, 106, 113, 133). This conclusion

is also supported by the reports that stomatal closure could be triggered in Com-

melina by ABA released inside guard cells from caged ABA microinjected into

the cytosol (2), and by ABA microinjected into guard cells in the presence of

1 M extracellular ABA (133).ABA closes stomatal pores by inducing net efflux of both K+ and Cl from

the vacuole to the cytoplasm, and from the cytoplasm to the outside of guard


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cells (85, 86). Tracer flux studies, measuring rate of loss of86Rb+ from isolated

Commelina guard cells, also indicate multiple actions of ABA, both inside and

outside the cell. It has been proposed that internal receptors regulate tonoplast

ion channels for release of vacuolar ions, whereas external receptors mediatethe stimulation of ion efflux at the plasma membrane (85, 86).

ION CHANNELS REGULATED BY ABA Electrophysiological studies, either by

whole cell impalement or by patch clamping of the plasma membrane of guard

cell protoplasts or of isolated vacuoles, have identified a number of ionic chan-

nels present in these guard cell membranes. The nature of the tonoplast ion

channels involved in the regulation of stomatal aperture by ABA is not yet

clearly identified (159). Attention is thus focused here on ion transport mech-

anisms across the plasma membrane of guard cells.

ABA causes a depolarization of the plasma membrane (149). Two types of

anion channels, which may reflect different states of a single channel protein

(26, 132), have been identified in the plasma membrane of guard cells (50, 129).

One of these types of anion channels (S-type) shows slow and sustained acti-

vation properties and is active over a wide range of voltage. Inhibitor studies

(132) and the recent demonstration that ABA activates S-type anion channels

(43, 114) indicate that these channels can account for the membrane depolariza-

tion and prolonged anion efflux required for ABA-mediated stomatal closure.

Inactivation of the plasma membrane H+-ATPase by ABA may, however, also

contribute to the membrane depolarization during stomatal closure (40).

The guard cell plasma membrane contains two types of K+ channels. The

inward-rectifying K+ channel is responsible for K+ influx to the guard cell

and is inhibited by ABA (76, 130, 149). The outward-rectifying K+ channel

is active only at membrane potentials positive to the equilibrium potential for

K+, and mediates K+ efflux required for stomatal closure (14, 130, 149). The

ABA-induced membrane depolarization mentioned above will thus activate

outward-rectifying K

+

channels, and the magnitude of this outward K

+

currentis further enhanced by ABA in a largely voltage-independent manner (14, 76).

SECOND MESSENGERS One of the earliest responses of guard cells to ABA is

an increase in the concentration of cytosolic free Ca2+ ([Ca]i) (2, 34, 61, 88a, 89,

128). The origin of the Ca2+ required to elevate [Ca]i in response to ABA is

unclear (90), but evidence favoring influx across the plasma membrane (128) or

release from internal stores mediated by inositol 1,4,5-trisphosphate (17, 37, 75)

and/or cyclic ADP-ribose (3, 90) has been presented. Experimental elevation

of [Ca]i is sufficient to induce stomatal closure (37) and mimics several of theabove-mentioned effects of ABA on ion channels in the plasma membrane of

guard cells. Increased [Ca]i activates the S-type anion channel and inhibits


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ABA SIGNALING 211

the inward-rectifying K+ channel (50, 127). These results thus provide strong

evidence for the involvement of Ca2+-coupled signal transduction cascade(s)

in the regulation of guard cell turgor by ABA.

Several observations, however, indicate that Ca2+-independent signalingpathways are also involved. The extent of ABA-induced increase in [Ca]iis variable, and stomata could even close in response to ABA without any

detectable change in [Ca]i (2, 34, 89). It remains, however, unclear whether

ABA always produces a local increase in [Ca]i that does not systematically

translate into global cytoplasmic change (89), or whether ABA can produce

stomatal closure using only Ca2+-independent signaling pathways (2). In any

case, the fact that the outward-rectifying K+ channel is insensitive to increases

in [Ca]i (76, 127) indicates that other second messengers must be involved in

the regulation of plasma membrane ion channels by ABA.

In particular, ABA increases the cytoplasmic pH (pHi) of guard cells (15, 61).

Studies in which guard cells were loaded with pH buffers or pHi was experi-

mentally modified support that the ABA-induced cytoplasmic alkalinization can

account for ABA activation of the outward-rectifying K+ channel and also con-

tribute to ABA inactivation of the inward-rectifying K+ channel (15, 42, 76, 98).

The ABA regulation of ion channels in guard cells thus appears to involve, at

least, both Ca2+-dependent and pHi-dependent signaling pathways.

DOWNSTREAM SIGNALING ELEMENTS How the ABA-induced Ca2+ and pHisignals are then decoded and relayed by other cellular components in the trans-

duction chain is not clear. The recent report that pHi regulates the outward-

rectifying K+ channel in isolated membrane patches from Vicia faba guard cells

suggests that the signaling elements acting downstream of the pH i signal are

membrane associated (98). The Ca2+ signal is possibly relayed by particular

protein kinases and phosphatases. Both Ca2+-dependent protein kinases (115)

and phosphatases (4, 23, 84) have been inferred to be potential candidates in

modulating the activity of various ion channels in guard cells, but their precise

roles in ABA signaling have not been clearly established at present.

There is nonetheless incontrovertible evidence that protein phosphorylation

is a critical component in the ABA signal transduction controlling stomatal

closure. In Vicia faba and Commelina, stomatal closure induced by ABA

was abolished by kinase inhibitors while enhanced by inhibitors of the protein

phosphatases PP1 and/or PP2A (125). These protein phosphatases, whose

actions are Ca2+-independent, have been implicated in the regulation of the

inward-rectifying K+ channel (82, 148), outward-rectifying K+ channel (148),

and S-type anion channel (114, 125) in Vicia faba and Commelina guard cells.Recent reports described two protein kinases whose activities are rapidly

stimulated by ABA in protoplasts from guard cells but not mesophyll cells in


14/24


Vicia faba (81, 99). Although they have a similar apparent molecular weight,

the two protein kinases are probably distinct because they differ in their ability

to autophosphorylate and in their substrate specificity in vitro (81, 99). The

cellular targets of these kinases are not yet known, but because their activitiesare detectable minutes after induction by physiological concentrations of ABA,

these kinases may be involved in the modulation of ion channel activities that

are essential for rapid stomatal responses.

Genetic dissection of ABA signaling in guard cells has been rather limited

despite an initial and promising report of the barley mutant cool, which dis-

played excessive transpiration and ABA-insensitive guard cells (118). Mutants

with specific impairment in ABA-induced stomatal closure have not yet been

identified in A. thaliana.

However, among the gca1-gca8 (Growth Control by ABA) mutants that wereisolated based on their reduced sensitivities to the inhibition of seedling growth

by exogenous ABA, the gca1 and gca2 mutants display a disturbed stomatal

regulation (10). The abi1-1 and abi2-1 mutants mentioned earlier also have

ABA-insensitive guard cells as part of their pleiotropic phenotypes (114, 122).

What are the roles of the Ca2+-independent (12) ABI1 and ABI2 protein

phosphatases 2C (PP2Cs) in stomatal regulation by ABA? Taking advantage of

the fact that the abi1-1 mutation is dominant (72, 78), the mutant A. thaliana

gene was introduced into the diploid tobacco Nicotiana benthamiana to facil-

itate electrophysiological measurements (6). Voltage clamp studies found that

the mutant abi1-1 transgene had no detectable effect on anion channels, but de-

creased ABA sensitivity of both the inward- and outward-rectifying K+channels

in the guard cell plasma membrane (6, 43). ABA sensitivity of these K+ chan-

nels and ABA-induced stomatal closure could be partially reestablished in the

abi1-1 transgenic plants by adding protein kinase inhibitors (6). Recent and im-

portant advances made in single-cell techniques have permitted measurements

of ion channel activities in A. thaliana guard cells, which had previously posed

technical difficulties because of their small sizes (114, 123). In patch-clamp

studies ofA. thaliana guard cell protoplasts, the ABA response of S-type anion

channels was impaired in the abi1-1 mutant. However, consistent with the re-

versibility of the effects of the abi1-1 transgene in tobacco (6), treatment with

the protein kinase inhibitor K-252a could partially restore ABA regulation of

the anion channel and ABA-induced stomatal closure in the A. thaliana abi1-1

mutant (114). The abi2-1 mutation also suppressed the ABA activation of S-

type anion channels, but curiously, this inhibition could not be counterbalanced

by K-252a treatments (114). In addition, the abi2-1 mutation also diminished

the background activity of inward-rectifying K

+

channels (114), an effect ob-served neither in the abi1-1 A. thaliana mutant(114)norintheabi1-1 transgenic

tobacco plants (6). The differential effects of the abi1 and abi2 mutations on


15/24

ABA SIGNALING 213

the background activity of inward-rectifying K+ channels, and the differential

sensitivity of these mutants to K-252a, thus indicate that, although the ABI1

and ABI2 proteins are homologous PP2Cs, the functions of these proteins in

stomatal regulation, as in other physiological responses, are nonredundant.The apparent discrepancies between the above-mentioned results on abi1-1

transgenic tobacco and abi1-1 mutant A. thaliana plants may be indicative of

the functional flexibility of ABI1 in maintaining an integrated ABA regulation

of both K+ and anion channels, depending on the imposed (environmental or

experimental) conditions. This would be analogous to the relative importance

of the ABA-induced increase in [Ca]i, which is subjected to both experimental

(89) and environmental (2) influences. A more serious problem in terms of

formulating a unified model of stomatal regulation, however, is how many

species-dependent differences exist in ABA signaling. For instance, various

pharmacological studies indicate that PP1/PP2A protein phosphatases (distinct

from the ABI1 and ABI2 PP2Cs) act as positive regulators of anion currents inA.

thaliana guard cells (114), but as negative regulators in Vicia, Commelina, and

N. benthamiana (43, 125). Therefore, it seems that detailed comparative studies

with different species would be necessary before a fuller understanding of the

signaling cascades that mediate stomatal regulation by ABA would emerge.

ABA SIGNALING TO GUARD CELL NUCLEUS As described above, stomatal

guard cells have been used extensively as a cellular system to explore the trans-duction pathways that target the ABA signal to ion channels during stomatal

closing. These responses are rapid and are thus thought to be operationally

distinct from long-term ABA responses that require RNA and protein synthesis

(166). However, hormone induction of gene expression at the transcriptional

level can also take place within minutes, as has been observed in the case of

auxin (93). The relevance of ABA in gene activation in guard cells has only

been explored recently. Several promoters or mRNAs have been shown to be

induced in guard cells by exogenous ABA treatment (53, 110, 137, 147, 157).

These studies demonstrate that guard cells are competent to relay ABA signals

to the nucleus. Initial attempts are being made to analyze whether identical or

distinct signaling pathways mediate the ABA regulations of ion channels and

gene expression in guard cells (53). It also remains to be established whether

there are gene products directly implicated in controlling stomatal movements

that are regulated by ABA transcriptionally.

CONCLUSION AND PERSPECTIVES

In recent years, the research into the molecular and physiological action of

ABA has benefited tremendously from cross-fertilization of different expertise


16/24


and ideas. Studies with optically pure ABA analogues revealed that the stereo-

chemical requirements of ABA are not identical in all ABA responses (21, 156),

indicating that higher plants may contain several types of ABA receptors. Ev-

idence for ABA perception sites located both at the cell surface and intracel-lularly has come from analyses on the regulation of ion fluxes in guard cells.

Whether this model is applicable to other cell types, and to more long-term

responses involving changes in gene expression, are important questions to be

explored (for instance, see 36). In addition, even though locations of these

reception sites can be detected physiologically, we have no firm idea whether

they are functionally distinct, and if so, what physiological relevance they may

harbor. Cloning genes of ABA receptors and creating corresponding mutants

would thus constitute a major step toward dissecting these initial events of ABA

action.Besides reception sites, we also need to improve further our understand-

ing of the more downstream events in the transduction cascades triggered by

ABA signals. Recent advances point to the central role of reversible protein

phosphorylation in mediating several of the physiological responses to ABA.

Much work, however, is clearly needed to identify the pertinent kinases and

phosphatases, and cellular targets of their action.

The ectopic expression of the seed-specific ABI3 gene in leaves resulted in

activation of several otherwise seed-specific genes. This observation and the

pleiotropic phenotypes of some ABA response mutants raise the question of

the degree of similarity between signaling cascades in the different tissues (or

cell types in the same tissue). It might be possible that many core components

of the ABA signaling network exist in different tissues. A simple model would

be that these central cascades are then regulated by cell-specific factors such

as the VP1/ABI3-like proteins. In this light, ABA-stimulated protein kinases

with apparent specificity to guard cells may play similar deterministic roles in

activating a branch of the ABA pathway in this cell type.

Isolation of additional mutants by judicious genetic screens (taking into ac-

count the potential for redundancy in signaling components) will no doubt

enrich our knowledge about regulatory points in ABA action, which might

include modulation in the activity of synthetic enzymes and transport of the

hormone (150). These two latter points have not been the subject of intense

investigations. Furthermore, mutations that simultaneously alter the respon-

siveness to ABA and other hormones or developmental clues, will permit a

fuller appreciation of cross-talk between ABA and other cellular signals. Sev-

eral mutants of this type have already been isolated in A. thaliana (Table 1).

The eventual cloning of the genes identified by mutational analyses will revealtheir molecular identity and, in some cases, suggest possible functions based on

homologies with known proteins. Genes with unexpected structural features


17/24

ABA SIGNALING 215

(such as CDT-1 in C. plantagineum) will divulge novel signal transduction

mechanisms, to the delight of researchers with iconoclastic tendencies.

The isolation of genes responsive to ABA, coupled with promoter analyses,

are vital to our understanding of how combinatorial and synergistic actions ofcis-acting elements and trans-acting factors are involved in the versatile control

of the output response. Other approaches, such as biochemical purification and

those based on interaction cloning, will undoubtedly complement the more

established technologies in unraveling additional signaling elements or even

signaling complexes.

Investigators with a pioneering spirit have already attempted to reconstruct

particular signaling cascades by reassembling individual components suspected

to play the relevant roles. These are much welcome advances, since these

cellular systems will complement the more established guard cell model. It iscertain that continuation of such a multidisciplinary approach will yield ever

deeper insight not only into ABA signal transduction itself but also into how

ABA and other signaling molecules jointly coordinate physiological responses.

ACKNOWLEDGMENTS

Work in the authors laboratory is supported by the Centre National de la

Recherche Scientifique and grants from the International Human Frontier

Science Program (RG-303/95), the European Community BIOTECH program

(BIO4-CT96-0062), and the Groupement de Recherches et dEtudes sur les

Genomes (Decision 9-95).

Visit the Annual Reviews home page at

http://www.AnnualReviews.org.

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