Plant Molecular Biology 26: 1557-1577, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1557

Current advances in abscisic acid action and signalling

J6r6me Giraudat*, Francois Parcy, Nathalie Bertauche, Frangoise Gosti, Jeffrey Leung, Peter-Christian Morris, Michelle Bouvier-Durand and Nicole Vartanian Institut des Sciences VOgOtales, Centre National de la Recherche Scientifique UPR 40, 91198 Gif-sur- Yvette Cedex, France (* author for correspondence)

Received and accepted 21 June 1994

Key words: abscisic acid, regulation, mutants, , stomata, stress

Abstract

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion trans- porters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of . The promoter analysis of ABA-responsive has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Introduction the underlying regulatory pathways. The first part of this article is thus devoted to an update of Abscisic acid (ABA) is a naturally occurring plant certain aspects of ABA physiology and in par- (or growth regulator) that was identified ticular of the information derived from the char- in the 1960s (see [2] for a review of the early acterization of ABA-biosynthetic mutants. This studies on ABA). ABA is probably present in all brief overview is only meant to provide the nec- higher plants, and has been implicated in the con- essary background to studies on ABA signalling; trol of a wide range of essential physiological pro- further details on ABA functions can be found in cesses including seed development and plant ad- several books and reviews [1, 18, 64, 166, 175]. aptation to environmental stress. In the second part, we then analyse more exten- The present review emphasizes the recent ad- sively the respective contribution of various ex- vances made in elucidating the ABA signal trans- perimental approaches to our present knowledge duction pathways. Identification of plant re- of the molecular cascades mediating ABA effects sponses that are regulated by ABA in vivo is a at the cellular level. prerequisite to studies aiming at characterizing

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Recent advances from the analysis of ABA- deficient mutants I V Famesyl pyrophosphate (C 15) As for many other biologically active substances, I vp2 the possible roles of ABA in plants were initially I vp5 investigated by monitoring endogenous ABA [] I vp7 vp9 contents and by analysing the effects of exog- enously applied ABA or biosynthetic inhibitors (C40) (reviewed in [166]). Mutants deficient in ABA [ aba synthesis provide a means to more directly assess the roles of ABA under physiological conditions. aba all-trans- As will be described below, such mutants lead us to a reevaluation of the extent of ABA action 9'-cis- (C40) during seed development and in the response of vegetative tissues to various stress conditions. XantSoxin (C15) Deficient mutants have also contributed exten- sively to the clarification of the biosynthetic path- ABA-aldehyde flacca ways of ABA. A"~ ~ sitiens ABA-alcohol droopy .~ abal "~ "~ nar2a The ABA biosynthetic pathway in plants ~"~ ABA

Mutants that are deficient in ABA have been iso- O~COOH lated in a variety of species (see Table 1). Among the best characterized are the maize viviparous Fig. I. Simplified pathway of ABA biosynthesis in higher ( vp) [ 114, 134], tomato flacca, sitiens and notabilis plants. The metabolic blocks in various ABA-deficient mu- [153, 156], Arabidopsis aba [79], potato droopy tants are indicated. Adapted from [155, 167, 175, 176]. [131], pea wilty [168] and Nicotianaplumbagini- folia abal [ 123, 139] mutants. These mutants dis- play various abnormalities that, as expected for ing zeaxanthin to antheraxanthin and most likely mutants deficient in the hormone, can be restored in the subsequent one leading to violaxanthin [26, to wild type by exogenous supply of ABA. 137]. The tomato flacca and sitiens [ 156], potato ABA is a sesquiterpenoid with mevalonic acid droopy [27 ] and N. plumbaginifolia abal [ 123, 139] as its precursor. While phytopathogenic fungi mutants are all blocked in the final step(s) of ABA synthesize ABA by a direct pathway (also known biosynthesis, oxidation of ABA aldehyde to ABA. as the C15 pathway) from , This step might be catalyzed by an that numerous distinct lines of evidence now support requires a molybdenum cofactor, as suggested in that higher plants rather synthesize ABA by an particular by the barley nar2a mutant [ 165]. De- indirect (or C40) pathway from (see fects in such a cofactor might conceivably affect [ 155, 167, 175, 176] for detailed reviews). In par- additional reactions unrelated to ABA biosynthe- ticular, the biosynthetic defects identified in sis and thus possibly explain the pleiotropic effects ABA-deficient mutants are congruent with the in- of the barley nar2a [165], tomato flacca [152] direct pathway schematically depicted in Fig. 1. and N. plumbaginifolia abal [ 123, 139] mutations. The maize vp2, vp5, vp7 and vp9 mutants are In addition, the existence of a minor shunt path- blocked in the early stages of biosyn- way that involves reduction of ABA aldehyde to thesis [108, 114]. The Arabidopsis aba mutants ABA alcohol and oxidation of ABA alcohol to are impaired in the epoxidation reaction convert- ABA (most likely via a cytochrome P-450 mono-

[3221 1559 oxygenase) was uncovered by the tomato flacca Roles of ABA during seed development and sitiens [136] and other mutations (reviewed in [ 136, 155, 176]). Other ABA-biosynthetic mu- ABA has been proposed to be an essential regu- tations have not yet been unambiguously related lator of various processes occurring during to a particular step of the pathway (discussed in roughly the last two thirds of seed development, [155]). i.e. during the 'maturation' and 'post-' As illustrated below for some ABA-response phases in the nomenclature proposed by Galau et mutants, techniques are now available to clone a al. [38]. In a number of mono- and di- gene simply on the basis of its associated mutant cotyledonous species, endogenous ABA content phenotypes. The available ABA-biosynthetic mu- has indeed been shown to peak during this period tants might thus lead in the near future to the before returning to low levels in the dry seed (for identification of genes encoding various reviews see [6, 132]). involved in ABA biosynthesis. These would be When removed from the ovule after the end of valuable tools to improve our understanding of pattern formation, most embryos display the abil- the regulation of ABA biosynthesis but also to ity to germinate precociously on culture medium. better identify which cells synthesize ABA. Such precocious could be prevented

Table 1. Characteristics of the various ABA mutants. Additional details on the mutant phenotypes can be found in the text. The loci that have been cloned are in bold, and the potential function of the encoded protein is indicated.

Species Name Defect References

ABA-deficient mutants Z. mays vp2 carotenoid biosynthesis [ 108, 114, 134] vp5 carotenoid biosynthesis [108, 114, 134] vp7 carotenoid biosynthesis [108, 114, 134] vp9 carotenoid biosynthesis [ 108, 114, 134] vp8 ? [108, 114, 134] A. thaliana aba epoxidation of xanthophylls [26, 79, 137] L. esculentum notabilis ? [ 153] flacca oxidation of ABA aldehyde to ABA [153, 156] sitiens oxidation of ABA aldehyde to ABA [ 153, 156] S. phureja droopy oxidation of ABA aldehyde to ABA [27, 131] P. sativum wilty ? [27, 1681 N. plumbaginifolia abal/ckrl oxidation of ABA aldehyde to ABA [ 123, 139] H. vulgare nar2a oxidation of ABA aldehyde to ABA [ 165] (deficient in molybdenum cofactor)

ABA-insensitive mutants A. thaliana abil ABA responsiveness [8O] (Ca2 +-modulated protein phosphatase) [86, 1061 abi2 ABA responsiveness [801 abi3 ABA responsiveness, seed-specific [80, 112, 119] (transcription activator) [451 abi4 ABA responsiveness (seed-specific?) [341 abi5 ABA responsiveness (seed-specific?) [34] Z. mays vpl ABA responsiveness, seed-specific [100, 134, 135] (transcription activator) [102] H. vulgare cool ABA sensitivity in guard cells [133]

[323] 1560 in many cases by adding ABA to the culture me- marker mRNAs both in vivo [68] and under vari- dium, thereby suggesting that the increased seed ous culture conditions [69], Hughes and Galau ABA levels play a similar role in vivo (for reviews have identified several classes of coordinately ex- see [6, 132]). The various nonallelic ABA- pressed mRNAs. Their expression patterns can biosynthetic viviparous mutants identified in maize be explained as unique combinations of a few indeed display precocious germination on the temporal programs ofgene expression. However, mother plant, i.e. vivipary [114, 134]. During these programs appear to be mainly controlled wild-type Arabidopsis seed development, there is by as yet unidentified 'maturation' and 'post- only a transient period during which freshly har- abscission' developmental factors distinct from vested developing seeds can readily germinate variations in ABA levels [38, 69]. upon water imbibition. This germination capacity While no ABA-mutant is available in cotton, is then lost in later stages: seeds develop primary the effect of ABA-biosynthetic mutations on seed . In contrast, seeds of the ABA- gene expression has been analysed in Arabidopsis deficient aba mutants remain non dormant until and maize. Most strikingly, such mutations in- ripeness [75, 79]. Reciprocal crosses between hibit only slightly, if at all, the in vivo accumula- wild type and the aba mutant further demon- tion of storage protein mRNAs both in Arabidop- strated that embryonic rather than maternal ABA sis ([121]; F. Parcy, C. Valon and J. Giraudat, participates in dormancy induction [ 75 ]. Reduced unpublished results) and in maize [120]. These has also been reported for N. mutant seeds nevertheless contain severely re- plumbaginifolia ABA-deficient mutants [ 139]. duced ABA levels and in particular do not display At the molecular level, seed developmental the peaks of ABA content observed during wild- stages are characterized by the accumulation of type seed development [75, 114, 120]. In agree- distinct sets of mRNAs and corresponding pro- ment with the conclusions of Hughes and Galau, teins in the embryo and endosperm (see [22, 49, the wild-type variations in bulk ABA content thus 68, 132, 159] for reviews). At least in dicots, stor- do not appear to be the major developmental sig- age proteins are characteristic markers of the nal controlling expression of the storage protein 'maturation' stage [38]. Their accumulation is fol- genes ('maturation' program). Although the ac- lowed by that of various classes of late embryo- cumulation of various maize [ 120, 128, 129, 164, genesis-abundant (LEA) proteins thought to par- 169] and Arabidopsis ([33]; F. Parcy, C. Valon ticipate in desiccation tolerance (for reviews on and J. Giraudat, unpublished results) LEA the structural characteristics of LEA proteins, see mRNAs is significantly reduced in ABA-biosyn- [28, 29]). Since several of these molecular mark- thetic mutant seeds, in most cases the extent of ers have been reported to be precociously induc- this inhibition does not seem to be linearly cor- ible by exogenous ABA in cultured embryos, de- related to the reduction in ABA content. It has velopmental variations in seed ABA levels have also been outlined that in wild type, LEA mRNAs been considered as candidate endogenous signals reach their maximal abundance at the very end of controlling the corresponding gene expression seed development whereas ABA content simul- programs (see [69, 132] for references). However, taneously decreases [68]. Expression of LEA several genes from rapeseed [35], sunflower [48] genes is thus probably controlled by some addi- or wheat [109, 110] are inducible in excised im- tional developmental factor(s) (such as the 'post- mature embryos by both exogenous ABA and abscission factor' of Hughes and Galau), even osmotic stress, but in the latter case without any though endogenous ABA levels seem to play a significant increase in endogenous ABA content. significant role in modulating the intensity of this The role of endogenous ABA has been further expression, challenged by a systematic analysis of gene ex- Similarly, the aba mutation alone does not pre- pression during cotton embryogenesis. By moni- vent the acquisition of desiccation tolerance dur- toring the expression pattern of a large set of ing Arabidopsis seed development. Endogenous [324] 1561

ABA nevertheless appears to participate in this not yet been traced at the cellular level, for in- developmental process since seeds of the aba,abi3 stance by electrophysiological recordings in guard digenic mutant (see below) remain desiccation- cells. ABA-biosynthetic mutants have so far only intolerant, a phenotype that can be reversed to rarely been analysed for other global stress re- wild type by exogenous supply of ABA [78, 105]. sponses. The Arabidopsis aba mutation, however, The above data on ABA-biosynthetic mutants affects the development of freezing tolerance [41, indicate that wild-type developmental variations 62] and impairs the production of characteristic in bulk ABA content appear to control the in- structures in response to progressive drought duction of seed dormancy but are not the primary [163]. regulators of the other responses analysed. Since In the past few years, the possible role of ABA ABA-dependent regulatory pathway(s) appear in mediating stress responses has been further nevertheless involved, these latter responses strengthened by molecular studies. A large num- might possibly be controlled by developmental ber of genes which are similarly regulated by stress variations in their sensitivity to ABA [ 160]. Such and exogenous ABA have indeed been identified possible variations in ABA sensitivity are how- in a variety of plant species (for reviews see [ 13, ever apparently not attributable to developmental 22, 70, 149]). Although the documented or puta- variations in the expression of the maize VP1 tive molecular functions of the proteins encoded [102] and Arabidopsis ABI3 (F. Parcy, C. Valon by these stress-regulated genes is obviously of and J. Giraudat, unpublished results) genes which great biological interest, this point is beyond the encode putative elements of seed ABA-signalling scope of the present review. The use of ABA- pathways (see below). biosynthetic mutants and/or ABA biosynthesis inhibitors has demonstrated that endogenous ABA indeed contributes to the regulation of sev- Ro&sofABA in response to environmental stress eral ABA-responsive genes by desiccation or condit~ns drought [16, 50, 83, 117, 128, 129], cold [83, 116, 117] and salt [11]. Interestingly, ABA also par- During vegetative growth, ABA has been pro- ticipates in the accumulation of ferritin mRNA in posed for a long time to be an essential mediator response to iron stress [88]. in triggering plant responses to various adverse The characterization of ABA-biosynthetic mu- environmental conditions such as drought, high tants however also revealed that the ability of a salinity or cold. This conclusion was initially sup- given gene to respond to exogenous ABA under ported by the observed stress-induced increases non-stress conditions does not necessarily imply in endogenous ABA levels [15, 16, 151] and by that this gene is actually regulated by endogenous the ability of exogenously applied ABA to mimic ABA upon stress. Several Arabidopsis genes many of the plant morphological and physiologi- which are inducible by exogenous ABA are regu- cal responses to these environmental stimuli (for lated by cold in an ABA-independent manner reviews see [19, 94, 161, 175]). [41, 116]. In particular, ABA is thought to be responsible Additional evidence demonstrate the existence for triggering stomatal closure under conditions of ABA-independent pathways mediating gene of water deficiency. The effects of exogenous ABA regulation in response to stress. For instance, on stomatal guard cells are now documented by genes unresponsive to ABA represent a signifi- a wealth of electrophysiological studies (see cant proportion of those recovered from screens below). Most of the available ABA-deficient mu- on the basis of differentially expressed mRNA tants display an increased tendency to wilt and/or upon water stress [50, 55, 171]. Although ABA- enhanced water loss in excised aerial parts, sug- independent regulation might take place prior to gestive of a defect in stomatal regulation [79, 113, the stress-induced de novo synthesis of ABA [54- 123, 131, 153, 168]. These defects have however 56, 173], such ABA-independent regulation does

[3251 1562 not occur exclusively in the early stages following the Solanaceae are now known to possess a sys- the onset of stress conditions [50]. A cis-acting temic wound response (reviewed in [140]). element involved in ABA-independent stress Several lines of evidence support that ABA regulation of gene expression has been recently contributes to transducing the wound response in characterized [174]. As shown in Table2, this potato and tomato [126, 127]. In the absence of DRE element does not resemble any of the known wounding, applied ABA results in high levels of ABA-responsive elements (see below). protease inhibitor II (Pin II) gene induction in In conclusion, available data do support a role both species. Mechanical wounding of potato for ABA in mediating various environmental increases ABA levels both locally and sys- stress. However, instead of acting as the central temically. More importantly, ABA-deficient mu- co-ordinator of all aspecfs of the plant response, tants of tomato (sitiens) and potato (droopy) show ABA seems required for only some of the regu- much lower local and systemic levels of Pin II latory pathways involved. These pathways are gene induction in response to wounding [126, most likely integrated into a more complex regu- 127]. Similar observations were later made for latory network. Diverse types of experiments in- additional mRNA markers of the potato wound deed support that ABA-dependent and ABA- response [65]. Conditions of water stress (shown independent pathways interact in regulating the to enhance endogenous ABA levels) did not in- expression of certain genes in response to drought duce Pin II nor other wound-responsive genes, [116, 173] or osmotic [11] stress. which suggests that in potato plants two indepen- dent transduction mechanisms regulate the ABA- dependent wound and water stress responses re- Roles of ABA in wound response spectively [65]. More recently it has been demonstrated that the potato Pin II promoter is Recently, ABA has also been implicated in me- inducible by wounding and exogenously applied diating local and systemic wound response. After ABA in transgenic rice, demonstrating that the mechanical wounding, a specific set of proteins basic induction machinery is conserved between (thought to be defense-related) accumulate both monocots and dicots [170]. at the local wound site and systemically through- Although the results summarized above sup- out the plant. By far the best characterized of port a role for ABA in mediating the systemic these induced proteins are protease inhibitors I wound response, definitive proof of ABA as the and II of tomato and potato (reviewed in [140]). systemic messenger is lacking. Furthermore, a However, other proteins such as those with number of other molecules or signals seem to homology to cathepsin D inhibitor, threonine participate in promulgating the systemic wound deaminase, or leucine aminopeptidase are also response (reviewed in [140]). In particular, jas- induced [65] and many plant families other than monic acid (which has effects similar to those of

Table 2. Sequence motifs of the various cis-acting elements mentioned in the text.

Element Gene Reference Sequence

DRE RD29A [ 173] -167 TACCGACAT EmlA Em [95] -153 GGACACGTGGC Motif I rab16A [ 111 ] - 186 CCGTACGTGGCGC hex-3 (synthetic) [ 82 ] GGACGCGTGGC Sph C1 [60] -145 TCCATGCATGCAC ? CDeT27-45 [ 115] -361 AAGCCCAAATTTCACAGCCCGATAACCG GARE Amyl/6-4 [ 150] - 148 GGCCGATAACAAACTCCGGCC GARE Amy32b [ 138] -120 GTAACAGAGTCTGG

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ABA on various other physiological processes re- closure. As already mentioned, the 'wilty' pheno- viewed in [124]) appears to be of central impor- type displayed by most ABA-deficient mutants tance. Exogenous applications of provide suggestive evidence for an action of en- (JA) [31, 65] or of its biosynthetic intermediates dogenous ABA on stomatal regulation. [32] induce wound-responsive genes. JA seems volume is controlled osmotically, to act downstream of ABA in regulating the mainly by large influx (stomatal opening) or effiux wound response [65, 125]. Several lines of evi- (stomatal closure) of K + , balanced by flux of dence suggest that wounding induces lipase ac- anions. Tracer flux studies on isolated epidermal tivity which releases linolenic acid, the precursor strips revealed that externally applied ABA to JA, from the plasma membrane and then sev- evokes the efflux of K + and anions from the guard eral oxygenation steps subsequently lead to JA cells; the released ions originating both from the biosynthesis [32, 125]. One might then envisage cytoplasm and the vacuole [90-92]. These stud- that in the first stage of the wound response, JA ies also provided evidence for the involvement of synthesis is ABA-dependent and that ABA, for second messengers in the ABA effects since a 2 instance, controls the early induction of one of the min ABA pulse was sufficient to elicit the full K + JA biosynthetic enzymes [ 104]. (86Rb + ) effiux response which lasted ca. 20 min, and the rate of K + efftux continued to rise after ABA washout [91]. Signal transduction Electrophysiological techniques further dis- sected the above fluxes into their various ionic The ultimate objective of studies on signal trans- current components. The considerable progress duction is a molecular description of the regula- made towards the deciphering of the electrical tory network that coordinates perception of the responses triggered by ABA in the plasma mem- signal to cellular responses. Chemical compounds brane of guard ceils are detailed in several recent such as ABA are generally thought to elicit a reviews [9, 92, 143]. Although several aspects cascade of events by interacting with a specific such as the exact time sequence of events still receptor site(s). Saturable ABA binding sites have remain to be clarified, a general scheme starts to been described [66, 67], but the corresponding emerge as briefly summarized here. The first elec- proteins have not been further identified. In con- trical change detected after exposure to ABA is trast, significant progress has been made in the an initial depolarization which reflects a net influx identification of more downstream elements that of positive charges [ 158]. The respective contri- contribute to the ABA regulation of ionic currents butions of anion efflux and of Ca 2 + influx to this in stomatal guard cells or of gene expression in early depolarization response are still a matter of various tissues. controversy [9, 147, 158]. Nevertheless, in either scenario this first event would then lead to the activation of two types of CaZ+-sensitive and ABA signalling pathways in stomatal guard cells voltage-dependent anion conductances which is the basis of the long-term depolarization and large The aperture of stomatal pores is controlled by anion efflux observed in response to ABA. These changes in the turgor of the two surrounding guard two anionic currents are carried by two distinct cells. In order to optimize CO2 and water vapour channels (or two functional modes of a single exchanges with the atmosphere, guard cell vol- channel type): R- and S-type [61,144, 145]. The ume responds within minutes to a variety of sig- characteristics of the S-type anion channel iden- nals (reviewed in [77]). In particular, during con- tified in the plasma membrane of Viciafaba guard ditions of water stress, the increased ABA levels cells make it a good candidate for mediating most in guard cells [58] are thought to reduce water of the long-term anion efflux [146]. The above loss through by promoting stomatal depolarization generates the driving force for K +

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efflux through outward rectifying K + channels, tributed both by an influx of external Ca 2+ which provide the predominant pathway for long- (through a non-specific cation channel) as well as term K + efflux. These K + currents are activated by Ca 2 + release from intracellular stores [42, 97, by ABA in a largely voltage-independent manner 147]. Inositol (1,4,5)-trisphosphate (IP3) is an [7]. Conversely, ABA inhibits K + influx through attractive intermediate for triggering this intra- inward rectifying K ÷ channels that represent the cellular Ca 2 + mobilization. When released in the major pathway for K + uptake into guard cells cytoplasm, IP3 induces a rise in Ca~ (apparently during stomatal opening [7, 8, 158]. from internal stores) followed by stomatal closure Both Ca 2 + and H + most likely participate as [44], and IP 3 inactivates the inward rectifying intracellular secondary messengers in mediating K + channel [10]. Activators of G-proteins have the above ABA effects on stomatal aperture also been shown to inactivate these same chan- and/or plasma membrane channels. An elevation nels, in a Ca 2 ÷ -dependent manner in the case of of the cytoplasmic Ca 2+ concentration (Cai) GTP-y-S [30]. above ca. 600 nM by photolysis of caged Ca 2 + These various observations, together with evi- suffices to produce stomatal closing [44]. Along dence suggesting that ABA acts from the outside with this observation, increases in Ca~ inhibit the of guard cells [4, 59], are of course reminiscent of inward rectifying K + channels and activate the hormone-receptor/G-protein linked trans- voltage-dependent anion channels [61, 144]. The duction cascades (see [23, 76] for reviews). One Ca 2 +-induced inactivation of the inward rectify- could thus speculate that by binding to a trans- ing K + channels appears to be mediated by a membrane receptor at the plasma membrane [ 67], Ca 2 +-dependent protein phosphatase related to ABA activates a G-protein, which then triggers the animal calcineurin [89]. ABA has been shown the release of IP3. IP3 would then evoke effiux of to induce an increase in guard cell Cai, as revealed Ca 2+ from the vacuole [3] and/or endoplasmic by fluorescent indicators, which precedes sto- reticulum into the cytosol. Numerous aspects of matal closure [96]. There is however a consider- this minimal framework for the Ca 2 +-mediated able variability in the reported ABA-induced el- ABA effects however clearly await experimental evations of Ca~, which might simply reflects confirmation. This analysis might be difficult since technical limitations in monitoring Ca~ [98], or several lines of evidence suggest that guard cells alternatively indicates that ABA treatment is not contain another G-protein linked cascade which systematically accompanied by an increase in Cai plays an antagonistic regulatory role, i.e. pro- [42]. The existence of an additional, Ca 2+- motes stomatal opening [30, 84, 85]. independent, pathway in ABA action is supported Another key aspect of the above model remains by several observations including the Ca 2+- uncertain, namely the location of the ABA recep- independent activation of outward rectifying K + tion site. Recent data indeed support that an ex- channels (reviewed in [9, 42, 143]). These K + tracellular reception site is critical in the ABA channels are in contrast affected by the cytoplas- inhibition of stomatal opening [4] but that ABA mic pH (pHi). ABA was shown to evoke an al- acts from within guard cells to promote stomatal kalinisation of the cytoplasm of guard cells [72] closure and the associated inhibition of inward which is a necessary intermediate in the ABA K + currents [ 148]. The molecular identification activation of the outward rectifying K + channels of these sites of ABA action (receptors) clearly [ 8 ]. Both of these pathways (Ca 2 + and H + ) thus represents an exciting challenge for the future. appear to be essential in mediating ABA-evoked At least some parts of the ABA signalling path- stomatal closure; the details of their mutual in- ways identified in guard cells might also be present teractions however remain to be determined. in other cell types. For instance, ABA-induced The cellular origin of the ABA-induced cyto- membrane depolarizations have been observed in solic alkalinisation is unknown (discussed in [8 ]). radish seedlings and in epidermal and mesophyll The ABA-evoked rise in Cai is most likely con- cells from tobacco (reviewed in [92]). Also, ABA- [328] 1565 induced increases in Ca~ associated with an in- the expression of these chimeric constructs were crease in pH i have been observed in cells of corn analysed by transient assays in protoplasts. Pro- , corn coleoptiles and parsley hypocotyls moter regions involved in the induction by ABA [40]. These various cellular events have however were initially delimited by 5' deletion analysis, not yet been unambiguously related to ABA- and found to contain various sequence motifs regulated physiological processes. (Em 1a/b and Em2, Motif I and Motif IIa/b)con- In contrast, as described above, ABA actions served in the promoters of other ABA-responsive both in seeds and in vegetative tissues involve genes [95, 111 ]. Additional evidence was then modifications of gene expression. Analysing the obtained that at least the Emla and Motif I ele- promoter of such ABA-responsive genes thus re- ments directly contribute to ABA responsiveness. presents a bottom-up approach to the character- A 75 bp fragment of the Em promoter (contain- ization of the corresponding ABA regulatory ing the Emla/b and Em2 motifs) confers ABA pathways. responsiveness to a truncated (-90) derivative of the cauliflower mosaic virus (CaMV) 35S pro- moter whereas two single-basepair changes in the Promoter analysis of ABA-responsive genes Emla motif decrease ABA induction from 12- to 2-fold [57]. Six tandemly repeated copies of the Over 150 genes from various species are known rab16A Motif I fused to a minimal (-46) CaMV to be inducible by exogenous ABA (reviewed in 35S promoter provide induction by ABA [ 150]. [13, 22, 70, 149]. Most of these genes were origi- Gel retardation DNA binding assays and foot- nally identified as being expressed during late seed printing experiments demonstrated that the Em 1a development and/or in the vegetative tissues of and Motif I sequences interact with nuclear pro- plants exposed to environmental stress. As dis- teins [57, 111 ]. Complementary DNA clones that cussed above, their responsiveness to applied enc ode protein s with binding affinity for the Em la ABA does not necessarily imply that all these (wheat EmBP-1 protein [57]) and Motif I (to- genes are primarily regulated by endogenous ABA bacco TAF-1 protein [ 118 ]) elements respectively content in vivo. Nevertheless, these target genes have been isolated. Although their in vivo roles are useful tools to investigate the cellular compo- in ABA responses await further analysis, the nents involved in their ABA induction. In most EmBP-1 and TAF-1 proteins display the two cases ABA responsiveness has been monitored adjacent domains characteristic of the bZIP only by northern blot analysis of steady-state (basic region-leucine zipper) transcription factors mRNA levels. Several genes have however been family. further demonstrated to be regulated by ABA at As shown in Table 2, the Emla and Motif I least in part at the transcriptional level. Analysing elements are similar to each other and to the pal- the promoters of these genes provide a powerful indromic CACGTG motif known as the G-box means to identify the terminal components of the [46, 73 ]. This observation was initially intriguing ABA regulatory cascade, namely the cis-acting since several motifs related to the G-box were element(s) and trans-acting factor(s) involved in known to participate in mediating the regulation ABA responsiveness. of unrelated plant genes by a diversity of stimuli The best characterized class ofcis-acting ABA- distinct from ABA such as light [24] or anaero- responsive elements (ABRE)is exemplified by the biosis [21]. It is now clear that the nucleotides Emla element from the wheat Em gene [95] and flanking the ACGT core play a critical role in the Motif I element from the rice rab16A gene controlling the bZIP DNA binding specificity. [ 111 ]. These two elements were identified and Sequence elements with a ACGT core have been characterized by roughly similar experimental accordingly subdivided into three categories (G-, strategies. Upstream sequences from the Em or C- and A-box) and bZIP proteins classified into rab16A genes were fused to a reporter gene and three groups depending on their respective affin- [329] 1566 ity for G-box and C-box elements [73]. These Endogenous coupling elements from native ABA- differential binding specificities provide a possible responsive promoters remain to be identified. explanation of how the diverse bZIP proteins and Additional types of cis-acting ABREs distinct ACGT-containing elements are integrated into from the above G-box-related ones have been different in vivo regulatory networks. These results characterized. A combination of deletion and also outline that, as discussed for TAF-1 [ 141], point mutation analyses identified an element a bZIP protein identified on the basis of its in vitro named Sph (Table 2) which is critical for the binding to a particular ACGT-containing element ABA-activation of the maize C1 promoter [60]. is not necessarily the endogenous factor that in- Related motifs occur in the promoter regions of teracts in vivo with this element. several other ABA-regulated genes but their func- The experimental evidence summarized above tional significance is unknown [60]. Tetramers of support that the Emla and Motif I elements at a synthetic element named hex-3 (Table 2) confer least contribute to the ABA responsiveness of the transcriptional activity upon a truncated (-90) wheat Em and rice rabl6A genes, respectively. CaMV 35S promoter, and this activity can be Although related sequence motifs with a ACGT enhanced by ABA [82]. An element which ap- core have been found in a number of other ABA- pears required, although alone insufficient, to responsive genes [ 70 ], their biological significance confer responsiveness to ABA has been recently remains uncertain until functionally assessed. The identified in the Craterostigma plantagineum CCACGTGG element seems to indeed partici- CDeT27-45 gene [ 115]. Binding of this element pate in the ABA induction of the maize rab28 (Table 2) to nuclear factor(s) is ABA-inducible gene [130]. In contrast, the Emla-like motifs [115], which is not the case for the binding ac- present in the maize C1 and Craterostigma plan- tivities to the G-box related Emla and Motif I tagineum CDeT27-45 genes are not major deter- elements [ 57, 111 ]. Finally, preliminary evidence minants of the ABA responsiveness of these genes suggests that the ABA-inducible ATMYB2 ho- [60, 115]. mologue of the MYB [162] Promoter deletion analysis showed that al- might contribute to the induction of the Arabidop- though the Em 1a and Motif I elements are clearly sis RD22 gene [172] by ABA. necessary components, they do not account alone All elements described so far participate in for the full transcriptional ABA induction of the transcriptional activation by ABA. In contrast, a Em and rab16A genes [95, 111]. Also, in gain of few other elements are known to mediate tran- function experiments several tandem copies of scriptional repression by ABA. In particular, the Motif I were needed to confer ABA responsive- -responsive elements (GARE) shown ness to the minimal (-46) 35S promoter (that in Table2 are essential for the gibberellin- contains only a functional TATA box) whereas inducible, ABA-repressible expression of the bar- Motif I is not tandemly repeated in the native ley ~-amylase Amy1/6-4 [ 150] and Amy32b [ 138] rab16A promoter [150]. Individual ABREs may genes. Recent data support that in barley aleu- thus require other element(s) to couple hormone rone protoplasts, ABA can regulate gene expres- effects to the transcriptional apparatus. Interest- sion by acting from the external face of the plasma ingly, such an abscisic acid responsive complex membrane [43]. This indicates the existence of (ABRC) was artificially built by substituting a intracellular cascade(s) linking this external site single copy of the Motif I ABRE to the gibberellin to nuclear transcription. responsive element in a promoter fragment of the In conclusion, molecular dissection of ABA- Amy32b barley ~-amylase gene [ 138]. The single regulated promoters has already revealed a diver- copies of the ABRE and of the otherwise non sity of cis-acting sequences that represent likely ABA-responsive O2S element were both required end-points of ABA-regulatory pathways. This in- to provide ABA responsiveness, which indicates ventory is possibly still far from complete. Iden- that the O2S-ABRE unit functioned as an ABRC. tifying the endogenous trans-acting factors that

[330] 1567 bind to these c/s elements, and understanding how The seed-specific Arabidopsis ABI3 and maize they are connected to more upstream elements of VP1 genes the ABA-signalling cascade(s) will be the next The Arabidopsis ABA-INSENSITIVE-3 (ABI3) logical (and probably most difficult) steps in this and maize VIVIPAROUS-1 (VP1) loci are both bottom-up approach. As in many other cases (re- active only in seeds. Phenotypically, no alteration viewed in [71]), protein kinases and/or phos- was detected in the vegetative tissues of abi3 mu- phatases (see ABI1 gene below) are likely to be tant plants [37, 41, 50, 80, 116]. Also, vpl muta- involved in the ABA-pathways regulating tran- tions were shown to inhibit the anthocyanin bio- scription. Additional potential candidates start to synthetic pathway only in embryo and emerge. Putative transcription activators such as tissues [25, 134]. Molecular cloning of the VPI the VP1 and ABI3 (see below), and GF14 [20] [102] and ABI3 [45] genes provided a simple proteins can potentially participate in ABA- explanation for this specificity in that neither gene related transcriptional complexes. Also, the maize is found to be expressed in vegetative tissues [45, Rabl7 protein might play a role in the ABA regu- 101, 102, 122]. The ABI3 gene is transiently ex- lation of nuclear protein transport [47]. pressed beyond seed germination in young seed- During the past few years, molecular techniques lings, but this expression is strictly confined to the have emerged to clone genes identified only on the organs of embryonic origin (cotyledons and hy- basis of their associated mutant phenotypes. pocotyl) [122]. This residual expression might Characterization of mutants defective in ABA re- possibly explain the few abnormal phenotypes de- sponsiveness thus potentially represents a pow- scribed for young abi3 mutant seedlings [37]. erful approach to dissect the molecular nature of The Arabidopsis abi3 and maize vpl mutants ABA-signalling cascades, as illustrated in the next display some common ABA-related phenotypes. section. Abi3 mutants were initially recovered by selecting for seeds capable of germinating in the presence of inhibitory ABA concentrations [80]. Mature Characterization of ABA-response mutants seeds of the severe abi3-3 [ 112], abi3-4 and abi3-5 [ 119] mutant alleles are several orders of magni- Mutants that are impaired in their responsiveness tude less sensitive to the inhibition of germination to ABA have been described in several plant spe- by exogenous ABA. Unlike in the Arabidopsis aba cies, including maize [134], Arabidopsis [34, 51, biosynthetic mutants, the endogenous ABA levels 80, 112, 119] and barley [133] (see Table 1). are not reduced in the abi3 (nor in the non-allelic These mutants are distinct from ABA-biosyn- abil and abi2) mutants [80]. Developing abi3 em- thetic mutants in that they do not have reduced bryos fail to become dormant [80, 112, 119], simi- endogenous ABA levels and their phenotypes lar to the phenotype described for the aba mutant cannot be reversed to wild type by exogenous [75, 79]. This deficiency however does not lead to supply of ABA. These ABA-response mutants vivipary unless Arabidopsis plants are grown are generally pleiotropic in phenotypes and are under high-humidity conditions [112]. In con- thus believed to unravel components of signal trast, like other maize viviparous (vp) mutants, the transduction chains. Since only the Arabidopsis vpl mutations were originally identified as leading abil, abi2 and abi3 [80] and the maize vpl [ 134] to precocious germination of the embryo while mutants have already been analyzed to a substan- still attached to the mother plant (vivipary) [ 134]. tial extent, and are targets for molecular studies, Whereas most of the other VP loci affect early they will be the main subjects of the discussion steps in the biosynthesis ofcarotenoids and ABA, below. vpl embryos do not have reduced ABA content [ 114]. Excised immature vpl embryos rather ex- hibit somewhat reduced sensitivity to growth in- hibition by exogenous ABA in culture [ 135].

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Further evidence supporting that the ABI3 and istic of late seed development since several VP1 proteins can participate in ABA signalling mRNA markers retained near wild-type expres- was obtained with the use of the cloned genes. A sion patterns in abi3-4 [ 122]. Maize vpl mutant transcriptional fusion between the CaMV 35S embryos have been examined at various selected promoter and the VP1 cDNA was electroporated time points and found to similarly contain se- into maize suspension culture protoplasts to- verely reduced endogenous levels of several gether with a construct carrying the GUS reporter globulin storage protein and LEA-type mRNAs placed under the control of the wheat Em pro- [101, 120, 129, 169]. moter [95]. In this transient assay system, VP1 In developing seeds of both species however, overexpression synergistically enhances the tran- the abundance of at least some of these marker scriptional activation of the heterologous Em pro- mRNAs remains markedly higher in ABA- moter by exogenous ABA [102]. More recently, deficient mutants than in abi3 or vpl [33, 129, Arabidopsis plants were stably transformed with 169]. When the abundance of four marker transcriptional fusions between the double en- mRNAs that are totally repressed in vpl mutant hanced CaMV 35S promoter and the ABI3 embryos was systematically compared in the vari- cDNA(2 × 35S::ABI3).Inthese2 x 35S::ABI3 ous viviparous biosynthetic mutants, substantial transgenic lines, ectopic expression of ABI3 con- correspondence between transcript level and ferred to plantlets both an increased sensitivity to ABA content was observed only for the Em the inhibition of root growth by exogenous ABA mRNA [120]. Similarly, the Arabidopsis napin and the ability to accumulate the normally seed- At2S3 and cruciferin CRC mRNA levels are specific At2S3 [53], CRC [121] and AtEml [39] slightly if at all reduced in the ABA-deficient aba-1 endogenous mRNAs in response to applied ABA mutant whereas the abi3-4 mutation markedly in- (F. Parcy and J. Giraudat, unpublished results). hibits the accumulation of both mRNAs (F. The ectopically expressed ABI3 protein can thus Parcy, C. Valon and J. Giraudat, unpublished functionally interact with ABA-regulatory cas- results). As discussed above, the expression of cade(s) present in differentiated vegetative tissues. such genes is most likely controlled primarily by Both abi3 and vpl mutations inhibit the in vivo developmental factors distinct from variations in accumulation of various endogenous mRNA spe- ABA levels. Mutant phenotypes nevertheless in- cies characteristic of developmental stages occur- dicate that the ABI3 and VP1 proteins are essen- ring during the last two thirds of seed develop- tial for the regulation of gene expression by these ment, as discussed above. In Arabidopsis seeds, unidentified factors. these ABI3-dependent mRNAs were initially Additional observations suggest that ABI3 and shown to include cruciferin and napin storage VP1 roles are not confined to ABA signalling. protein mRNAs as well as the late embryogenesis- Accumulation of seed storage lipids is inhibited in abundant (LEA) AtEm6 mRNA [33, 37, 112, abi3-1 but not in aba-1 Arabidopsis mutants [37]. 121 ]. In a recent and more systematic analysis, 19 Unlike aba mutants, embryos of the strong abi3 cDNA probes have been used to compare the mutant alleles fail to lose chlorophyll and to ac- kinetics of expression of the corresponding quire desiccation tolerance during seed develop- mRNAs throughout silique development in the ment [ 112, 119]. Maize vpl seeds are defective in wild-type and in the severe abi3-4 mutant [122]. anthocyanin accumulation, a phenotype dis- This study demonstrated that the abi3-4 mutation played by none of the ABA-deficient vp mutants markedly inhibits the accumulation of multiple [ 134]. This colorless phenotype results from the transcripts (including the above At2S3, CRC and failure to express the C1 regulatory gene in vpl AtEml mRNAs) throughout the last two thirds of seed tissues and interestingly, partially distinct Arabidopsis seed development. This mutation cis-acting sequences mediate activation of the C1 however does not globally disrupt the various promoter by VP1 and exogenous ABA, respec- temporal programs of gene expression character- tively [60].

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Altogether the above data indicate that in vivo that the ABI3 and VP1 proteins play a much the ABI3 and VP1 proteins interact with ABA- more complex role than initially anticipated. In signalling cascades (for instance those controlling the future, combined genetic and molecular dormancy) but also with distinct regulatory path- approaches should unravel further details about ways. Available evidence support that these pro- the exact function of these proteins in the regu- teins are transcriptional activators. As shown in latory networks controlling seed development in Fig. 2, the primary structures of the VP1 [102] mono- and dicotelydonous species respectively. and ABI3 [45] proteins display a similar arrange- In this respect, several recent Arabidopsis mutants ment of domains with distinct biochemical char- represent promising tools since they share several acteristics. Some of these domains further corre- phenotypes with abi3 mutants but apparently do spond to discrete regions of remarkable amino not display reduced ABA responsiveness [ 14, 81, acid conservation [45]. No significant sequence 103]. similarities to other known proteins were found, Additional Arabidopsis ABA-insensitive mu- and no typical motifs associated with DNA- tants have been recently isolated by similar means binding were detected. However, several regions [36]. These mutants correspond to at least four of the polypeptide chains present features previ- new Arabidopsis loci, two of which (named ABI4 ously described in transcriptional activation do- and ABI5) have been characterized to some ex- mains [45, 102]. In particular, experimental evi- tent [34]. Available abi4 and abi5 mutant alleles dence support that the N-terminal acidic domain display rather weak phenotypes, some of which of VP1 can indeed participate in transcriptional (for instance reduced AtEm6 mRNA levels in dry activation [102]. As transcriptional activators, seeds) are also found in abi3. From these data the ABI3 and VP1 proteins could control the in- and from the characterization of various abi di- tensity of gene expression during seed develop- genic mutants, it has been proposed that ABI4 ment by interacting with various transcription and ABI5 might act in the same pathway than factors related to distinct regulatory pathways. ABI3 [34]. Further experiments and/or stronger Such molecular interactions however remain to alleles might help to firmly assess the direct con- be experimentally demonstrated. tribution of ABI4 and ABI5 to ABA-signalling From the wealth of data accumulated in the per se as well as their exact relationship with past few years on these two systems, it emerges ABI3.

The Arabidopsis abil and abi2 mutants 1 S/T 720 ABI3 ~//4¢///~ ~/////.4¢////////~//////.¢¢/////////1~ ~ I A. thaliana is the only plant species where ABA- response mutants displaying phenotypes both in VP1 I seeds and in vegetative tissues have been identi- fied. Like abi3, the abil and abi2 mutants were initially selected for their reduced sensitivity to 1 Ca2+ Phosphatase2C-like 434 the inhibition of seed germination by exogenous ! ABI1 ABA [80]. These mutants also share the reduced Fig. 2. Schematic diagrams of the architecture of the ABI3, seed dormancy phenotype [80], similar to that VP1 and ABI1 proteins. Top: the Arabidopsis ABI3 and maize VP 1 proteins display a similar arrangement of acidic (-), basic described for the aba mutants [75, 79]. However, ( + ) and serine/threonine rich (S/T) domains. The three basic available abil and abi2 mutant alleles do not dis- domains further correspond to regions of high amino acid play the additional abi3 seed phenotypes de- sequence identity. See [45, 102] for additional details. Bottom: scribed above [33, 37, 78]. the Arabidopsis ABI 1 protein displays a novel amino-terminal Do the ABI1 and/or ABI2 proteins interact domain containing an EF-hand Ca 2 + -binding motif (Ca 2 + ), and a carboxy-terminal domain homologous to the 2C class with the same seed ABA-regulatory cascade(s) as of serine/threonine protein phosphatases. See [86, 106] for ABI3? In a germination assay, the abi3-1,abil-1 additional details. and abi3-1,abi2-1/+ digenic mutants are mark-

[3331 1570 edly more resistant to ABA than any of these 106], is more resistant to ABA inhibition than monogenic mutants [37]. Also, whereas in vivo wild type. In particular, root meristematic cells of accumulation of the AtEm6 mRNA level is inhib- abil plantlets retain their ability to passage ited in abi3-1, but not abil-1 nor abi2-1 mature through the S phase of the mitotic cycle in the seeds, this mRNA level is further reduced in abi3- presence of inhibitory ABA concentrations [86]. 1,abil-1 and abi3-1,abi2-1/+ digenic mutants Abil and abi2 mutations impair the root hair de- [33]. These additive effects are ambiguous in formation induced by applied ABA [142]. These terms of epistatic interactions since a single phe- mutations also inhibit ABA-induced proline ac- notype was scored in each set of data and none cumulation and changes in protein synthesis [37]. of the mutant alleles used has been proven to be The abil mutation has been further shown to in- null (abi3-1 in particular is definitely a much terfere with the ABA-induced accumulation of all weaker allele than e.g. abi3-4) [5]. Homozygous identified mRNAs tested [41, 50, 83, 116, 117, abi2-1 and abi3-1 monogenic mutants are viable 173]. Unfortunately, the abil and abi2 mutants but the homozygous abi2-1,abi3-1 double mutant have been only rarely [41, 50] compared in these appears lethal [37]. This synthetic lethal pheno- studies. Interestingly, the ABA-induced accumu- type [52] might indicate that the ABI2 and ABI3 lation of various cold-responsive genes is im- proteins belong to the same seed response path- paired in abil but not abi2 [41]. way(s). Although essentially all the above phenotypic In addition to the above seed phenotypes, the analyses have been performed on single abil and abil and abi2 mutants are defective in numerous abi2 mutant alleles, available data already indi- ABA-responses during vegetative growth. Like cate that the ABI1 and ABI2 proteins contribute the aba biosynthetic mutants [79], abil and abi2 to many ABA-regulated responses in vegetative plants display disturbed water relations as shown tissues. The relationship between the ABI1 and by their increased tendency to wilt [80]. In the ABI2 proteins in the ABA-signalling network case of abil-1 (hereafter simplified as abil) which nevertheless remains unclear. The common mu- has been analysed in more detail, this wilty phe- tant phenotypes suggest that these proteins may notype has been traced to improper regulation of both regulate certain processes. However, the dif- stomatal aperture on the abaxial (lower) surface ferential effects of the abil and abi2 mutations on of the , which is on average twice as wide in several responses suggest that the ABI1 and ABI2 the mutant as that in the wild type [86]. proteins might belong to distinct branches of the Whereas aba mutants are impaired in cold ac- ABA-signalling network. Additional Arabidopsis climation, abil nor abi2-1 (hereafter simplified as loci have been recently identified by selecting for abi2) mutation s do not alter this proce s s [ 41, 116 ]. mutants with reduced sensitivity to the ABA in- In contrast to these common phenotypes, abil hibition of seedling growth [51]. Some of these and abi2 mutations differentially affect the ABA- mutants also display reduced seed dormancy dependent morphological [163] and molecular and/or disturbed regulation of leaf water status, [50] responses of Arabidopsis plants to progres- and should thus help to further decipher the sive drought stress. The abil mutation also af- branching of the ABA-signalling network. fects the ABA-dependent accumulation of other The ABII locus has been cloned recently inde- mRNAs in response to rapid desiccation and/or pendently by us [86] and by Meyer et al. [106]. cold [83, 117, 173], the abi2 mutant was not The sequence of this gene predicts that it encodes analysed in these studies. a protein of 434 amino acids that shares sequence In addition to the above in vivo ABA-dependent similarity (35~o identity, 55~o similarity) in its processes, the abil and abi2 mutants have also carboxyl-terminus with the 2C class of serine/ been characterized for various responses to ex- threonine protein phosphatases (PP2Cs) identi- ogenously applied ABA. Abil and abi2 seedling fied in rat [154] and yeast [93]. However, in con- growth [37, 80], including root development [ 86, trast to these classical PP2Cs, which are Mg 2+-

[334] 1571 or Mn 2 + -requiring enzymes [ 17 ], the ABI 1 pro- of the protein suggest that it may have a more tein is appended with a novel amino-terminal do- versatile role. The protein might serve to cross- main containing an EF-hand Ca 2+-binding site talk and integrate ABA and other Ca 2 +-depen- [107]. The combination of these two motifs dent stimuli that converge on phosphorylation- (Fig. 2) suggests that the ABI1 protein is a modi- regulated signalling pathways. The nature of these fied phosphatase 2C which may have acquired an integrated pathways should now become acces- ability to interact with Ca 2 +. sible for systematic investigation with the cloned The structural features of the ABI1 protein gene available. evoke several intriguing possibilities regarding its role in ABA signalling, particularly with regards Conclusion to stomatal aperture and cellular division in the root meristem. However, the direct involvement We are still ignorant of the identity and functions of the ABI 1 protein in these processes would still of many of the elements involved in ABA signal- need to be verified by further biochemical, physi- ling, but have arrived at an exciting edge where ological and genetic analysis. ABA is known to pieces of the puzzle are emerging at an increas- induce an increase in cytoplasmic Ca 2 + in a va- ing rate. The impressive progress made in the last riety of cell types [ 99]. Moreover, exogenous ABA few years already provide conceptual frameworks inhibits cell division by arresting nuclei preferen- for further studies. The combined use of physi- tially in the GI phase [ 12, 87]. The p34 cd°2 gene, ological, genetic and molecular approaches will which is required for the G1/S transition and the undoubtedly continue to unravel exciting and entry into mitosis has been cloned from Arabi- possibly unexpected aspects ofABA-signal trans- dopsis [63]. Its expression was found to be com- duction pathways in plant cells. pletely inhibited in the lateral root tips and de- creased over the vascular cylinder of the entire root by exogenous ABA. Although these results Acknowledgements are not directly comparable to ours because of We thank Dorothea Bartels, Hrl~ne Barbier- different experimental criteria employed, it is con- Brygoo and Michel Delseny for critical comments ceivable that ABI1 in response to ABA or asso- on this manuscript, and the numerous colleagues ciated Ca 2 + changes could antagonize the phos- who provided us with reprints and preprints of phorylation events necessary for the synthesis and their publications. Work in our laboratory is activity of similar cell cycle components control- funded by the Centre National de la Recherche ling entry into S-phase [63, 74]. Scientifique, the European Economic Community As mentioned above, Ca 2 + is alSO strongly im- and the Minist+re de la Recherche et de la Tech- plicated as one of the second messengers involved nologie. in stomatal response [9, 99, 143]. Recent physi- ological studies with kinase and phosphatase in- hibitors further suggest that stomatal movements References as well as some of the electrogenic units involved (for example, plasma membrane H + pump, 1. Addicott FT (ed): Abscisic Acid. Praeger Scientific, New voltage-independent inward- and outward-recti- York (1983). 2. Addicott FT, Carns HR: History and introduction. In: fying K + channels) are sensitive to protein phos- Addicott FT (ed) Abscisic Acid, pp. 1-21. Praeger phorylation [85, 89, 157]. ABI1, as a potential Scientific, New York (1983). calcium-modulated phosphatase, could couple 3. Alexandre J, Lassalles JP, Kado RT: Opening of Ca 2 + ABA-stimulus response by modifying the phos- channels in isolated red beet root vacuole membrane phorylation states of these target proteins. Fur- by inositol 1,4,5-trisphosphate. Nature 343:567-569 (1990). ther, although the analysis of abil mutant has so 4. Anderson BE, Ward JM, Schroeder JI: Evidence for far been focused on ABA sensitivity, the features an extracellular reception site for abscisic acid in Com-

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