Interaction with ethylene: changing views on the role of abscisic acid in root and shoot growth responses to water stress

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1 Blckwell Science, LtdOxford, UK PCEPlnt, Cell nd Environment16-825Blckwell Science Ltd ABA, ethylene nd root nd shoot growth R. E. Shrp Review ArticleBEES SGML Plnt, Cell nd Environment (22) 25, Interction with ethylene: chnging views on the role of scisic cid in root nd shoot growth responses to wter stress R. E. SHARP Deprtment of Agronomy, Plnt Sciences Unit, 1 87 Agriculture Building, University of Missouri, Columi, MO 65211, USA ABSTRACT Shoot nd root growth re differentilly sensitive to wter stress. Interest in the involvement of hormones in regulting these responses hs focused on scisic cid (ABA) ecuse it ccumultes in shoot nd root tissues under wter-limited conditions, nd ecuse it usully inhiits growth when pplied to well-wtered plnts. However, the effects of ABA cn differ in stressed nd non-stressed plnts, nd it is therefore dvntgeous to mnipulte endogenous ABA levels under wter-stressed conditions. Studies utilizing ABA-deficient mutnts nd inhiitors of ABA synthesis to decrese endogenous ABA levels, nd experimentl strtegies to circumvent vrition in plnt wter sttus with ABA deficiency, re chnging the view of the role of ABA from the trditionl ide tht the hormone is generlly involved in growth inhiition. In prticulr, studies of severl species indicte tht n importnt role of endogenous ABA is to limit ethylene production, nd tht s result of this interction ABA my often function to mintin rther thn inhiit shoot nd root growth. Despite erly specultion tht interction etween these hormones my influence mny of the effects of wter deficit, this topic hs received little ttention until recently. Key-words: Ascisic cid; ethylene; root growth; shoot growth; wter stress. INTRODUCTION Shoot growth is very sensitive to wter-limited conditions. Much evidence over the pst 2 yers indictes tht the inhiition of growth is metoliclly regulted, rther thn eing direct consequence of ltered plnt wter sttus rising from soil drying. For exmple, studies hve shown tht stem nd lef growth cn e severely inhiited t low wter potentils despite complete mintennce of turgor in the growing regions s result of osmotic djustment (e.g. Michelen & Boyer 1982). Furthermore, shoot growth cn Correspondence: Roert E. Shrp. Fx: ; e-mil: ShrpR@missouri.edu e so sensitive to soil drying tht sustntil inhiition cn occur efore the development of decresed wter potentils in the eril plnt prts (S & Shrp 1989; Gowing, Dvies & Jones 199), nd such oservtions hve led to much interest in the involvement of non-hydrulic regultory signls from the roots (Dvies & Zhng 1991; Dvies et l. 2). Root growth is usully less inhiited thn shoot growth, or even promoted, in plnts growing in drying soil, which is of ovious enefit to mintin n dequte plnt wter supply (Shrp & Dvies 1989). An importnt feture of the root system response is the ility of some roots to continue elongtion t wter potentils tht re low enough to completely inhiit shoot growth. For exmple, this occurs in nodl (dventitious) roots of mize, which must penetrte through the often dry surfce soil (Westgte & Boyer 1985), nd in primry roots of rnge of species, which helps seedling estlishment under dry conditions y ensuring supply of wter efore shoot emergence (Shrp, Silk & Hsio 1988; Spollen et l. 1993; vn der Weele et l. 2). Figure 1 shows for severl importnt gronomic species tht the primry root mintins sustntil elongtion rtes t wter potentils s low s 1 6 MP, wheres shoot growth is inhiited completely t out 8 MP. The mechnisms tht determine the different sensitivities of root nd shoot growth to wter stress re not well understood. Although hormones re likely to ply importnt regultory roles, nd despite considerle erly ttention to this topic (e.g. Iti & Ben-Zioni 1976; Brdford & Hsio 1982), the involvement of these compounds hs not een elucidted. Most interest in this question hs concerned the role of scisic cid (ABA). This review evlutes the evidence for the involvement of ABA in root nd shoot growth responses to wter stress, with focus on dvnces in understnding gined from studying effects of endogenous ABA deficiency. In prticulr, recent studies hve reveled tht n importnt role of endogenous ABA is to limit ethylene production, nd tht this interction is involved in the effects of ABA sttus on root nd shoot growth. Despite erly specultion tht interction etween these hormones my influence mny of the effects of wter deficit (Wright 198; Brdford & Hsio 1982), this topic hs received little ttention until recently. 22 Blckwell Science Ltd 211

2 212 R. E. Shrp Elongtion rte (% control) Mize Primry Root Shoot Cotton Vermiculite wter potentil (MP) Figure 1. Elongtion rtes of the primry root ( ) nd shoot ( ) of seedlings of four species t vrious wter potentils. Seeds were germinted for 36 h t high wter potentil. The seedlings were then trnsplnted to vermiculite t different wter potentils (otined y thorough mixing with different mounts of wter) nd grown t 29 C in drkness nd ner sturtion humidity to minimize trnspirtion. For roots, dt were evluted t root lengths of 5 cm, when elongtion rtes were pproximtely stedy. For shoots, dt represent mximum elongtion rtes otined fter trnsplnting. Dt re mens from 1 to 4 seedlings, nd re plotted s percentge of the men rte t high wter potentil. (From Spollen et l Copyrighted y BIOS Scientific Pulishers nd reprinted with permission). APPROACHES TO STUDY THE ROLE OF ABA IN GROWTH RESPONSES TO WATER STRESS Soyen Sqush Interest in the involvement of hormones in regulting growth responses to wter stress hs focused on ABA ecuse () it ccumultes to high concentrtions in shoot nd root tissues under wter-limited conditions, often correlting with growth inhiition, nd () it usully inhiits growth when pplied to well-wtered plnts. Bsed on these findings, commonly proposed function of incresed ABA concentrtions in wter-stressed plnts is growth inhiition (reviewed in Trewvs & Jones 1991; Munns & Shrp 1993; Munns & Crmer 1996). Two of the most compelling exmples of this type of study re those y Creelmn et l. (199) in soyen nd y Zhng & Dvies (199) in mize. In the former study, reltionships etween root nd shoot elongtion nd the ABA contents of the respective growth zones were compred in seedlings whose growth ws inhiited y trnsfer to vermiculite of low wter potentil ( 3 MP) or to vermiculite sturted with vrious concentrtions of ABA. Similr to the responses for soyen shown in Fig. 1, shoot growth ws more inhiited thn root growth in the wter-stressed seedlings. In the well-wtered seedlings treted with ABA, growth ws lso more inhiited in the shoot thn in the root t ll ABA concentrtions tested. The reltionships of growth inhiition to ABA content suggested tht vrition in endogenous ABA ccumultion, together with differing sensitivity to ABA, could lrgely explin the differentil inhiition of shoot nd root growth t low wter potentil. In the study of mize y Zhng & Dvies (199), the reltionship etween inhiition of lef elongtion rte nd xylem sp ABA concentrtion ws compred in plnts grown in drying soil nd in well-wtered plnts to which rnge of ABA concentrtions ws supplied to prt of the root system. The similrity etween the two dt sets suggested tht the limittion of lef growth resulting from soil drying could e explined entirely y the increse in endogenous ABA in the xylem sp. Similrly to the results of these studies, primry root growth of well-wtered mize seedlings is inhiited progressively when incresing concentrtions of ABA re pplied, nd comprison of the reltionship of growth inhiition with growth zone ABA content to the vlues otined in seedlings grown t low wter potentil would suggest tht endogenous ABA ccumultion could explin the inhiition of root growth during wter stress (Fig. 2). However, interprettion of these nd other similr studies relies on the ssumption tht when ABA is pplied to well-wtered plnts nd increses to internl levels similr to those of wter-stressed plnts, the effects on growth re the sme s those resulting from endogenous ABA ccumultion during wter stress. Incresing evidence indictes tht this is not necessrily the cse. Decreses in plnt wter sttus hve een shown to chnge the comprtmenttion (Hrtung, Rdin & Hendrix 1988; Bcon, Wilkinson & Dvies 1998), pprent sensitivity (Trdieu & Dvies 1992; Dodd & Dvies 1996) nd response (Shrp et l. 1994) to ABA in vrious orgns. To void these concerns, it is of ovious dvntge to mnipulte endogenous ABA levels under wter-stressed conditions. However, despite the vilility of ABA-deficient mutnts (nd recently, trnsgenics) in severl species, nd of severl inhiitors of ABA iosynthesis, very few studies hve used this pproch to study the role of ABA in growth responses to wter stress. A prticulr difficulty for the study of ABA in growth regultion is its effect on stomtl ehviour nd, therey, on plnt wter lnce. Hence, ABA-deficient mutnts re typiclly wilty due to high stomtl conductnce even under well-wtered conditions (Qurrie 1987), nd this prolem is excerted under soil drying conditions. The decrese in wter sttus of ABA-deficient reltive to control plnts could inhiit growth independently of direct growth-regultory properties of ABA. Jones, Shrp & Higgs (1987) sujected severl ABA-deficient tomto mutnts to soil drying, nd reported greter decreses in lef nd root iomss thn in the wild 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

3 ABA, ethylene nd root nd shoot growth 213 Root elongtion rte (% control) A A ψ w = 3 MP F 2 F ψ w = 1 6 MP F Root tip ABA content (ng g 1 H 2 O) Figure 2. Primry root elongtion rte s function of root tip (picl 1 mm, encompssing the elongtion zone) ABA content for vrious mize genotypes growing in vermiculite t high wter potentil (ψ w ) ( 3 MP, open symols) or low wter potentil ( 1 6 MP, closed symols). Seedlings were grown s descried in Fig. 1. At high wter potentil, the root ABA content of hyrid (cv. FR27 FRMo17) seedlings ws rised ove the norml level y dding vrious concentrtions of ABA (A) to the vermiculite. At low wter potentil, the root ABA content ws decresed elow the norml level y tretment with fluridone (F) or y using the vp5 or vp14 mutnts. Dt re plotted s percentge of the rte for the sme genotype t high wter potentil. Elongtion rtes of the mutnts t high wter potentil were similr to their respective wild types. (Modified from S et l. 199; Shrp et l nd unpulished results of I.-J. Cho, B.-C. Tn, D.R. McCrty & R.E. Shrp.) type. However, lef wter potentils lso decresed more in the mutnts, so interprettion of the role of ABA deficiency in the greter growth inhiition of the mutnts ws not possile. Reduced root iomss compred to the wild type of ABA-deficient nd ABA-insensitive mutnts of Aridopsis in drying soil hs lso een reported (Vrtnin, Mrcotte & Girudt 1994). However, plnt wter reltions were not mesured nor were comprtive dt for wellwtered controls included, so the cuse of the smller root system development in the mutnts could not e ssessed. Accordingly, studies of the effects on growth of endogenous ABA deficiency require experimentl strtegies tht void vrition in wter sttus etween plnts with different levels of ABA. A few such studies hve now een pulished, nd the results re chnging the view of the role of ABA from the trditionl ide tht the hormone is generlly involved in growth inhiition. ABA ACCUMULATION MAINTAINS MAIZE PRIMARY ROOT GROWTH AT LOW WATER POTENTIALS A When mize seedlings re grown in vermiculite t wter potentil of 1 6 MP, t which primry root ut not shoot A Hyrid Vp5 vp5 Vp14 vp14 elongtion continues (Fig. 1), the ABA content of the root growth zone increses out five-fold (Fig. 2). Three pproches hve een used to study the effect on root growth of reducing the ccumultion of ABA: (i) the inhiitor fluridone, which locks crotenoid synthesis (t the conversion of phytoene to phytofluene) nd, therey, inhiits ABA synthesis lthough t n erly step of the pthwy; (ii) the vp5 mutnt, which hs defect t the sme step s tht locked y fluridone; (iii) the vp14 mutnt, which hs defect in the synthesis of xnthoxin (Tn et l. 1997). Xnthoxin is converted to ABA vi ABA-ldehyde, nd its synthesis is considered key regultory step in wter stressinduced ABA production (Qin & Zeevrt 1999). Initil studies used the fluridone nd vp5 mutnt pproches (S et l. 199; S, Shrp & Pritchrd 1992; Shrp et l. 1994). Recently, similr studies of vp14 were undertken to strengthen the conclusion tht the results otined with fluridone nd vp5 were due to ABA deficiency nd not to other effects of their ction (I.-J. Cho, B.-C. Tn, D.R. McCrty & R.E. Shrp, unpulished results). To circumvent vrition in plnt wter sttus with ABA deficiency, seedlings were grown under conditions of ner-zero trnspirtion (miniml shoot development, drkness nd nersturtion humidity). The results otined with the three pproches were very similr. At high wter potentil, root elongtion rtes (nd ABA contents) were minimlly ffected. At low wter potentil, in contrst, reduced ABA ccumultion ws ssocited with more severe inhiition of root elongtion thn in wild-type or untreted seedlings (Fig. 2). The three methods produced vrying degrees of root tip ABA deficiency, ut yielded common reltionship of inhiition of root elongtion rte to root tip ABA content. In ll cses, root elongtion rte fully recovered when the ABA content of the elongtion zone ws restored to norml levels with exogenous ABA. Similr results were otined using fluridone-treted nd vp5 mutnt seedlings grown t wter potentil of 3 MP (Shrp et l. 1994). These studies demonstrted tht ccumultion of ABA is required for the mintennce of mize primry root elongtion t low wter potentils. Additionl experiments t wter potentil of 3 MP showed tht when the root tip ABA content of fluridonetreted seedlings ws incresed to twice (or more) the level in untreted seedlings, recovery of root elongtion ws decresed. Also, promotion of root elongtion ws not oserved with ny pplied ABA concentrtion in untreted seedlings; gin, slight inhiition of elongtion resulted when the root tip ABA content ws incresed to twice the nturlly occurring level (Shrp et l. 1994). Thus, the norml increse in ABA content in roots t low wter potentil is optiml for growth mintennce (lso see Fig. 7). It is importnt to note tht the conclusion tht the ccumultion of ABA in wter-stressed roots helps to mintin growth cnnot e inferred y pplying ABA to wellwtered seedlings to simulte the increse in content under wter stress (Shrp et l. 1994). Figure 2 shows tht in wellwtered seedlings treted with ABA, root growth ws su- 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

4 214 R. E. Shrp stntilly inhiited t the root tip ABA content tht occurs in wter-stressed roots. These results illustrte tht the mintennce of root elongtion t low wter potentil y ABA is not solely function of the increse in ABA content, ut lso requires the chnge in environmentl conditions tht modifies the growth response to ABA. ABA mintins root growth t low wter potentils y restricting ethylene production 3 c 2 () Cntl +AOA +FLU +FLU +AOA Cntl +AOA +FLU +FLU +AOA Figure 4. Primry root length increse () nd ethylene evolution rte () of control (Cntl) nd fluridone (FLU)-treted mize seedlings with or without tretment with minooxycetic cid (AOA) to inhiit ethylene synthesis. Mesurements were mde t 42 h () nd 2 h () fter trnsplnting to vermiculite t wter potentil of 1 6 MP; seedlings were grown s descried in Fig. 1. Within ech pnel, rs with different letters re significntly different t the 5 level. (Modified from Spollen et l. 2.) ll cses the root tip ABA content of the ABA-deficient seedlings t low wter potentil remined higher thn in well-wtered seedlings (Fig. 2), indicting tht n incresed concentrtion of ABA is necessry to prevent excess ethylene production under wter stress. Third, it ws shown tht root elongtion could e lrgely restored y ech of three inhiitors of ethylene synthesis or ction (Spollen et l. 2). Significntly, when ethylene evolution ws restored Root elongtion rte (% inhiition) Hyrid + FLU vp 5 vp Root elongtion 4 6 Ethylene evolution Ethylene evolution (% promotion) Extending the study of ABA-deficient mize seedlings, recent work hs shown tht n importnt function of endogenous ABA in the mintennce of primry root growth t low wter potentil is to prevent excess ethylene production. First, it ws oserved tht the roots of ABAdeficient seedlings t low wter potentil not only re shorter ut lso exhiit pronounced rdil swelling primrily eyond the picl 2 mm (Fig. 3). Exogenous ethylene inhiits elongtion nd cuses similr pttern of swelling in mize primry roots t high wter potentil (Moss, Hll & Jckson 1988; Whlen & Feldmn 1988). Second, it ws shown tht under wter stress, ethylene production of ABA-deficient seedlings ws sustntilly greter thn in control plnts (Figs 4 & 5), nd this effect ws completely prevented when the root ABA content ws restored with exogenous ABA, together with restortion of root elongtion (Spollen et l. 2). Among the three methods used to reduce ABA ccumultion (fluridone, vp5, vp14), the mgnitude of the increse in ethylene evolution correlted oth with the degree of ABA deficiency nd the inhiition of root elongtion rte (Fig. 5). It is importnt to note tht in () Ethylene evolution rte per seedling (pmol h 1) Root length increse (mm) Root tip ABA content (% inhiition) Figure 5. Primry root elongtion nd seedling ethylene Figure 3. The picl 11 mm of primry roots of () untreted nd fluridone (FLU)-treted hyrid, nd () wild-type nd vp5 mutnt mize seedlings 48 h fter trnsplnting to vermiculite t wter potentil of 1 6 MP. Seedlings were grown s descried in Fig. 1. Br = 1 mm. (From Shrp et l Copyrighted y the Americn Society of Plnt Biologists nd reprinted with permission.) evolution rte s function of root tip (picl 1 mm) ABA content for fluridone (FLU)-treted hyrid, vp5 mutnt nd vp14 mutnt mize seedlings growing in vermiculite t wter potentil of 1 6 MP. Dt re plotted s percentge of vlues for untreted or wild-type seedlings growing t the sme wter potentil. (Modified from Spollen et l. 2 nd unpulished results of I.-J. Cho, B.-C. Tn, D.R. McCrty & R.E. Shrp.) 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

5 ABA, ethylene nd root nd shoot growth 215 to norml levels [using the inhiitor of ethylene synthesis minooxycetic cid (AOA)], root growth ws completely restored (Fig. 4), suggesting tht ABA deficiency did not lso increse the sensitivity of growth to ethylene. Importntly, since none of the inhiitors of ethylene synthesis or ction sustntilly incresed root elongtion when ABA-deficiency ws not imposed (results with AOA re shown in Fig. 4), ethylene does not pper to e n importnt cuse of the inhiition of elongtion in wterstressed roots tht ccumulte norml levels of ABA (Figs 1 & 2). In other words, the ccumultion of ABA in wter-stressed roots is sufficient to prevent excess ethylene production, in greement with the ove-mentioned finding tht roots t low wter potentil exhiit n optimum ABA content for growth mintennce. The possile involvement of ethylene in the inhiition of growth during wter stress is long-stnding question (El-Beltgy & Hll 1974), ut there is no previous informtion in reltion to root growth. This question is considered further elow in reltion to the inhiition of shoot growth in wter-stressed plnts. The discovery tht incresed levels of endogenous ABA in roots t low wter potentil re required to limit ethylene production confirms ides first suggested y Wright (198) nd developed further y Brdford & Hsio (1982) ut which hd not een tested. These suggestions were sed on the finding tht pre-tretment with exogenous ABA prevented the increse in ethylene production cused y wilting of excised whet leves. It should e noted tht lthough severl other studies reported tht ABA tretments of well-wtered plnts inhiited ethylene production (e.g. Gertmn & Fuchs 1972; Yoshi & Imseki 1981; Tn & Thimnn 1989), there re lso mny reports of ABA-stimulted ethylene production (Riov et l. 199 nd references therein). Interprettion of these results is complicted y uncertinty tht effects of pplied ABA in well-wtered plnts re predictive of the role of endogenous ABA ccumultion in wter-stressed plnts, s discussed ove. The pproch of using chemicl nd genetic mens to mnipulte endogenous ABA levels in wter-stressed plnts voided these concerns. ABA ACCUMULATION CAN INHIBIT AND PROMOTE MAIZE SEEDLING SHOOT GROWTH AT LOW WATER POTENTIALS As discussed ove, ABA is generlly regrded s n inhiitor of shoot growth (Trewvs & Jones 1991; Dvies 1995; Munns & Crmer 1996). Initil studies of the effect of decresing endogenous ABA levels in mize seedlings grown t low wter potentil (under conditions of ner-zero trnspirtion) were consistent with this expecttion (S et l. 199, 1992). Seedlings were grown in vermiculite t wter potentil of 3 MP, t which slow rtes of shoot growth continue (Fig. 1), nd fluridone nd the vp5 mutnt were gin used to decrese endogenous ABA ccumultion. Studies were confined to the first 5 h fter trnsplnting to low wter potentil, nd showed tht ABA-deficiency resulted in sustntil promotion of shoot elongtion. Similr results of experiments with fluridone re shown in Fig. 6 nd Tle 1 (first 4 h fter trnsplnting). These experiments indicted n involvement of ABA in the inhiition of shoot growth of wter-stressed mize seedlings, in contrst to its role in root growth mintennce. However, experiments of longer durtion reveled greter complexity in the reltionship of shoot growth to ABA sttus t low wter potentil (Feng 1996). After 5 h Shoot length increse (mm) 8 () ψ w = 3 MP () Control +STS Control +ABA +FLU +FLU,+ABA ψ w = 3 MP ψ w = 3 MP Hours fter trnsplnting Figure 6. Shoot length increse of mize seedlings (cv. FR27 FRMo17) fter trnsplnting to vermiculite t high ( 3 MP) or low ( 3 MP) wter potentil (ψ w ). Seeds were germinted for 55 h t high wter potentil, then seedlings were trnsplnted to high or low wter potentil nd grown t 29 C in drkness nd ner sturtion humidity. () Fluridone (FLU, 1 µm) nd ABA were supplied during germintion nd fter trnsplnting. The ABA concentrtion supplied during germintion (1 µm) prtilly restored (y pproximtely 45%) the ABA content of fluridone-treted shoots erly fter trnsplnting; higher concentrtions were not used ecuse they severely delyed germintion. The ABA concentrtion supplied fter trnsplnting (2 µm) ws optiml for growth promotion during the second phse of the experiment. () Silver thiosulphte (STS, 6 mm) ws supplied during germintion to inhiit ethylene ction; the STS concentrtion ws optiml for growth promotion during the second phse of the experiment. (STS ws not supplied fter trnsplnting ecuse this resulted in less growth promotion, presumly due to toxicity from long-term exposure.) Dt re mens ±SE, n = 24. (Modified from Feng 1996.) 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

6 216 R. E. Shrp Tle 1. Effects of tretments with ABA, fluridone (FLU) nd silver thiosulphte (STS) on shoot elongtion rte of mize seedlings during erly nd lter periods fter trnsplnting to vermiculite t wter potentil of 3 MP. Elongtion rtes were clculted from the shoot length increses shown in Fig. 6. (Modified from Feng 1996) Tretment Shoot elongtion rte (mm h 1 ) 41 h h Control ABA FLU FLU, +ABA h h Control STS from trnsplnting to wter potentil of 3 MP, the effect of ABA-deficiency reversed so tht shoot growth ecme inhiited reltive to the control (Fig. 6). Tle 1 shows tht shoot elongtion rte etween 72 nd 135 h ws inhiited y over 7% in fluridone-treted compred to control seedlings. Both the fluridone-induced promotion of shoot growth erly fter trnsplnting nd the inhiition during the lter period could e prevented with exogenous ABA. Moreover, when the sme ABA tretment ws pplied without tretment with fluridone, shoot growth ws further inhiited in the erly phse nd further promoted during the lter phse (Fig. 6), such tht the shoot elongtion rte ws more thn two-fold greter thn in the control seedlings fter 7 h from trnsplnting (Tle 1). These results indicte tht the norml ccumultion of ABA in wter-stressed mize seedlings functions to inhiit shoot growth only t n erly stge of development, nd susequently helps to mintin growth, s in roots. In contrst to the roots, however, in which ABA levels were optiml for growth mintennce, endogenous ABA ccumultion ws pprently insufficient for mximl shoot growth during the second phse of the experiment. In the experiment shown in Fig. 6, supplementl ABA ws supplied oth during germintion nd fter trnsplnting, nd the resulting growth promotion fter 7 h ws lrgely ttriutle to growth of the mesocotyl. Since it is lso the mesocotyl (nd coleoptile) tht is promoted y ABA deficiency during the first 4 h (S et l. 199, 1992), the chnge in response of shoot growth to ABA from inhiition to promotion ws not due to differentil sensitivity mong orgns ut occurred within the sme orgn. The shift to growth promotion ws not restricted to the mesocotyl, however. In other experiments in which ABA ws supplied only vi the low-wter-potentil vermiculite into which the seedlings were trnsplnted, overll shoot elongtion ws similrly promoted fter 5 h ut the response ws specific to lef growth (Fig. 7). This difference proly reflects the different developmentl stge of the shoots reltive to the timing of ABA uptke. Importntly, there ws no effect of the supplementl ABA tretment on root growth (Fig. 7), demonstrting tht the promotion of shoot growth ws not ttriutle indirectly to root growth inhiition (see shoot nd root dry weight dt in Fig. 7, legend), nd in greement with the ove-mentioned conclusion tht the endogenous ABA content of wter-stressed roots is optiml for growth mintennce. As ws the cse for root growth (Fig. 2), shoot growth of seedlings grown t high wter potentil ws inhiited progressively with incresing concentrtions of pplied ABA (Feng 1996). Thus, the promotive effect of supplementl ABA on shoot growth t low wter potentil ppered to e specific to the wter-stressed condition. Effects of ABA on shoot growth re mimicked y inhiiting ethylene ction The chnge with time fter trnsplnting to low wter potentil in the response of shoot growth to supplementl ABA, from inhiition to promotion, ws closely mimicked y tretment with silver thiosulphte (STS) to inhiit ethylene ction (Fig. 6, Tle 1). Thus, tretment with STS inhiited shoot growth erly fter trnsplnting nd then mrkedly promoted shoot growth reltive to the control. Similr results were otined using AOA to inhiit ethylene synthesis (Feng 1996). Consistently, preliminry exper- Figure 7. Representtive mize seedlings 29 h fter trnsplnting to vermiculite t wter potentil of 3 MP tht contined either µm (control) or 1 µm ABA. Seedlings were grown s descried in Fig. 6. Dry weights (mg, mens ±SE, n = 24) were: control shoot, 24 ± 4; control root, 69 ± 6; +ABA shoot, 33 ± 3; +ABA root, 7 ± 7. (From Feng 1996.) 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

7 ABA, ethylene nd root nd shoot growth 217 iments showed tht shoot growth could e incresed during the erly phse, nd inhiited during the lter phse, y pplying either ethylene or the ethylene precursor 1- minocyclopropne-1-croxylte (ACC), simulting the effects of ABA deficiency. Tken together, the results in Fig. 6 nd Tle 1 suggest tht, s in roots, n importnt role of ABA in shoot growth of wter-stressed mize seedlings is to restrict ethylene production nd/or sensitivity. ABA ccumultion therey plyed either growth-inhiitory (erly phse) or growthmintining (lter phse) role in the response of shoot growth to wter stress, due to chnge in the effect of ethylene from promotive to inhiitory. The sustntil promotion of shoot growth t lter stges of development y tretment with STS indictes tht the norml ccumultion of ABA in the shoot ws suoptiml for growth ecuse it ws insufficient to fully prevent ethylene-induced growth inhiition. This contrsts with the pprent sufficiency of ABA ccumultion in the roots, nd this difference ccounted t lest prtly for the differentil ility of the roots nd shoots to grow t low wter potentil. It should e noted tht the extent to which ethylene cused the inhiition of shoot growth in wter-stressed seedlings t lter stges of development, nd therefore the extent to which growth could e promoted y supplementl ABA, ws influenced y nutritionl sttus. It ws susequently discovered tht the seedlings used for the experiments illustrted in Figs 6 nd 7 nd Tle 1 were grown with suoptiml C 2+ vilility. When seedlings were grown with different culture protocol involving supplementl C 2+ (ecuse the properties of the vermiculite hd chnged; Spollen et l. 2), responses to STS nd ABA fter 5 h from trnsplnting were less pronounced (M.A. Else & R.E. Shrp, unpulished results). Such n interction with nutrient sttus is not unexpected, ecuse nutrient vilility cn mrkedly lter plnt ethylene reltions (e.g. He, Morgn & Drew 1992). GENERALITY OF THE PROMOTIVE EFFECT OF ABA ON SHOOT GROWTH As discussed ove, most of the reserch underlying the view tht ABA is n inhiitor of shoot growth during wter stress hs involved pplictions of ABA to non-stressed plnts or correltions of growth inhiition to incresed endogenous ABA levels during stress. Indeed, the erly phse of the mize seedling studies descried ove (Fig. 6; S et l. 199, 1992) remins the only demonstrtion of enhnced shoot growth in response to endogenous ABA deficiency t low wter potentils. The pprent chnge in response to ethylene during shoot development in those experiments, from promotive to inhiitory, is consistent with reports tht ethylene stimultes mesocotyl growth in some species (Suge 1971; Cornforth & Stevens 1973), wheres it is usully inhiitory to shoot growth of terrestril plnts t lter stges of development (Aeles, Morgn & Sltveit 1992; Lee & Reid 1997; Hussin et l. 1999). The following sections summrize recent studies which suggest tht restriction of ethylene production my e common function of ABA nd therefore tht endogenous ABA my often ct to mintin rther thn inhiit shoot growth. Ressessment of the role of ABA in shoot growth of tomto nd Aridopsis under wellwtered conditions Prdoxiclly to the long-stnding view tht ABA is generlly inhiitory to shoot growth, it hs een oserved for over 3 yers tht ABA-deficient mutnts re often shorter nd hve smller leves thn the corresponding wild types, nd tht lef nd stem growth cn e sustntilly restored y pplying ABA (Imer & Tl 197; Brdford 1983; Qurrie 1987). As lredy mentioned, in ddition to reduced growth, ABA-deficient mutnts re typiclly wilty even when the soil is well supplied with wter. This results from high stomtl conductnce nd hs lso een shown to involve decresed root hydrulic conductnce. These effects cn lso e prevented y pplying ABA (Imer & Tl 197; Tl & Nevo 1973; Koornneef et l. 1982; Brdford 1983). Accordingly, the inhiited shoot growth of ABAdeficient mutnts of tomto nd Aridopsis hs een ttriuted to shoot wter deficits (Brdford 1983; Neill, McGw & Horgn 1986; Ngel, Konings & Lmers 1994; Léon-Kloosterziel et l. 1996). The growth-promotive effect of pplied ABA in these cses hs therefore een ssumed to result from improvement of plnt wter lnce. Consequently, these findings hve generlly not een regrded s evidence ginst direct inhiitory role of ABA in lef nd stem growth, lthough the oservtions hve led some uthors to question this view (Jones et l. 1987; Qurrie 1987; Tylor 1987; Zeevrt & Creelmn 1988). Alterntively, endogenous ABA my e required to mintin shoot growth of well-wtered plnts independently of its effect on plnt wter lnce. Consistent with the finding tht ABA restricts ethylene production in wterstressed mize seedlings, it ws reported tht under wellwtered conditions, ethylene production ws greter in shoots of the flcc (flc) mutnt of tomto (Tl et l. 1979) nd in whole plnts of the 1 mutnt of Aridopsis (Rkitin et l. 1994) thn in the corresponding wild types. In flc, it ws lso shown tht ethylene production could e reduced to norml levels with exogenous ABA. In ddition, the ABA-deficient mutnts of tomto exhiit morphologicl symptoms chrcteristic of excess ethylene such s lef epinsty nd dventitious rooting (Tl 1966; Ngel et l. 1994). Despite these oservtions, the possiility tht ethylene is cuse of reduced shoot growth in ABA-deficient mutnts ws not considered until recently. To distinguish etween these possiilities, it is necessry to ssess the growth of ABA-deficient mutnts t the sme plnt wter sttus s the wild type. If the inhiition of growth normlly oserved is cused y dverse wter reltions, then under such conditions growth should e restored 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

8 218 R. E. Shrp Lef re (cm 2 ) c c () WT flc flc flc (sc) +ABA Lef ABA content (nmol g 1 FW) () WT flc flc flc (sc) +ABA Ethylene evolution (pmol g 1 FW h 1 ) (c) WT flc flc flc (sc) +ABA Figure 8. Totl lef re (, 35 d fter emergence), lef ABA content (, 21 d) nd lef ethylene evolution rte (c, 21 d) of wild-type (WT) tomto (cv. Rheinlnds Ruhm), flcc (flc), flcc spry control (sc), nd flcc spryed with 1 µm ABA dily from dy 9. Spry control plnts were spryed with deionized wter contining the sme concentrtions of ethnol nd Tween 2 s in the ABA solution. All plnts were well wtered, nd were grown under controlled-humidity conditions so tht throughout development, lef wter potentils of untreted flcc were equl to or higher thn those of the wild type, nd lef wter potentils of flcc treted with ABA were equl to or lower thn those of the flcc spry control. Within ech pnel, rs with different letters re significntly different t the 5 (lef re, ABA) or 1 (ethylene) level. (Modified from Shrp et l. 2.) or even enhnced reltive to wild-type plnts. In contrst, if endogenous ABA is required to mintin shoot growth independently of effects on plnt wter lnce, then the mutnts should remin smller. Jones et l. (1987) grew flc nd two other ABA-deficient mutnts of tomto, notilis (not) nd sitiens (sit), under mist in greenhouse, nd oserved tht stem height ecme greter thn in the wild type, consistent with ABA plying n inhiitory role in stem elongtion. In contrst, lter-formed leves remined smller nd totl lef iomss remined sustntilly decresed. However, lef wter potentils lso remined considerly lower thn in the wild type, so interprettion of the role of ABA in lef growth ws not possile. Tylor (1987) reported tht the inhiition of shoot growth in doule mutnts of flc, not nd sit ws prtilly llevited when the plnts were grown t high humidity, ut informtion on plnt wter reltions ws not included so, gin, full interprettion ws not possile. In other studies in which flc ws grown t high humidity, effects on growth were not reported (Puri & Tl 1977; Tl et l. 1979). To ssess whether the reduced lef nd stem growth of well-wtered flc nd not re ttriutle to wter deficits, in recent study plnts were grown under controlled-humidity conditions in growth chmer, such tht the lef wter potentils of the mutnts were equl to or higher thn those of wild-type plnts throughout development (Shrp et l. 2). Most prmeters of shoot growth remined mrkedly impired; for exmple, totl lef re in flc ws 48% less thn in the wild type (Fig. 8). Root growth ws lso gretly reduced. Consistent with the oservtion of Jones et l. (1987), the stems of the mutnts initilly elongted more rpidly thn those of the wild types; however, this effect ws not sustined, nd the mutnts ecme shorter t lter stges of development. Shoot growth sustntilly recovered when wild-type levels of ABA were restored y tretment with exogenous ABA (Fig. 8,), even though the experimentl strtegy prevented improvement in lef wter potentil. The ility of pplied ABA to increse growth ws gretest for lef expnsion, which ws restored y 75%. The ethylene evolution rte of growing leves ws douled in flc compred with the wild type (Fig. 8c), nd tretment with STS to inhiit ethylene ction prtilly restored lef, stem nd root growth (Shrp et l. 2). Similr results hve een otined in preliminry studies of Aridopsis. When grown t the sme lef wter potentils s well-wtered wild-type plnts throughout development, totl lef re of the 2 mutnt (ABA-deficient) remined gretly inhiited, nd ws restored y out 5% in doule mutnt of 2 nd etr1 (ethylene-resistnt) (M.E. LeNole, W.G. Spollen & R.E. Shrp, unpulished results). The lef ethylene evolution rte of 2 ws pproximtely twice tht of the wild type under these conditions. These results demonstrte tht () norml levels of endogenous ABA re required to mintin shoot development, prticulrly lef expnsion, in well-wtered tomto nd Aridopsis plnts independently of effects on plnt wter lnce, nd () the impirment of lef nd shoot growth cused y ABA deficiency is t lest prtly ttriutle to incresed ethylene production. It should e noted tht these findings do not negte the necessity of voiding differences in plnt wter sttus mong genotypes in future studies of the effects of ABA mnipultion on growth. Differences in wter reltions, when they do occur, might lso contriute directly or indirectly to growth responses. It will e prticulrly criticl (nd chllenging) to extend the strtegy of circumventing vrition in plnt nd lso soil wter sttus etween control nd ABA-deficient (or ABA-overproducing) genotypes to ddress the function of ABA ccumultion in the responses of plnt growth to soil drying. The conclusion with well-wtered tomto nd Aridopsis tht endogenous ABA is required to mintin shoot 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

9 ABA, ethylene nd root nd shoot growth 219 growth contrsts with the mny exmples in the literture where ABA hs cused shoot growth inhiition when pplied to well-wtered plnts. The explntion for this difference might e simply explined y suggesting tht the effects of supplementl ABA in well-wtered plnts re non-physiologicl. However, Bcon et l. (1998) pulished compelling evidence tht the norml levels of ABA in wellwtered rley cn cuse lef growth inhiition. They showed tht the lef elongtion rte of wild-type plnts declined s the ph of rtificil xylem sp fed to the leves ws incresed, ut tht this response did not occur in the ABA-deficient mutnt Az34 unless norml well-wtered concentrtion of ABA ws supplied in the sp. (The phinduced reduction in lef elongtion is suggested to result from incresed ccumultion of ABA in the poplst.) This result is difficult to reconcile with the inhiited growth of the ABA-deficient mutnts of tomto nd Aridopsis. It is possile tht whole plnt ABA-deficiency leds to n overriding inhiitory effect on growth of incresed ethylene production. However, the ove-descried experiments with wter-stressed mize seedlings indicte tht, for root growth t lest, this is not the cse. In ABA-deficient seedlings in which ethylene evolution ws restored to the norml wter-stressed rte y tretment with AOA, root growth recovered ut only to the rte of the control seedlings (Fig. 4). In other words, preventing the effect of ABAdeficiency on ethylene production did not uncover growth-promotive effect of ABA deficiency. Resolution of the differing conclusions on the role of ABA in lef growth of well-wtered plnts from the studies of rley (Bcon et l. 1998) nd of tomto nd Aridopsis will require further investigtion. Role of ABA in shoot growth response to compction Plnts growing in compcted soil often exhiit reduced shoot growth. Evidence tht endogenous ABA plys role in shoot growth mintennce rther thn inhiition in compction-stressed rley ws reported y Mulhollnd et l. (1996, ). Lef growth of plnts growing in compcted soil ws more inhiited in Az34 thn in the wild type, nd the evidence suggested tht chnges in lef wter reltions were not the cuse. Recent studies from the sme group demonstrted tht ethylene ws mjor cuse of the inhiition of shoot growth in tomto plnts grown with their root system divided etween pots of uncompcted nd compcted soil (Hussin et l. 1999, 2). As shown in Fig. 9c, ethylene production incresed gretly in wild-type plnts under compction. This response ws lmost fully prevented in the ethylene-deficient trnsgenic ACO1 AS (ACC oxidse ntisense; Hmilton, Lycett & Grierson 199), s ws the inhiition of lef growth (Fig. 9). The increse in ethylene production in the wild type occurred despite n increse in xylem sp ABA concentrtion (Fig. 9). Interestingly, when supplementl ABA ws supplied to the wild type, lef ethylene production ws prtly reduced nd lef growth ws prtilly restored. The sme ABA tretment hd miniml effect on growth or ethylene evolution in ACO1 AS, indicting tht the effect of ABA on growth in the wild type ws likely n indirect effect vi inhiition of ethylene production. These results indicte tht the norml increse in ABA in compction-stressed plnts ws insufficient to fully prevent excess ethylene production in the leves. In ddition, Hussin et l. (2) ttempted to use the ABA-deficient not mutnt to demonstrte tht the increse in endogenous ABA in the wild type helped to mintin lef growth during compction y limiting the production of ethylene. However, ethylene production ws similr nd inhiition of shoot growth ws ctully less in the mutnt thn in the wild type in compcted reltive to noncompcted plnts. Interprettion of these findings ws confounded, however, ecuse growth of the mutnt ws lredy sustntilly inhiited in uncompcted control plnts. The inhiition in the control plnts ws ssocited with, nd proly cused y, n lredy-incresed rte of lef ethylene evolution, consistent with the results for flc shown in Fig. 8. The prolem of impired growth of ABAdeficient control plnts is discussed further elow. Lef re (cm 2 ) Uncompcted Wild type ACO1 AS Compcted () Compcted+ABA Xylem ABA concentrtion ( mol m 3 ) µ Uncompcted Compcted () Compcted+ABA Ethylene evolution (pmol g 1 FW h 1 ) Uncompcted Compcted (c) Compcted+ABA Figure 9. Totl lef re (), xylem sp ABA concentrtion (), nd lef ethylene evolution (c) of wild-type (cv. Ails Crig) nd ACO1 AS trnsgenic tomto plnts t 21 d fter emergence. Plnts were well wtered, nd were grown in split-pot system in which either oth comprtments contined uncompcted soil or one comprtment contined uncompcted soil nd the other contined compcted soil. The comprtment contining compcted soil ws supplied either with wter or with 1 nm ABA (compcted +ABA) twice dily from dy 5. (Modified from Hussin et l. 2.) 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

10 22 R. E. Shrp DOES ABA MAINTAIN SHOOT GROWTH IN WATER-STRESSED PLANTS AT LATER STAGES OF DEVELOPMENT? As descried ove, in well-wtered tomto nd Aridopsis plnts, the norml low levels of ABA re sufficient to prevent excess ethylene production. In contrst, in roots of wter-stressed mize seedlings, ABA deficiency resulted in incresed ethylene production lthough ABA levels were still sustntilly higher thn in well-wtered plnts (Figs 2, 4 & 5). Similrly, in tomto plnts exposed to soil compction, lef ethylene production incresed to growth-inhiitory levels despite n increse in xylem sp ABA concentrtion (Fig. 9). Tken together, these results indicte tht, for resons unknown, stressed reltive to nonstressed plnts require n incresed concentrtion of ABA to prevent excess ethylene production. Furthermore, in oth wter-stressed mize seedlings nd compctionstressed tomto plnts, norml increses in endogenous ABA were insufficient to fully prevent ethylene-induced shoot growth inhiition. In contrst, ethylene ws not n importnt cuse of the inhiition of elongtion in wterstressed mize roots tht ccumulted norml levels of ABA. Bsed on these findings, it seems resonle to hypothesize tht incresed levels of endogenous ABA function to mintin rther thn inhiit shoot growth of wterstressed plnts t lter stges of development, ut tht the ccumultion of ABA my e insufficient to fully prevent ethylene-induced growth inhiition, hence contriuting to the greter sensitivity of shoot reltive to root growth. Consistent with this hypothesis, there re mny reports tht ethylene production cn e incresed y plnt wter deficits (e.g. El-Beltgy & Hll 1974). However, Morgn et l. (199) nd Nryn, Llonde & Sini (1991) reported tht in intct cotton, en nd whet plnts sujected to slow soil drying, ethylene production rtes decresed. They cutioned tht mny of the erlier oservtions might hve een erroneous due to the use of excised plnt prts nd rpid drying. Restriction of ethylene production resulting from ABA ccumultion provides possile explntion for decresed ethylene production under wter deficits. Definitive experiments to test whether incresed ethylene production in drying soil, when it occurs, is cuse of the inhiition of lef growth re lcking. In prticulr, despite the vilility of ethylene deficient or insensitive mutnts nd trnsgenic plnts of severl species, there re no reports of the growth responses of these genotypes to soil drying. To ssess the role of ABA in the responses of growth to soil drying, system in which ABA levels re dequte for norml growth under control conditions is essentil. Otherwise, it my not e possile to distinguish the role of stress-induced increses in ABA concentrtion from the function of norml ABA levels. As descried ove, shoot growth is lredy sustntilly inhiited in ABA-deficient tomto nd Aridopsis mutnts under non-stressed conditions independently of effects on plnt wter lnce, t lest prtly due to excess ethylene production. This would confound interprettion of the effect of decresing ABA ccumultion on growth under soil drying conditions. It is lso essentil tht root s well s shoot development is norml under non-stressed conditions. Otherwise, fter withholding wter, the ptterns of soil drying nd root stress exposure would differ etween genotypes. Such n effect would proly influence the shoot growth response ecuse of the likely involvement of non-hydrulic signls from roots in drying soil. To void the prolem of impired growth in control plnts, prtilly complemented line of the not mutnt of tomto hs recently een identified tht exhiits norml ABA levels nd shoot nd root growth when well wtered yet is deficient in wter stress-induced ABA ccumultion (E. T. Thorne, A. C. Jckson, A. Buridge, A. J. Thompson, I. B. Tylor & R. E. Shrp, unpulished results). This genotype is eing utilized to exmine specificlly the role of incresed ABA concentrtions in the response of shoot growth to soil drying. CONCLUSIONS The role of ABA in determining plnt growth responses to wter stress is long-stnding question. The studies of ABA-deficient genotypes descried in this review hve provided compelling insight into this issue. The commonlity of the findings in mize, tomto nd Aridopsis suggests tht restriction of ethylene production my e widespred function of ABA, nd tht, s result, endogenous ABA my often function to mintin rther thn inhiit plnt growth. In ddition to root nd shoot growth regultion, this hormonl interction is likely to e relevnt to other stress responses tht re thought to involve ethylene, for exmple erly lef senescence nd lef, flower nd fruit scission (Morgn & Drew 1997). Moreover, ecuse increses in ABA concentrtion nd ethylene production occur in response to rnge of environmentl stress conditions, the findings my e of wide pplicility. ACKNOWLEDGMENTS I thnk Dr Mry LeNole for useful discussions nd helpful comments on the mnuscript. The work ws supported y the Ntionl Science Foundtion, the Ntionl Reserch Inititive Competitive Grnts Progrm of the U.S. Deprtment of Agriculture, nd the University of Missouri Food for the 21st Century Progrm. Contriution no. 13,171 from the Missouri Agriculturl Experiment Sttion Journl Series. REFERENCES Aeles F.B., Morgn P.W. & Sltveit M.E. Jr (1992) Ethylene in Plnt Biology, 2nd edn. Acdemic Press, Inc., Sn Diego, CA. Bcon M.A., Wilkinson S. & Dvies W.J. (1998) ph-regulted lef cell expnsion in droughted plnts is scisic cid dependent. Plnt Physiology 118, Blckwell Science Ltd, Plnt, Cell nd Environment, 25,

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Effect of scisic cid nd wter content on RNse ctivity nd RNA. Plnt Physiology 59, Qin X. & Zeevrt J.A.D. (1999) The 9-cis-epoxycrotenoid clevge rection is the key regultory step of scisic cid iosynthesis in wter-stressed en. Proceedings of the Ntionl Acdemy of Sciences USA 96, Qurrie S.A. (1987) Use of genotypes differing in endogenous scisic cid levels in studies of physiology nd development. In 22 Blckwell Science Ltd, Plnt, Cell nd Environment, 25,