Plant Growth Regulation 23: 79–103, 1997. 79c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

The involvement of cytokinins in plant responses to environmental stress

P.D. Hare�, W.A. Cress & J. van StadenNatal University Research Unit for Plant Growth and Development, Department of Botany, University of NatalPietermaritzburg, Private Bag X01, Scottsville, 3209, South Africa (�author for correspondence)

Received 15 May 1997; accepted in revised form 24 July 1997

Key words: cytokinin, environmental stress, MAP kinase cascade, signal transduction

Abstract

Cytokinin (CK) levels tend to decrease under adverse environmental conditions. A general view has emergedthat during stress, a reduction of CK supply from the root alters gene expression in the shoot and thereby elicitsappropriate responses to ameliorate the effects of stress. However, recent studies have indicated that transcriptionof many stress-inducible genes can also be caused by CK application. This review attempts to highlight someof these apparently paradoxical findings and to suggest potential approaches for future research in this area.Changes in patterns of gene expression associated with responses to adverse environmental conditions are mostprobably the end products of hierarchical changes in regulatory controls exercised at hormonal, developmentaland morphological levels. We consider possible roles for CKs in affecting overall hormonal balance following theimposition of environmental stresses, and thereby playing a role in systemic responses to stress. Particular attentionis devoted to the interactions of CKs with ABA, ethylene, salicylic acid and jasmonates, all of which are knownto be involved in plant stress responses. The recent emergence of MAPK-type cascades in mediating responses toseveral environmental modifications is discussed in relation to their possible modulation by CKs.

Since CKs are likely to elicit their effects at the level of gene expression and stress-induced alterations ingene expression are usually rapid and repeatable, molecular analysis of CK-regulated stress-inducible genes maycontribute to enhanced understanding of CK-mediated signal transduction in plants. In this regard, examinationof stress responses of transgenic plants with altered levels of endogenous CKs as well as mutants altered in CKmetabolism or signalling may be informative. Emerging evidence that CKs may impact on methylation reactionsis discussed in relation to the apparent importance of methyl group transfers in the biochemical acclimation toenvironmental stress.

Abbreviations: ABA = abscisic acid; BA = N6-benzyladenine; [Ca2+]c = concentration of cytosolic free calcium;CAM = Crassulacean acid metabolism; CK = cytokinin; DAG = diacylglycerol; DHZ = dihydrozeatin; HR= hypersensitive response; IAA = indoleacetic acid; iP = N6-(�2-isopentenyl)adenine; IP3 = inositol 1,4,5-trisphosphate; JA = jasmonic acid; KIN = kinetin; MAP = mitogen-activated protein; MAPK = MAP kinase;MAPKK = MAPK kinase; MAPKKK = MAPKK kinase; (OG)Z = O-glucoside of Z; (OG)[9R]Z = O-glucosideof [9R]Z; PEPCase = phosphoenolpyruvate carboxylase; PIP2 = phosphatidylinositol 4,5-bisphosphate; PR =pathogenesis-related; [9R]Z = Z ribonucleoside; SA = salicylic acid; SAH = S-adenosyl-L-homocysteine, SAM =S-adenosyl-L-methionine; SAR = systemic acquired resistance; TMV = tobacco mosaic virus; TRSV = tobaccoringspot virus; Z = trans-zeatin

1. Introduction

The involvement of CKs in the regulation of manyaspects of growth and differentiation, including cell

division, apical dominance, nutrient mobilisation,chloroplast development, senescence and flowering iswell documented [67, 193]. Plants exhibit consider-able plasticity in their development and the course of

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their developmental programmes is largely determinedby environmental factors. In the light of the importantregulatory role played by CKs in modulating develop-ment, it seems feasible to also anticipate their involve-ment in responses to adverse environmental conditions.At present, the mechanisms of perception of environ-mental stress by plants and the conversion of physicalphenomena such as water deprivation or temperatureextremes into physiological responses largely remainenigmatic. However, plant hormones are likely candi-dates for playing a role in the transformation of stress-related signals into changes in gene expression neededto effect appropriate adaptation to suboptimal environ-mental conditions.

For many years, ABA and to a lesser extent, eth-ylene, have captured the attention of most hormonephysiologists interested in plant responses to bioticand abiotic stresses. Much has been elucidated con-cerning the involvement of these two growth regu-lators in stress responses [34, 197] and recent stud-ies have begun to unravel some of the mechanismswhereby these two phytohormones act [59, 149]. Ana-lyses of the promoters of drought- and cold-induciblegenes have revealed a novel cis-acting element thatis involved in the ABA-dependent response to dehy-dration, low temperature and high salt concentra-tion [208]. Although a role for ABA in mediat-ing many physiological responses to environmentalstress is now well-established, evidence has been pre-sented for the existence of both ABA-dependent andABA-independent dehydration-related [62, 84] andcold-induced [85] signal transduction pathways. Ofat least five signal transduction pathways that regu-late drought- and cold-inducible genes, only two aredependent on ABA action [169]. Furthermore, addi-tional factors are necessary to account for the involve-ment of ABA in responses to wounding [138]. Thedominant role played by ABA in mediating responsesto water deprivation has also been called into ques-tion by investigations at the physiological level whichfailed to correlate inhibition of shoot growth or leafturgor pressure with ABA levels [187, 199]. ABAlevels frequently peak shortly after the imposition ofstress, but thereafter decline to unstressed levels ifthe stress is sustained. Accumulation of ABA understressful conditions has been found to lag behindstomatal closure [13, 187] and upon relief fromstress, a decline in leaf ABA levels preceded stomatalopening [13, 111].

Cytokinins are generally considered to be antago-nists of ABA, with the two hormones having opposing

effects in several developmental processes includingstomatal opening [21], cotyledon expansion and seedgermination [183]. In contrast to ABA, we still havea poor understanding of the precise role of CKs inplant stress responses. In many respects this is surpris-ing, considering that there was much early speculationconcerning a role of this class of growth regulator inmediating plant responses to drought and high salinity[81, 190]. In this review, we aim to evaluate our currentunderstanding of the involvement of CKs in respons-es to adverse environmental stimuli such as waterdeprivation, temperature extremes, salinity, flooding-induced anaerobiosis, nutrient deficiency, woundingand pathogen infection. Although their contribution tostress responses cannot be ignored, detailed examina-tion of the effects of growth regulators such as auxin[51, 107, 110, 144], gibberellins [2, 76], brassinos-teroids [177] and polyamines [56] is beyond the scopeof this article.

2. General stress response signalling in plants

The sessile habit of plants requires that they must showconsiderable capacity to respond to their environment,particularly under adverse conditions. All environ-ments are in a constant state of flux and environ-mental conditions that may impact on plant growth andreproductive capacity throughout the life cycle includedrought, flooding, temperature extremes, nutrient defi-ciencies, heavy metal toxicity, herbivory, excessiveirradiance or infection by pathogens. Often thesestresses do not occur in isolation, but are coupled. Fre-quently, the level of any single stress factor (e.g., mois-ture availability, temperature or threat from pathogens)may not be adequate to constitute stress by itself, butin combination with other equally marginal factor(s),plant vitality may be considerably reduced. Therefore,plants continuously need to adapt to new combinationsof stresses they may encounter.

Increasing evidence suggests that there may be abasic physiological framework involved in the regu-lation of plant responses to environmental stress [35].Not only do environmental stresses frequently occur inconcert, but they are often linked by common aspectsof their effects, the signal systems whereby they aredetected, or the response to them. Ecologists havenoted that reduced growth rate, a low capacity to cap-ture resources and a high investment in reserve storageare consistently found amongst plants indigenous tohostile environments [35]. Furthermore, the multitude

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of adverse stimuli that impact on plant growth caninduce, besides very specific responses, many similareffects at the cellular level. Widespread consequencesof the imposition of different stresses include osmot-ic challenge, oxidative damage, changes in mem-brane chemistry and ion transport, alterations in cel-lular redox potential and protein denaturation [72].Accordingly, gene products commonly induced fol-lowing imposition of a diversity of suboptimal con-ditions include enzymes involved in the synthesis ofputative osmoprotectants [25, 71], enzymes associatedwith detoxification of reactive oxygen intermediates[78], lipid desaturases for membrane modification [25,169], proteases [88, 196] and cyclophilins [115]. Thefrequent association of these effects with the induc-tion of common subsets of genes and the activation ofspecific metabolic pathways has led to the proposalthat different stress responses may be regulated byoverlapping and interacting cellular signal transductionpathways [72]. The well-documented phenomenon ofcross-tolerance to stresses is consistent with this pro-posal. Activation of appropriate responses to one stresshave frequently been shown to ameliorate the impact ofsubsequent imposition of a different stress. For exam-ple, previous exposure to water stress, salinity or ABAat non-hardening temperatures increases subsequentcold-hardiness [151] and irradiance of tobacco plantswith UV light or fumigation with ozone induced resis-tance to a subsequent challenge with TMV [207].

Several recent studies of stress-induced gene induc-tion augment this idea. For example, the pattern ofgene expression observed in ozone-treated Arabidopsisthaliana overlaps significantly with the pattern of geneexpression observed during a hypersensitive responseto pathogen attack [166]. Transcripts of several maizegenes induced by treatment with HgCl2 also accumu-lated in response to heat stress, NaCl, UV-irradiation,polluted rainwater, wounding, cold stress and pathogeninfection [49]. Despite their responsiveness to severalabiotic extremes, these did not always elicit similartranscriptional changes in the genes studied. This con-firms the conclusions of others [196] that several inter-acting signalling pathways are likely to regulate plantresponses to biotic and abiotic stresses. While over-lap between genetic responses to distinct stress condi-tions most likely arises from their common effects oncellular physiology, different stresses must also elicitunique genetic responses in order to enable the plantto distinguish between stress treatments.

The likely complexity of such a system of partial-ly overlapping stress response patterns has led to the

suggestion that the integration of plant stress responsesis dependent on an overall shift in the relative activ-ities of several growth regulators. Of these, ABAand CKs have been proposed to be the most likelycandidates [35], although other growth regulators arealso likely to play a crucial role in controlling plantresponses to adverse environmental conditions. Whilethe mechanisms of hormonal balance in plants remainpoorly understood, CKs and other growth regulatorsare known to mutually affect their absolute concentra-tions at the levels of their synthesis and metabolism aswell as interacting at the level of differential inductionor repression of gene expression [67].

3. Changes in endogenous CK levels with stressand the effects of exogenous CKs on stressresponses

Traditionally, the most common experimental designsused by CK physiologists have been observation of theeffects of exogenous application of CKs and attemptsto correlate these effects with changes in endogenouslevels of the phytohormone. The former approachhas the disadvantage that uncertainties of interpre-tation arise from unknown quantities of amount ofCK absorbed, its distribution and the extent to whichendogenous levels are altered. Furthermore, it is notknown whether effects elicited by the exogenouslyapplied hormone reflect the normal physiological rolesof endogenous CKs [193]. The latter approach pro-vides only inferential evidence of the involvement ofCKs in any response. Nonetheless, both strategies haveyielded evidence consistent with a role for CKs in adap-tation to environmental changes.

3.1 Water deprivation and salinity stress

A limitation of water availability affects almost allplant processes. In field-grown plants, the two condi-tions contributing most frequently to water deficit aredrought and soil salinity. Although a direct and insep-arable relationship exists between salinity stress andwater deprivation, it is important to note that salinitystress also has an ionic component, which may intro-duce stress effects not found in plants which have sim-ply been deprived of water. Early studies indicated thatroot exudate from water stressed sunflower plants con-tained significantly less KIN-like activity than exudatefrom control plants [81], thus suggesting that modifica-tion of shoot physiology during stress may result from

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decreased supply of CKs from the root system. Theeffects of osmotic stress on CK transport is covered indetail in Section 5. Here we consider only investiga-tions of changes in bulk CK concentrations in wholeplants or different plant parts.

In leaves of salt-stressed tomato plants, a transientrise in [9R]Z levels for the first 2 d following the impo-sition of stress was followed by a depression in concen-tration also observed for Z [199]. The reduction in CKactivity correlated with reduced growth, and returnedto a normal level within 4 d after relief from stress[199]. This study indicated that although water con-tents and osmotic potentials declined throughout the8 d stress period, a large peak in ABA levels after 2 dwas followed by its rapid decline to pre-stress levelsby the end of the stress period. Levels of CKs in theshoots of droughted sunflower plants were about halfthose in unstressed shoots and qualitative differencesoccurred in the roots [76]. A reduction in growth rateand reduced shoot-to-root ratio following salinisationof a salt-resistant variety of barley was accompanied byrapid reductions in Z and [9R]Z contents in both rootsand shoots [98]. In contrast, salt-sensitive varietiesmaintained their growth rates and root:shoot ratios forat least 10 d after exposure to NaCl and displayedno change in levels of endogenous CKs [98]. It wassuggested that a decrease in CK content was an earlyresponse to salt stress, but that the effects of NaCl onsalt-sensitive varieties is not mediated by CKs since areduction in growth rate preceded any decline in CKlevels. However, endogenous levels of Z-type CKsremained unaltered in both roots and leaves duringsalt-stress in the facultative halophyte Mesembryan-themum crystallinum [181]. A single study indicatedno significant change in the [9R]Z content of Lavan-dula stoechus leaves throughout a water stress treat-ment, although [9R]Z levels in leaves of Rosmarinusofficinalis increased during the first 3 d of treatmentand then slowly decreased during the remaining exper-imental period [110].

Similar contradictory evidence has arisen frominvestigations involving the exogenous applicationof CKs. Exogenous application of KIN overcamethe effects of salinity stress on the growth of wheatseedlings [133] and treatment of potato plants withKIN prior to salt stress diminished salt-related growthinhibition [1]. However, earlier studies reported thatapplication of KIN to bean plants during salinity stressexacerbated its effects [95]. Addition of BA inhibitedgrowth during stress of a salt-sensitive variety of bar-ley, but overcame the decline in growth rate, shoot:root

ratio and internal CK content in a salt-tolerant vari-ety [98]. Under drought conditions, CK-treated barleyand wheat seedlings performed poorly compared withthose treated with ABA, and under severe drought,only ABA-treated plants survived and produced grains[131]. However, the phenylurea-type CK thidiazuronsignificantly increased the yield of salinity-stressedwheat [15], an effect that may arise in part from inac-tivation of CK oxidation [68].

Exogenous CKs have been shown to increase thetranspiration rate of leaves [16, 19]. ABA-mediatedstomatal closure and the responsiveness of guard cellsto dehydration stress have been studied extensively[6, 59]. In both stressed an unstressed leaves, addi-tion of KIN reduced ABA content [16]. Whereas KINprevented a decrease in transpiration in excised wheatleaves placed in water and stimulated transpiration ratein excised water stressed leaves, intact seedlings whichwere stressed before leaf excision displayed only aslight stimulative effect of KIN on transpiration rate[16]. Nonetheless, treatment of turgid lettuce leaveswith KIN had no effect on stomatal aperture, althoughit considerably retarded stomatal closure in desiccatingleaves, thereby accelerating wilting [2].

3.2 Temperature extremes

The antagonistic interaction between ABA and CKs inprocesses such as seed and bud dormancy, senescenceand transpiration have led several workers to inves-tigate whether their relative levels may be importantin abrogation of stress effects. A brief heat treatmentto roots of bean seedlings caused a 6-fold decreasein CK content and a 4-fold increase in ABA levelsin xylem exudates [82]. Treatment of tobacco leaveswith KIN after heat shock enhanced recovery, althoughapplication prior to the stress increased heat damage[83]. Pretreatment with ABA neither prevented norreduced heat damage while post-treatment enhancedrecovery, albeit to a lesser extent than KIN. It was con-cluded that KIN enabled faster recovery upon relieffrom heat stress [83]. Consistent with this role for CKsin recovery, the inhibition of photosynthetic activityand chlorophyll accumulation in photosynthetic tis-sues when the roots of maize seedlings were heat-ed correlated with reduced CK levels and could bereversed by application of BA [30]. At the concen-trations used, BA was ineffective in stimulating bothof these parameters in unstressed green leaf segments.A similar ameliorative effect of BA was observed forheat-induced inhibition of chloroplast development in

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etiolated leaf segments [30]. Disruption of maize ker-nel development by heat stress during the endospermdivision stage is associated with an imbalance betweenCK and ABA levels. Stem infusion of BA to the earsof heat-stressed maize enhanced thermotolerance ofdeveloping kernels, as indicated by increased averagekernel dry weight and greatly reduced kernel abortion[36]. The data obtained from the same study indicat-ed that CKs play a more pivotal role than ABA inthe establishment of maize kernel sink potential andthermostability of kernel development.

Temperature extremes are frequently associatedwith water stress. Not only do elevated tempera-tures and a reduction in water supply frequently occurtogether, but chilling and freezing injury may also beassociated with a reduction in water availability.Levelsof endogenous ABA rise in several species upon expo-sure to low temperatures and ABA application is alsocapable of inducing cold hardiness in certain species[34, 151]. Similarly, CK activity increased in sap dur-ing the cold storage of excised grape cuttings [170]and in both the sap and buds of poplar during chillingand bud burst [73]. Application of BA improved chill-ing resistance in peas and several other plant species,albeit in an apparently season-dependent manner [100].However, BA application to pot-grown winter wheatplants had no effect on cold-hardiness [64]. The sameworkers were only able to demonstrate a marginal hard-ening effect of ABA on winter wheat grown in nutrientsolution when BA was also included. In contrast, hard-ening in cultured alfalfa cells could only be induced bya combination of ABA and low temperature if KINwas removed from the media [136]. A study involv-ing monitoring changes in endogenous CK and ABAlevels of winter wheat during hardening and dehard-ening suggested that CKs did not play a role in thehardening process during winter, but were apparentlyimportant in the resumption of active growth in thespring [180].

3.3 Pathogen infection and wounding

Changes in CK activity in plants affected by sever-al diseases have been studied. In leaves of Phase-olus vulgaris and Vicia faba infected by Uromycesphaseoli (rust), “green islands” surrounding sitesof fungal infection were attributed to enhanced CKaction [94]. However, extracts from the leaves andstems of Verticillium albo-atrum-infectedcotton plantshad lower levels of CKs than healthy plants [127].Although a greater loss in CK activity was observed in

Verticillium-infected plants than in plants subjected towater stress, it could not be discounted that wilt symp-toms associated with fungal infection were responsi-ble for the decline; the reduction in CK activity in theinfected plants occurred only after the onset of wiltsymptoms [127].

Investigation of the effects of viral infection on CKactivity have provided conflicting data. Infection withTRSV reduced CK activity in root exudate and roottissue extracts from cowpea [101] and leaf extractsfrom Nicotiana glutinosa [179], while TMV infectionincreased CK activity in tobacco plants [178]. A sub-sequent study indicated that although the overall effectof TMV infection in tobacco leaves was to increase CKconcentrations above those found in uninfected plants,infection actually reduced the concentration of puta-tive active forms but increased the concentration ofCK-O-glucosides [203]. A reduction in concentrationof free CKs in “green islands” led to the conclusion thatincreased free CK concentration is neither involved intheir formation nor their resistance to virus. Treatmentwith ABA, known to reduce susceptibility of tobaccoto infection by TMV, decreased Z and [9R]Z concen-trations in the leaves of control plants and stimulatedformation of the corresponding O-glucosides [203].The enhancement of CK turnover by exogenous ABAwas more pronounced in infected leaves, suggestingthat ABA reinforced changes induced by TMV infec-tion.

Cytokinins have been implicated as playing rolesin both alleviating and exacerbating the HR of plantsto pathogen infection. When a plant is infected with apathogen to which it is resistant, induction of a widerange of biochemical and physiological responses pro-vide protection by restricting or even eliminating thepathogen and thereby limiting the damage it causes.This HR is characterised by tissue necrosis and isfrequently accompanied by the subsequent inductionthroughout the plant of SAR, which may provide pro-tection against a broad spectrum of pathogens for up toseveral months. It has been suggested that an increasein endogenous CKs may trigger the induction of SARto plant viruses [178, 203]. Pretreatment of older, butnot young leaves of tobacco with CKs suppressed theHR induced by Pseudomonas pisi [135], while theauxin/CK ratio was found to be important in resis-tance of tobacco callus to Phytophthora parasiticavar. nicotianae [65]. Inhibition of the HR at elevat-ed levels of BA or KIN was reversed by increases inlevels of IAA [65]. Exogenously applied CKs sup-pressed the induction of hypersensitive necrosis by

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viruses [8, 9]. Whereas TMV content in tomato leafdiscs was reduced at higher concentrations of BA orKIN and increased at lower concentrations [8], it wassuggested that in tobacco leaves systemically resis-tant to infection by TMV, the decrease in numbersof visible local lesions in resistant upper leaves re-sulted from suppression of necrosis, since virus pro-duction was not reduced [10]. Similarly, when cucum-ber leaves were sprayed with BA, the size of lesionscaused by Colletotrichum lagenarium decreased sub-stantially, although no decrease in the numbers oflesions occurred [126]. In leaves in which resistancehad been induced systemically by early inoculationwith the fungus, similar decrease in lesion size wasnoted, together with a 60–80% decrease in lesionnumber [126]. In contrast, application of KIN to bar-ley enhanced the HR induced by powdery mildewErysiphe graminis [109]. In this study, Z applicationhad no effect on HR [109]. Spraying intact tobac-co leaves daily with KIN (0.1 mg l�1) increased theinfectivity of TRSV, whereas treatment at levels 10-and 100-fold in excess of this decreased symptoms[179]. Treatment of cowpea primary leaves with KINbefore and after inoculation with TRSV reduced virusproduction in roots relative to untreated plants [101].Thus, although the literature provides conflicting evi-dence, involvement of CKs in the manifestation of theplant HR is evident. Increased CK content followingtreatment of tobacco and wheat leaves with exogenoushuman interferon 2 and 20–50 oligoadenylates was cor-related with suppression of the proliferation of plantviruses and the induction of PR and heat shock pro-teins [99].

Besides infection by viral, microbial and fungalpathogens, field-grown plants are frequently subjectto wounding stress resulting from mechanical injury,herbivory by insects and grazing by animals. Wound-ed potato tubers produced increased amounts of CKschromatographically similar or identical to Z and[9R]Z [128]. It was concluded that CKs were impor-tant for enhanced cell division accompanying wound-induced periderm formation in potato tubers.Enhancedcell division, chlorophyll formation and cotyledonexpansion displayed by wounded cucumber cotyledonsis consistent with the proposal that wounding enhancesendogenous CK activity [41]. The observation thatZ promoted growth of wounded cotyledons less thanthat of unwounded cotyledons suggests that wound-ing compensates partly for the requirement of exoge-nous CK [150]. Similarly, KIN was almost ineffectivein further delaying the reduced senescence in excised

oat leaves subjected to various wounding techniques[60].

3.4 Mineral nutrient deprivation

Changes in mineral nutrient availability strongly affectCK levels in vivo. Cytokinin levels (or activities whendetermined using bioassays) in roots, shoots and xylemsap decrease under conditions of low nitrogen avail-ability [43, 75, 97, 152, 158, 185]. Several studieshave indicated that phosphorus deficiency influencesCK levels in a similar way to nitrogen deficiency [75,124, 185], although when compared in the same study,phosphorus deficiency elicited slower and less pro-nounced changes in CK concentrations [75, 185]. Thegrowth stimulating effects of exogenous applicationsof CKs to plants grown in nitrogen-deficient mediumindicates that CKs mediate the response to changes innutrient supply [75, 97]. A stimulatory effect of BA onplant mineral nutrition was associated with an effecton the levels of endogenous CKs [97]. Symptoms ofCa2+ deficiency were mitigated and delayed by BAtreatment of cucumber plants before and during Ca2+

deprivation [52]. These workers noted parallel changesin Ca2+ and inorganic phosphate levels in the rootsand shoots of plants starved of Ca2+, possibly due totheir closely related transport. Cytokinin reversed thechanges in phosphate distribution resulting from Ca2+

deficiency [52].

4. Stress-induced changes in CK metabolism

In its simplest form, CK metabolism is a dynamicbalance between biosynthesis, formation of CK con-jugates which may retain some degree of CK activity,and the catabolic reactions resulting in a loss of biolog-ical activity. Cytokinin conjugates generally possesslow intrinsic physiological activity, and what activitythey do demonstrate invariably correlates with theirrate of hydrolysis on plant tissues [105]. They arethus considered to represent storage, inactivation ortranslocatory forms of the active hormone, which isgenerally regarded as the free base, although this hasnot yet been proven unequivocally [192]. In manyrespects, the existence of at least 20 naturally occur-ring CKs in plants [105] may have retarded effortsto evaluate changes in CK levels under stress condi-tions since the complexity of metabolic transforma-tions to which CKs can be subjected in the plant cellintroduces the question of which should be measured

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during a stress/recovery cycle. Virtually nothing con-clusive is known about the pathway(s) of CK biosyn-thesis in plants or of the enzymes that control it [142].Furthermore, changes in endogenous shoot CK con-tent following stress are subject not only to alterationsin localised biosynthesis and metabolism, but also bytransient accumulation from the vascular stream.

Among the processes identified as possibly play-ing a role in CK inactivation are the forma-tion of CK nucleosides and nucleotides from freebases, N-glucosylation, N-alanyl conjugation, O-glucosylation, O-acetylation, side chain reduction andside-chain oxidation [105]. Of these processes, degra-dation by CK oxidase has received the most atten-tion [69]. Few investigations have been devoted toestablishing whether perturbations in environmentalconditions alter the activities of other enzymes cur-rently known to be involved in CK metabolism [192].Although most of the evidence concerning the effectsof stress on CK metabolism is inferential, preliminaryfindings indicated as much as a 2-fold increase in theactivity of CK oxidase from heat-stressed maize ker-nels compared with that in non-stressed kernels [36]. Inthis regard, it is interesting to note that several studiesinvolving heat-shock-regulated expression of the CKbiosynthetic ipt transgene revealed a decrease in thecontent of CK metabolites in control tissues upon heattreatment [121, 161, 191]. Studies conducted usingtobacco leaves [121] and maize kernels [36] suggesteda difference in the thermal sensitivity of Z and [9R]Z,with Z levels apparently being more sensitive to heatstress than those of [9R]Z. However, inconsistencies inmetabolite levels following heat stress prevent gener-alisations concerning effects on CK metabolism. Manyof these are likely to be attributable to differences inthe rate of temperature increase and maximal temper-ature chosen for heat stress, the choice of tissue-typeanalysed or interference by other environmental para-meters. Levels of [9R]Z in potato were significantlylower in plants grown at higher temperatures with an18 h photoperiod than those grown at lower tempera-tures with a 10 h photoperiod [118] and [9R]Z levelsdeclined in both axillary shoots and tissue from thetenth internode of soil-grown tobacco plants subjectedto a daily 1 h heat shock for 4 weeks [191]. Levelsof Z were not measured in this study, although [9R]iPappeared to be insensitive to the heat treatment [191].Comparison of CK levels in rectangular areas of wild-type tobacco leaf 4 h after a 2 h heat shock at 42�C, withlevels in the rest of the leaf indicated that heat stressmarkedly increased levels of the ribotides of Z, DHZ

and iP. Levels of iP and the ribosides of Z, DHZ and iPwere unaffected, and heat treatment caused a greaterelevation in DHZ than Z [171]. The levels of these CKmetabolites reported in this study [171] appear to benegatively correlated to their susceptibility to degrada-tion by CK oxidase [69].

The reversible conjugation of CKs by their conju-gation is a likely mechanism whereby plants achievehormonal homeostasis and “fine-tune” CK actionunder adverse conditions. Infection of tobacco byTMV stimulated CK O-glucosylation even in dark-green areas of leaves known to contain little virus[203]. Whereas both dark-green and light-green areasof infected leaves contained low levels of [9R]Z sim-ilar to the concentrations found in uninfected leaves,Z concentration was consistently lower in dark-greenareas of systemically-infected leaves while the concen-tration in light-green areas was unchanged. Concentra-tions of (OG)Z and (OG)[9R]Z in dark areas of infectedleaves were markedly higher than in uninfected leaves,while their concentrations in light-green areas wereeither similar to or slightly elevated in comparison withhealthy control leaves [203]. An enhancement of glu-coside content by mineral shortage in Plantago majorhas also been reported [97]. This was accompanied byreduced levels of free bases, ribosides and nucleotides[97]. Nonetheless, in Betula pendula greater CK activ-ity was found in ribotide fractions of root extracts fromplants starved of nitrogen than from control plants[43], although no significant changes in CK levelsof the base, nucleoside or nucleotide fractions couldbe detected in leaf material of Acer pseudoplatanuseven after extensive nitrogen or phosphorus deficiencytreatments [43, 75].

5. Stress-induced changes in CK transport

Since higher plants, unlike animals, do not possessa nervous system, long-distance communication islargely dependent upon a complex hormonal systemto integrate physiology at the whole organism level.Although some CK biosynthesis has also been demon-strated in shoots [37], roots are the primary sites of CKbiosynthesis and CKs are commonly considered to bethe main developmental signals of the root [32, 193]. Ithas long been acknowledged that the spatial separationof CK biosynthesis and action is biologically importantas an internal control mechanism for plants to balanceroot and shoot growth. However, inter-organ commu-nication is also likely to be of particular significance

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during responses to adverse environmental conditionssince the preponderance of plant stresses occur becauseof environmental or biological factors impinging on theplant through the root system. Whereas biomass is pre-dominantly directed to shoot tissue under conditions ofadequate supply of soil-borne resources such as wateror nutrients, depletion of these resources is known toincrease allocation to the roots. Although conclusiveevidence is lacking, CKs have long been proposed tobe passively transported from the roots to the shootsthrough the xylem via the transpiration stream and thatthis constitutes an important mechanism of integrat-ing shoot and root physiology. Several studies involv-ing stress-induced CK effects have thus consideredwhether a stress-induced decline in CK biosynthesisin root tips and the resulting decrease in CK transportmight affect the physiology of the shoots.

A role for root-to-shoot signalling in elicitingresponses to osmotic stress is supported by severalstudies which have demonstrated that although leafwater potentials of droughted and well-irrigated plantscan be very similar, stomata are always more closed instressed plants [45]. This suggests that stomata respondto soil water status rather than to leaf water potentialand that shoot physiology is regulated independentlyof local osmotic influences by chemical informationoriginating in the root. A recent study involving split-root experiments to investigate the expression of fourABA-responsive genes demonstrated that changes ingene expression in the shoot might also be inducedby signals originating in the root [63]. Although themechanism whereby such signalling operates remainsunknown, ABA [12, 45, 116] and CKs [12, 122,201] have both been implicated as possible signalsinvolved in the process. Despite the well-documentedinvolvement of ABA in stomatal regulation, contro-versy surrounds a simple cause-and-effect relationship[45, 187]. One explanation is that stress-related alter-ations in CK levels may affect guard cell sensitivityto endogenous ABA and thereby modify the response.Stomatal closure without an increase in the concentra-tion of xylem ABA suggests that other signals,possiblyCKs or the pH or mineral composition of the sap, playa role [163].

A central, yet poorly resolved issue in assessingthe role of CK transport in shoot stress responses iswhether output from the roots depends exclusively onthe transpiration stream, exhibits a regulated dynamicof its own or is a product of both considerations. Inaeroponically-grown sunflower plants, drought stressreduced the root to shoot transport of CKs and it was

concluded that changes in CK synthesis were unlike-ly to be of significance [76]. The concentrations ofZ, iP and their ribosides in the xylem sap of ricedecreased significantly following water deprivationand increased again after rewatering [12]. Impairmentof root metabolism by flooding decreased CK activ-ity in the root exudate from sunflower [29]. Expo-sure of the root systems of Phaseolus vulgaris plantsto 47 �C for 2 min reduced root and shoot growthand was associated with a reduction in CK levels andincrease in ABA in xylem exudate [82]. Infection oftomato plants with Verticillium dahliae caused a reduc-tion in CK levels in xylem exudate after the onset ofsymptoms and was associated with chlorosis [137].It was proposed that a decrease in flower number intomato plants deprived of phosphorus may result froma reduced supply of CKs from roots to the shoots [124].Recent studies suggested a dependence of CK exportto the shoot of Urtica dioica on plant nitrogen status,with a significantly higher CK content being observedin roots from plants grown on an adequate nitrogensupply than those grown at low nitrogen [14, 198].Roots from plants grown at optimal nitrogen exportedapproximately twice as much Z and [9R]Z as rootsfrom plants grown at low nitrogen [14]. Furthermore,a close correspondence between nitrogen supply to theUrtica plants and their shoot to root ratio correlatedpositively with the estimated CK gain by the shoots,thus implicating an essential role for CKs in regulatingbiomass partitioning in response to nutrient depriva-tion stress [14, 198].

Nevertheless, reports in which the flux of CKs hasbeen quantified and compared to physiological changesinduced by stress are contradictory. Anaerobic stresshad no effect on the transport of KIN in cotton leafpetioles [44]. Although xylem sap contained less CKfollowing root hypoxia, [9R]Z, [9R]DHZ and theirequivalents were not reduced in the leaves of Phase-olus or poplar [134]. The observation that chilling at2 �C increased CK levels in buds and sap of excisedpoplar twigs suggested that roots are not a neces-sary source of supply of CKs for the developing bud[73]. Nonetheless, the extent of elevation in CK lev-els observed in excised twigs was less than that foundin intact plants subjected to chilling. Partial drying ofthe root system failed to alter CK concentrations inxylem sap of sunflower plants [116] and a correlationbetween plant water status and CK concentrations inxylem sap of Prunus dulcis could not be established[54]. It was concluded that in almond trees, CKs affectstomatal behaviour on a short-term basis as a result

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of diurnal fluctuations in their levels in xylem sap,although ABA provides an overriding signal indicat-ing long-term water deficit [54]. This interpretation isconsistent with the findings of others that exogenouslyapplied CKs cannot alleviate stomatal closure by highABA concentrations [21, 134]. However, supplemen-tary CKs prevented stomatal closure and amelioratedthe decline in photosynthetic assimilative capacity inflooded tomato plants [26]. Recent findings [167] mayhelp to reconcile some of these conflicting data.Moder-ate soil drying caused ABA concentration and deliveryrate to increase in sunflower plants. Although [9R]Zconcentration did not change, delivery rates decreasedsignificantly [167]. A decrease in [9R]Z concentra-tion was observed only following more severe droughtstress. Delivery rates rather than absolute hormoneconcentrations were proposed to be more informativesince they are less subject to change as a result of dif-ferences in sap flow. Not only does the volume of thetranspiration stream fluctuate throughout a day/nightcycle, but a decrease in transpiration following waterdeprivation may increase the CK concentration withinthe xylem sap simply as a result of reduced sap flowrate. Determination of CK concentrations alone mayresult in under-estimates of the negative influence ofdroughting on CK export from the roots, while relyingon ABA concentrations alone could over-estimate theinfluence of droughting on ABA transport from roots toshoots. A better reflection of the amounts of hormoneentering the transpiration stream can be obtained bymultiplying concentration with sap flow rate to gener-ate delivery rates. Flux of ABA or CKs may be moreimportant than bulk leaf concentrations of these growthregulators in explaining shoot responses to perturba-tions in the root environment [54]. Recent data suggestthat in oat, and possibly in wheat, Z-type bases, ribo-sides and O-glucosides but not nucleotides, suppliedto the leaf in xylem sap at approximately endogenousconcentrations, are likely to play a role in regulatingtranspiration [7]. Besides an over-reliance on inter-preting bulk concentration data, our currently poorappreciation of the possible modulation of the endeffect of CK transport in the transpiration stream byreflux of CKs from the shoot to the root via the phloem[202] or by CK synthesis in shoot meristems [37] mayalso account for conflicting evidence concerning theinvolvement of root-derived CKs in mediating stressresponses.

The observation that droughted mycorrhizal plantsfrequently maintain higher stomatal conductance thansimilarly-sized and -nourished non-mycorrhizal plants

has prompted several investigations into whethervesicular-arbuscular mycorrhizal colonisation of rootsmay alter non-hydraulic root-to-shoot communica-tion of soil drying. Mycorrhizal infection of flaxwas initially associated with a temporary decline in[9R]Z levels in roots, although there was an increased[9R]Z content in the shoots when compared to non-mycorrhizal plants [50].Once the symbiosis was estab-lished, colonised roots displayed significantly higher[9R]Z levels than those of non-mycorrhizal plants andimproved growth responses in both roots and shootsresulting from mycorrhizal infection were precededby higher CK levels in these tissues [50]. Althoughintroduction of Z into the vascular system on non-mycorrhizal flax did not affect stomatal opening, it wascapable of reversing decreased transpiration and CO2

assimilation rates caused by ABA application [50].Mycorrhizal alfalfa plants maintained higher leaf CKsduring drought and these were associated with stimu-lated shoot growth and an enhanced capacity to retardleaf senescence [61]. It was not established whetherthe smaller decrease in drought-stressed mycorrhizalplants was associated with improved root CK produc-tion or maintained CK xylem transport during drought.Together these findings indicate that irrespective ofplant nutrient status, elevated internal CK levels result-ing from mycorrhizal symbiosis may be a primary fac-tor causing improved plant growth, particularly underunfavourable conditions.

6. Molecular genetic analysis of CK-mediatedplant stress responses

For many years, the best means of assessing a role forCKs in plant stress responses was by exogenous appli-cation of CK. As detailed above, the results of suchstudies have been used to infer that changes in endoge-nous CK activity might have the same effect. Whilethis approach has shed much light on CK-mediatedresponses to plant stress, interpretation of these find-ings is hampered by our poor understanding of themechanisms of CK uptake, transport and metabolism.Furthermore, the paucity of data concerning subcel-lular compartmentation of CKs has introduced con-siderable speculation, especially when physiologicaleffects of applied CK have not correlated quantita-tively with data describing variations in endogenousCK levels. Although not without their own limitations,the advent of molecular genetic approaches to studiesof plant growth regulation over the past decade has

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presented a means to overcome many of these prob-lems. In particular, genetic engineering has enabledthe in vivo manipulation of CK levels. Many workershave exploited the use of the ipt gene from Agrobac-terium tumefaciens, which encodes an enzyme thatcatalyses the rate-limiting step in CK biosynthesis [67,142]. Several studies involving the constitutive or reg-ulated expression of ipt have demonstrated that theresulting plant phenotypes are similar to those associ-ated with exogenous application of CKs. These effectsinclude reduction in apical dominance, suppression ofroot growth, retarded senescence and disturbances inflower formation [3, 57, 67, 121, 161, 171, 182].

Consistent with the demonstration that exogenousBA can counteract the effects of heat stress [30], leavesexpressing ipt under the control of a soybean heatshock promoter were more tolerant of periodic heatshocks than untransformed leaf tissue [171]. In keep-ing with a role for CKs in mediating stomatal open-ing [19, 21, 22], transgenic tobacco plants whichoverproduced CK were characterised by wilting asobserved during salinity stress [182]. More attentionon the influence of ipt expression on environmentaltolerance has focused on the effects associated withenhanced pathogen resistance. Wound-inducible iptexpression in tobacco adversely affected insect feed-ing and development [172]. Another study demon-strated that ipt-mediated CK overproduction in tomatoincreased resistance to the fungal pathogen Fusariumoxysporum [175]. These findings are consistent withthe protective effect of exogenously-applied CK duringpathogen infection (Section 3.3) and the demonstrationthat ipt-expression in transformed, non-rooting tobac-co lines up-regulated the expression of defence-relatedmRNAs [123, 172]. It has been proposed that productsof secondary metabolic pathways affected by elevatedCK levels are also likely candidates as effectors of theobserved enhanced resistance of ipt plants to pathogeninfection [172].

The possible involvement of CKs in regulatingtrans-methylation reactions is discussed elsewhere [67,Section 9]. Transgenic tobacco expressing the anti-sense mRNA of SAH hydrolase contain 3-fold higherlevels of endogenous CKs than wild-type plants anddisplay resistance to infection by several plant viruses[117]. However, it was inconclusive whether or notthe observed resistance resulted exclusively from ele-vated CK levels. Since most plant viruses require amethylated cap structure at the 50 terminus of theirmRNA for viral replication, viral suppression throughSAH hydrolase inhibition may have resulted, at least

in part, from reduced methylation of the viral mRNAcap structure [117].

7. The paradox of CK-mediated induction ofstress-related genes

Cytokinins have been shown to affect the expressionof specific genes by both increasing and decreasingthe abundance of particular proteins or mRNAs [67].Most CK-regulated genes characterised thus far areknown to be controlled by other factors. Many areinvolved in response to light, stress or other environ-mental perturbations and are frequently regulated byother plant hormones [67]. In view of what has alreadybeen discussed regarding the possibility of generalstress response pathway(s), these findings are consis-tent with an important role for shifts in CK action inmediating plant responses to several stresses, regard-less of their precise nature. However, the large overlapbetween sets of CK- and stress-related genes is para-doxical in the light of the generally accepted decreasein CK activity in response to most stresses (Section 3).

Presently, among the major plant hormones, ourunderstanding of the mechanism of CK action is proba-bly the least understood. Efforts to identify the mode ofCK action have been frustrated largely by the fact thatclassical CK effects such as morphogenesis and senes-cence delay are typically slow and complex responses[67]. Since alterations in gene expression in response tostress are both rapid and repeatable,one might envisagethat study of stress-induced effects on CK activity mayprovide useful paradigms for elucidating the molecularmechanisms involved in CK signal transduction. Forexample, the demonstration that Arabidopsis plantstreated with exogenous CKs accumulate anthocyanins[46] suggests that this may be a useful marker for inves-tigating CK-mediated stress responses in this popularmodel plant system. The accumulation of flavonoidsis stimulated by several environmental stresses includ-ing exposure to UV irradiation, high intensity light,pathogen attack, nutrient deficiency and drought [46].Another metabolic product which frequently accumu-lates in response to a variety of adverse conditionsis free proline [71]. Induction of Arabidopsis pro-line biosynthetic genes by cold and osmotic stresses isindependent of the endogenous ABA level, althoughtheir expression can also be triggered by exogenouslyapplied ABA [159]. Cytokinins were reported to medi-ate proline accumulation and accumulation of an iso-form of PEPCase involved in the transition from C3 to

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CAM photosynthesis independently of osmotic stressin the facultative halophyte Mesembryanthemum crys-tallinum [181]. In contrast, exogenous ABA was a poorsubstitute for NaCl in inducing these responses, neitherof which were prevented by an inhibition of stress-induced ABA accumulation [181]. Interestingly, thefindings related to CK-induced proline accumulationcontrast with earlier reports [174, 200] where it wasshown that exogenous application of higher CK con-centrations prevented stress-induced proline accumu-lation in barley and sunflower respectively. Similarlythe conclusions concerning a stimulative effect of CKon PEPCase levels in M. crystallinum conflict directlywith those presented by others studying the same sys-tem [160]. Subsequent investigation of whether CKsignalling might overlap with stress-induced responsepathways in a glycophyte involved the study of stressresponses in tobacco transformed with ipt under thecontrol of a light-inducible promoter. Under inducingconditions, a 10-fold increase in levels of Z-type CKswas accompanied by increased levels of proline andisoforms of the PR protein, osmotin [182], which isresponsive to several stress factors [72].

These paradoxical findings concerning the involve-ment of CKs in stress responses are parallelled bymany other apparently contradictory effects of thispoorly understood class of phytohormone. For exam-ple, low concentrations of BA (4.4� 10�5 M) accele-rated senescence of cut carnation flowers, while higherconcentrations (1 –2 � 10�4 M) retarded the process[194]. Such observations of inhibition of CK actionat excessive concentrations are parallelled by find-ings at the level of gene induction. Accumulation ofrRNA transcripts and those encoding the small subunitof RuBisCo is stimulated by low concentrations ofKIN, but higher concentrations inhibited their accu-mulation [58]. Similarly, while high levels of CK(10�5 M) inhibit the development of lateral root pri-mordia and growth even in the presence of an opti-mal level of auxin, reduced levels stimulated lateralroot initiation and growth [20, 112, 204]. Cytokininis widely accepted to be a controlling factor in vas-cular differentiation by synergistic interaction withauxin in stimulating cambial initials to differentiateinto xylem cells. However, in one case, transgene-mediated CK overproduction inhibited xylem forma-tion [121], while in other instances, it promoted theformation of a thicker vascular cylinder with expandedxylem [3, 108]. Both enhanced greening [171] andreduced chlorophyll levels [3, 108, 182] have beenassociated with ipt expression in tobacco plants.

A large body of evidence indicates that the speci-ficity and magnitude of CK-induced effects maydepend largely on the nature of the target cell andthe developmental state of the plant [67]. Accordingly,PEPCase accumulation is not induced by salt in suspen-sion cultures of M. crystallinum [181] and CK inducesthe accumulation of proline in tobacco plants,but not intobacco suspension cells [182]. The latter findings areconsistent with the proposal that stress-induced prolineaccumulation may be dependent on systemic organi-sation of the plant [70]. Stomata of young leaves ofZea mays are unresponsive to exogenous CK, but inageing leaves they open more widely after applica-tion of KIN [22]. Together, these observations extendinto consideration of differential cellular sensitivity toCKs. The sensitivity component of hormone actionhas been proposed to be of particular importance inresponses to environmental conditions, since varia-tions in the response thresholds of individual cells mayconfer the necessary plasticity to adjust to short termvariations in environmental conditions in a manneranalogous to that whereby genetic variation buffersspecies against long-term climate change [27]. It isimportant to note that even if absolute levels of CKsdecline under adverse conditions, this does not nec-essarily imply a suppression of CK action in stressedplants, since environmental extremes may affect cel-lular sensitivity to CK. A simplistic model which maycontribute to resolution of the long-standing debateconcerning the relative importances of hormonal con-centration and tissue sensitivity to growth regulatorshas been presented elsewhere in this issue [67]. Ofrelevance to the present discussion however, is thelikelihood that both aspects of hormone action need tobe considered together and it may be the overall differ-ence between the concentration of active CK and thethreshold sensitivity under adverse conditions ratherthan the absolute value of either [27] that is the mostimportant determinant of differential CK action in thepresence and absence of stress. Furthermore, as dis-cussed elsewhere [71], genetic responses to adverseconditions at the cellular level are but one of manypossible aspects of stress tolerance. In many instances,morphological or specialised physiological avoidancestrategies may eliminate the need for sensitive shifts inhormonal balance in counteracting stresses. For exam-ple, anatomical and ultrastructural differences weresuggested to account for differences in hormonal shiftsfollowing water stress of two Mediterranean shrubsadapted to survive drought by tolerance or avoidance[110].

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8. Integration of CK action with stress-relatedsignal transduction pathways

Unlike animals, plants display considerable plasticityin their development. Since the pattern of developmentis to a large measure determined by environmental fac-tors, vascular plants must coordinate developmentalprocesses to produce physiological and morpholog-ical preparations in anticipation of and in responseto environmental eventualities, as well as adaptingto changing conditions on a more rapid time scale.The latter class of responses must be elicited by acoordinated signal transduction system(s) capable ofeffecting changes appropriate to the severity of thestress. Thus under adverse conditions, changes in geneexpression patterns modified by extrinsic cues need tobe superimposed upon intrinsic plant developmentalprograms. Plant hormones are likely candidates forintegrating these vastly different cues, most proba-bly by subtle modifications of flexible gene expres-sion patterns. The likely integration of developmentalpathways and those activated by stressful cues isexemplified by the observation that tissues subject-ed to stress invariably display symptoms of prematuresenescence. This highly coordinated, genetically pro-grammed sequence of biochemical and physiologicalevents which culminates in cellular destruction anddeath occurs irrespective of exposure to unfavourableenvironmental conditions, yet the same changes canbe recapitulated by cues such as extremes of tem-perature, water and nutrient availability or invasionby pathogens. An antagonistic effect of CKs on plantsenescence is well established [57, 171, 195]. Similar-ly, in addition to mediating stress responses of vege-tative tissues, ABA also participates in developmentalprocesses such as embryogenesis and seed dormancy[183].

8.1 Stimulation of ethylene production by CK

Adaptation to any unfavourable environmental condi-tion must involve a responsive and coordinated sig-nal transduction system capable of eliciting responsesappropriate to the severity of the stress. Mutual effectsbetween plant growth regulators at the levels of theirsynthesis and metabolism are an important mechanismwhereby plants integrate information from differentenvironmental and developmental signals. Many CKeffects have long been known to be mediated via stimu-lated ethylene production [33]. Ethylene is induced bya variety of stress factors including wounding, anaer-

obiosis, viral infection, chilling, mechanical injury,drought and exposure to heavy metal ions [197, 209].Cytokinin-mediated stimulation of ethylene produc-tion appears to be of particular importance duringwater stress [120, 206] and anaerobiosis [91], althoughthe promotive effect of KIN on aeranchyma forma-tion in non-aerated maize seedling roots is apparentlynot mediated via ethylene [92]. Cytokinin and ethyl-ene also act synergistically to alleviate the inhibitoryeffect of heat stress on lettuce seed germination [90].It was recently suggested that many phenotypes typi-cally associated with CK overproduction (e.g., reduc-tion in root formation as well as stem and leaf areaor shortened hypocotyl length) may in fact be ethyl-ene responses elicited indirectly by the action of CK[33]. Accordingly, the Arabidopsis ctr1 mutant, whichpossesses a constitutively activated ethylene responsepathway, is characterised by smaller rosette leaves,a less extensive root system and delayed flowering[93]. Consideration of a stimulative effect of CKson ethylene production warrants special attention withregard to CK-stimulated expression of genes normallyexpressed in response to stress. In particular, PR genesknown to be affected by CK action [67, 123, 172] areinducible by ethylene [146, 147]. A role for ethylene inresponse to biotic stress is consistent with the demon-stration that an ethylene insensitivity in Arabidopsisconferred by mutations at the EIN2 locus prevents thedevelopment of disease symptoms upon infection withvirulent Pseudomonas syringae pv. tomato and Xan-thomonas campestris [17]. Interestingly, mutation ofETR1, which acts earlier in the ethylene signal trans-duction pathway [149] did not increase tolerance to vir-ulent bacterial pathogens. It was suggested that etr1-3may have developed disease symptoms because it hasa less severe phenotype than the ein2 mutants, andmay thus have possessed greater sensitivity to ethyl-ene. Alternatively, the distinct pathways for sensitivityto pathogens and ethylene may share EIN2 as a com-mon participant [149].

8.2 Regulation of levels of salicylic acid andjasmonic acid by CKs

Besides ethylene, SA and JA are also known to beimportant signal molecules involved in the activationof plant defence responses. Results from a series ofstudies [155, 156, 157] suggest that CKs are indis-pensable for the temporal and differential biosynthe-sis of SA and JA, which in turn serve as endogenousinducers for distinct classes of PR proteins. Transgenic

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tobacco plants expressing a rab-related rice gene, rgp1that encodes a small GTP-binding protein in rice, pos-sess levels of endogenous CKs 6-fold higher than theirwild-type counterparts and display increased resistanceto TMV infection [156]. This modification also alteredthe response of the transgenic plants to wounding. Nor-mally, wounding signals are transmitted to JA whichinduces basic PR proteins, whereas pathogenic signalscause, in addition to JA, accumulation of SA, whichstimulates production of acidic PR proteins. Wound-ing of tobacco expressing a sense copy of rgp1 elicitedhigh levels of production of SA and salicylic acid-�-glucoside, and consequently acidic PR proteins [156].Additionally, the transgenic plants exhibited a highlysensitive wound-response, with JA production occur-ring at least 18 h earlier than in the wild-type [157].These effects could be simulated by wounding in thepresence of BA and were blocked by a CK antagonist[157]. Based on this observation that the signallingpathway for wounding had been modified to intersectwith the disease response signalling pathway, it wassuggested that CKs may act as a signal switch whichdirects the input signals to the appropriate transduc-tion channels [155]. On the basis of analysis of thewound responses of ipt transformants and the inabilityof exogenous CKs to suppress TMV multiplication, itwas concluded that CKs are essential, but not suffi-cient, for the induction of the defence response [155].Rather, through the action of G-proteins, they weresuggested to regulate SA and JA levels to ensure thatenvironmental stimuli are transmitted to the relevanttransduction pathways.

8.3 Oxidative burst: a common feature in plantresponses to stress

An oxidative burst is an early feature of plant responseto pathogen infection [205]. Many compounds thatinduce PR gene expression and disease resistance inplants inactivate catalases directly or indirectly. In par-ticular SA specifically inhibits catalase, and treatmentof leaves with compounds that generate H2O2inducedaccumulation of PR1 protein [38]. This led to thesuggestion that H2O2 plays a key defensive role inboth the HR and SAR. In this regard, it is interestingto note that exogenous application of BA decreasesthe level of a catalase mRNA transcript in etiolatedcucumber cotyledons [186]. The repression of a soy-bean Fe-containing superoxide dismutase gene by CK[42] is also consistent with this interpretation of a rolefor alteration of CK action in potentiating a transient

increase in the concentration of reactive oxygen inter-mediates produced during stresses such as drought,pathogen infection, temperature extremes, air pollu-tants, nutrient deficiency and heavy metal toxicity [78,173] in order to facilitate transcriptional activation ofstress-response genes, as in the HR to pathogen infec-tion.

8.4 The involvement of phototransduction pathwaysin plant stress responses

Cytokinin action is frequently associated with theaction of light and in some instances, CK can par-tially replace the light requirement for gene expres-sion [154, 184]. Since light quality and quantity areperhaps the most significant extrinsic cues affectingplant growth and development, it would not be sur-prising if many stress-related genetic responses haveconserved their light dependence throughout evolu-tion. The importance of light as an abiotic factor deter-mining the capacity for acclimation to adverse envi-ronmental conditions has recently been emphasised[77]. Photoinhibition is a feature associated with mostabiotic and biotic stresses [71] and is likely to causean imbalance between energy supply and its dissim-ilation by the plant [77]. It has been proposed thata chloroplastic redox sensor acts synergistically withother signal transduction pathways to elicit appropriateresponses under adverse conditions [77].

The light-dependence of stress-induced prolineaccumulation, which is also CK-inducible [181, 182]has been demonstrated in several higher plants [153].Although the induction of proline accumulation in bar-ley leaves by the synergistic action of ABA and KClwas recently suggested to be independent of photosyn-thesis [140], the stimulative effect of low levels of irra-diance with red light may implicate the involvementof phytochrome in mediating this well-characterisedstress response [140]. Since phytochrome can par-ticipate in the signal transduction pathway leading toCAM, pinitol accumulation and modified pigment con-tent in M. crystallinum, it was recently suggested thatphytochrome mediated effects and salt stress effectsmay act on the same signal transduction pathways inthis species [39]. Furthermore, BA does not cause anincrease in anthocyanin accumulation in Arabidopsisin the absence of light [46]. Although transcription ofwpk4, encoding a wheat protein kinase homologue wasinducible by nutrient deprivation only in the light, BAincreased transcript levels irrespective of light condi-tions and a CK antagonist prevented induction by either

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light or nutrient deprivation [154]. It was suggestedthat a common protein phosphorylation cascade mightcoordinate plant responses to light, CKs and nutri-tional cues. This proposal is consistent with evidenceimplicating CK action in both light [184] and nutrientassessment signal transduction pathways (Section 3.4).An ameliorative effect of increased mineral nutrientconcentration on tolerance of high salinity in Sorghumbicolor could be mimicked by addition of KIN [5].

Mutations that affect any one of 16 presentlyidentified de-etiolated (DET), constitutively photo-morphogenic (COP) and fusca (FUS) genes causedark-grown Arabidopsis seedlings to exhibit vary-ing degrees of developmental characteristics of light-grown plants [67, 184]. Although the simplest modelexplaining these phenotypes is that DET/COP/FUSgene products are negative regulators of photomor-phogenesis in the dark, the pleiotropic effects of det,cop and fus mutations in light-grown plants have longsuggested that these proteins identified by these muta-tions are not exclusively involved in light signallingpathways. The screening conditions used in the identi-fication of these mutants were not specifically related tophytochrome action and several factors influence pho-tomorphogenesis. Consistent with this interpretationthat the abnormal phenotypes of det and cop mutantsgrown in the light indicate other functions besidesthe suppression of photomorphogenesis in darkness,recent studies suggest that DET1, COP1 and COP9act in a coordinated manner to repress transcriptionof many classes of genes and apparently participatein a regulatory network that integrates informationfrom photoreceptors, transducers of other environ-mental stimuli and possibly from a developmental pro-gram. Genes encoding PR proteins are constitutivelyexpressed in det1, cop1 and cop9 and the alcohol dehy-drogenase and albumin 2S storage protein genes areinappropriately activated in vegetative tissues of cop1and cop9 respectively [119]. In contrast, det2, cop2,cop3 and cop4 mutants displayed a gene expressionpattern similar to that of the wild-type [119]. Nonethe-less, the constitutive activation of light-, hypoxiaand defence-regulated genes observed in many of themutants suggests that improved understanding of theroles of DET/COP products in growth and develop-ment is thus likely to contribute significantly to ourunderstanding of how adverse environmental stimuliare integrated with light perception duringplant growthand development.

8.5 The involvement of calcium and proteinphosphorylation in plant stress responses

The [Ca2+]c has been implicated as a factor criticalto the regulation of almost all cellular signal trans-duction pathways in plants, including those involvingphytochrome and CK [67, 184]. Increases in [Ca2+]c

are observed in response to the imposition of sev-eral environmental stresses, including anoxia [164],cold-shock [96, 132], chemical treatments that simu-late stress-induced enhancement of oxygen activation[141] and elicitor-stimulated oxidative burst, defence-related gene activation and phytoalexin production[211]. A disruption of cellular Ca2+ homeostasis hasbeen proposed to be the primary response to salt stress[148].

Rapid increases in [Ca2+]cafter osmotic stressmay be mediated by phosphoinositides [4]. In animalcells, IP3 is the primary membrane-derived effector ofthe release of intracellular Ca2+ [18]. Activation ofphosphoinositide-specific phospholipase C by occu-pancy of receptors results in cleavage of the phospho-diester bond of PIP2 in the inner leaflet of the plasmamembrane to release water-soluble IP3 and membrane-bound DAG. The binding of IP3 to receptors on intra-cellular Ca2+ storage sites releases Ca2+ into the cyto-plasm in a quantal fashion, while DAG remains in themembrane where it activates several subspecies of pro-tein kinase C, thereby regulating protein phosphoryla-tion. Thus the concomitant increase in cytosolic Ca2+

and protein kinase activation results in a bifurcated sig-nal that enables the possibility of high-fidelity cross-talk. Many of the components involved in this cascadehave been identified in plant cells, thus suggestingthis to be a likely mechanism whereby gene expres-sion is altered in response to stress. Transcription ofa phosphatidylinositol-specific phospholipase C genefrom Arabidopsis was inducible by dehydration, highsalinity, low temperature and ABA, although not byheat stress [74] and recent evidence is consistent witha role for IP3-mediated Ca2+ release from the vacuolein response to cold shock [96]. It was suggested thatinhibition of root growth in wheat by Al3+ may resultfrom an inhibitory effect on phospholipase C [87]. Thiscould conceivably have profound effects on cell divi-sion and elongation by either blocking IP3-mediatedCa2+transients needed for cellular growth or by inter-fering with growth-mediated cytoskeletal cytoskeletaldynamics that rely on the presence of PIP2 or IP3. Acti-vation of phospholipase C was suggested to be onepathway by which elicitors trigger the oxidative burst

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in soybean cells [104]. Therefore, transduction path-way involving phosphoinositide second messengersand Ca2+ signalling apparently may connect solubleor membrane-bound stress sensors to nuclear eventsvia phosphorylation, although a resultant change ingene expression is not an absolute requirement. Fur-thermore, Ca2+may enter the cell from the apoplastor be displaced from membranes without the necessityfor a phosphoinositide second messenger. The involve-ment of CKs in mediating stress-induced shifts Ca2+

homeostasis has not been investigated.The rapidity of stress responses suggests that

reversible protein phosphorylation cascades are likelycandidate mechanisms for participation in signaltransduction events associated with adaptation tounfavourable conditions. The extent to which changesin [Ca2+]c and alterations in protein phosphoryla-tion are coupled is not yet fully understood. Tran-scripts encoding two Ca2+-dependent, calmodulin-independent protein kinases from Arabidopsis arerapidly induced by dehydration and salinisation stress,but not by ABA, low temperature or heat stress [188].Nonetheless, both induction and repression of theaccumulation of transcripts encoding alfalfa Ca2+-dependent protein kinases by cold stress has beenreported [132]. Further evidence for widespread useof phosphorylation cascades in stress responses comesfrom the identification of rapid ABA-independent acti-vation of a protein kinase in the elongation of the maizeprimary root in response to water deficit [40]. Increas-ing evidence implicates protein kinases in mediat-ing plant responses to wounding and pathogen attack.A serine/threonine protein phosphorylation cascadeconstitutes part of the signal transduction pathwayenabling resistance of tomato plants to P. syringaepv. tabaci strains carrying the avirulence gene avrPto[210]. In view of the proposal that such phosphory-lation cascades involved in ‘gene-for-gene’ resistancemay be widely conserved throughout the plant king-dom [210] and the apparent involvement of CKs inpathogen response (Section 3.3) and phosphorylationcascades [67], it is interesting to note that infection ofa susceptible species of Eucalyptus with Phytophthoracinnamomi resulted in a significant reduction of Z-and iP-type CKs, although no reduction in CK levelsoccurred following infection of a resistant variety [31].

Transient increases in [Ca2+]c have been observedin response to ABA treatment [59], thus making[Ca2+]c a potential candidate for mediating interac-tions between ABA and CK. The Arabidopsis ABI1and ABI2 genes encode homologous serine/threonine

phosphatases which are intermediates in ABA sig-nal transmission [106]. ABI1 functions as part of akinase/phosphatase pathway that modulates the sensi-tivity of guard cell K+ channels to ABA [6]. ABA-induced increases in [Ca2+]c trigger stomatal closureby rapidly regulating ion transporters in guard cells.Similarly, both Ca2+ and phosphorylation events par-ticipate in the induction of PR protein expression byethylene [146, 147]. A synergistic interaction betweenCK and Ca2+ in eliciting CK-mediated ethylene syn-thesis was reported [103]. Determining the extent towhich influences such as light and other growth regu-lators modify CK action and how these stimuli interactwith each other and stress-related signalling pathwaysis likely to remain a crucial goal over the next fewyears.

9. The involvement of MAP kinase pathways inplant responses to environmental stress

In animal cells, MAPKs are involved in signal trans-duction pathways associated with growth factor depen-dent cell proliferation, differentiation and responses tovarious stresses. Activation of MAPK activity requiresphosphorylation on tyrosine and threonine residues(Figure 1) and is mediated by a dual-specific activatorprotein kinase, MAPKK, which is activated by serinephosphorylation by a MAPKKK. This module of threefunctionally associated protein kinases has also beenidentified in yeast and plants. In Arabidopsis, morethan 9 genes encode MAPKs, and phylogenetic analy-sis indicates at least 4 subfamilies of MAPK [130,169].

Considerable evidence which implicates hormonalaction on MAPK activity in plants has led to the pro-posal that MAPK-type cascades may participate in CKsignal transduction [67]. Current evidence stronglysupports an important role for MAPK action in eukary-otic stress responses (Figure 1). The observation thatthe mammalian MAPKs Jnk [55] and p38 [66] arealso rapidly activated in osmotically shocked cells andthat both can rescue the growth defect of the yeasthog1-�1 mutant grown on hyperosmolar media [55,66] argues strongly in favour of a highly conservedosmosensing signal transduction system between thesetwo biological kingdoms. Thus a similar mechanismmay function in higher plants exposed to water deficit.Circumstantial evidence implicates a role for MAPKinvolvement in signalling pathogen infection, sinceMAPK-like proteins were activated following treat-

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Figure 1. Generic MAP kinase module and homologues involved in stress responses in plants, yeast and metazoans.Following activation by the appropriate stimulus, the signal is transmitted to the canonical MAPK module comprising three protein kinaseswhich phosphorylate and thereby activate the next member in the sequence. The progression of events for all MAPK-type cascades is identical,although specific isoforms of each enzyme type and possibly the physical association of components related to the propagation of a single signalconfer a degree of specificity on individual pathways. MAPK kinase kinase (MEKK) homologues are serine(S)/threonine(T)-specific proteinkinases that activate MAPK kinase (MEK) enzymes by dual phosphorylation on two S or T residues with a S-X-X-X-S/T motif. ActivatedMAPK kinase homologues, which are mixed function S/T/Tyr(Y) protein kinases, phosphorylate MAPK(ERK) enzymes on both T and Yresidues within the T-X-Y consensus sequence. Phosphorylation at only one of the two positions does not activate the enzyme, but may primethe kinase domain for receipt of the second phosphorylation event. Specific protein phosphatases (PPs) can theoretically downregulate the signalby dephosphorylating either one or both of the phosphorylated residues. MAPK phosphorylation activates one or more nuclear transcriptionalfactors (TFs).

ment of tobacco cells with a fungal elicitor [176]and also by wounding [165, 189]. Of particularinterest is the demonstration that trans-inactivationof the endogenous tobacco WIPK gene was asso-ciated with inhibition of wound-inducedJA productionand the accumulation of wound-inducible gene tran-scripts [165]. In contrast, levels of SA and transcriptsencoding pathogen-inducible, acidic PR proteins wereincreased upon wounding [165]. This behaviour isreminiscent of that displayed by transgenic tobaccoexpressing the rab-related rgp1 gene from rice [156]and implicates CK involvement in WIPK action if CKs

signal flux through wounding and pathogen-induciblepathways [155, 157, Section 8.2].

10. A role for CKs in regulating methyl grouptransfer during stress?

Although several CK-binding proteins have been iso-lated from a variety of plants, the 130 kDa proteincomplex CBP130 from Nicotiana sylvestris is one ofonly a few for which extensive structural informationis available [129]. CBP130 consists of at least two

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subunits of 57 and 36 kDa (CBP57 and CBP36). Thelarger subunit CBP57 bears high homology to SAHhydrolase from other organisms has been shown to befunctional as an SAH hydrolase [129].

As discussed elsewhere in this isssue [67], SAHhydrolase catalyses the reversible cleavage of SAHinto adenosine and homocysteine. SAH is one of theproducts of methyl-transfer reactions from SAM andis a competitive inhibitor of SAM-dependent methyl-transfer reactions [145]. It has been proposed thatif CK regulates SAH hydrolase activity, then it mayaffect DNA and protein methylation and thereby influ-ence gene expression at either the transcriptional orpost-translational level [67, 129]. With regard to theinvolvement of the proposed mechanism of CK medi-ated regulation of methylation by modulation of theSAM/SAH ratio in stress responses, it is interestingto note that three SAM synthetase transcripts fromtomato are differentially regulated by osmotic stressas well as by ABA [53] and both SAH hydrolase andSAM synthetase transcript levels are influenced byfungal-elicitor treatments in parsley cell cultures [89].Three genes encoding isoforms of SAM synthetase inCatharanthus roseus are differentially expressed dur-ing growth of cell cultures and in response to elicitortreatment, nutritional down-shift and increased NaCl,although increases in transcript levels did not alwayscorrelate with levels of SAM synthetase protein andenzyme activity [162].

Further evidence for a close metabolic link betweenan activated methyl cycle and stress response comesfrom the observation that methyl group transfers areinvolved in several pathways that lead to the accumu-lation of quaternary amines such as glycine betaine,tertiary sulfonium compounds and methylated inosi-tols [25]. All of these compounds have frequently beenassigned functions in osmoprotection and/or osmoticadjustment. It is also interesting to note that removalof the roots of aeroponically grown barley prior tosalinity stress caused a reduction in the accumulationof betaine aldehyde dehydrogenase transcript in theleaves [79], thus providing some evidence for the exis-tence of a root-derived signal such as ABA or CK inmediating the accumulation of this organic osmolyte.In M. crystallinum, but not in Arabidopsis, the path-way from glucose-6-phosphate to inositol-1-phosphateis extended to enable accumulation of methylated inos-itols D-ononitol and D-pinitol, which serve as osmo-protectants in this halophyte [25, 80]. Transcription ofgenes encoding myo-inositol 1-phosphate synthase andmyo-inositol O-methyltransferase is strongly induced

by osmotic stress in M. crystallinum [80] and the SAM-dependent reaction catalysed by the latter gene productis strongly inhibited by SAH [145], thus suggesting thatthe methyl transferase activity depends on the intracel-lular ratios of SAM and SAH.

A role for methylation in adaptation to osmoticstress is also consistent with the demonstration thatoverexpression of the yeast gene HAL2, which encodesa phosphatase that recycles adenosine trapped in 30-phospho-adenosine-50-phosphate (after the transfer ofthe sulphate group), increases salinity tolerance inyeast [114]. Adenosine scavenging is connected tothe pathway producing sulphur-containingamino acidsand to methylation reactions that use SAM. A ricehomologue of HAL2 (named RHL) was shown to func-tion in a futile cycle (substrate cycle) in the sulphurassimilation pathway [139]. Although this plant genewas not induced by NaCl, activation of its product byK+ suggested that the RHL enzyme may respond tosalt stress at the protein level. Although it is not yetknown whether overexpression of RHL will increaseosmotic stress tolerance in plants, its contribution tomethionine biosynthesis, and thus potential activationof the activated methylation cycle [67] is one level atwhich this might occur. The modification of pathwayswhich may accommodate large fluxes in the absenceof stress, such as proline biosynthesis [71], has beensuggested to be a recurrent theme in the evolution ofstress tolerance mechanisms [80] and substrate cyclessuch as the one in which RHL participates, consti-tute extremely sensitive flux control systems [139].An Arabidopsis homologue of HAL2 (named SAL1)displayed both 30(20),50-bisphosphate nucleotidase andinositol 1-phosphatase activities [143]. These workerssuggested that the ability of SAL1 to hydrolyse inosi-tol 1,4-bisphosphate and inositol 1,3,4-trisphosphatemay implicate its involvement in phosphoinositidesignalling, which is consistent with the salinity-induced expression of an Arabidopsis gene encodinga putative phosphatidylinositol-specific phospholipaseC [74].

Since SAM is an intermediate in ethylene biosyn-thesis, it is tempting to speculate that a CK-mediatedalteration in SAM levels may account for the stimu-latory effect of CK on ethylene synthesis. However,SAM is not believed to be rate limiting in the ethyl-ene biosynthetic pathway [209]. SAM also participatesin the biosynthesis of the polyamines spermine andspermidine and there is a well-documented quantita-tive interplay between polyamines and ethylene, whichis widely believed to result in part from competition

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for the common precursor SAM. Cytokinins increasepolyamine biosynthesis and titre in lettuce and cucum-ber cotyledons and in red-irradiated etiolated pea buds[56]. However most stress conditions lead to increasedproduction of putrescine but not other polyamines, andthe decarboxylation of SAM is generally accepted tobe the rate-limiting step in the synthesis of spermineand spermidine. It has been suggested that the mostlikely explanation for increased SAM synthetase activ-ity in stressed tissues is to meet an increased demandfor SAM used in lignin biosynthesis and methylationof other derivatives of the phenylpropanoid pathway[53].

11. Conclusions

Until now, studies of hormonal influences on stressphysiology have been dominated by investigations ofthe role of ABA in plant responses to suboptimal envi-ronmental conditions.Despite their importance, little isunderstood concerning ABA-independent responses tomany stresses. With the comparatively recent advent ofmolecular analysis of CK action, several studies haveprovided many new insights into the involvement ofCK in coordinating plant responses to environmentalstress. Nonetheless, experimentally-observed effectson the expression of any stress-regulated gene by CKsis not a priori evidence of the involvement of CKs inregulation of its expression, just as induction of a geneby adverse conditions is not necessarily indicative ofa role for the gene product in conferring stress toler-ance. Shifts in hormonal balance accompanying envi-ronmental stress do not necessarily indicate a role inadaptation at the genetic level. Current evidence indi-cates that in M. crystallinum, increases in ABA con-tent and the presently-characterised changes in geneexpression under stress occur as parallel responses andare not causally linked [80]. Moreover, the effects ofexogenous application of CKs or transgene-mediatedalterations in CK levels on stress responses do not nec-essarily reflect the normal physiological roles of CKsunder these conditions. Transgenic strategies towardsincreasing CK levels may challenge transformants withCK levels so excessive that in themselves, they con-stitute a severe stress. However, the apparent abilityof CKs to increase plant tolerance to environmentalstress may make CK-overproducing plants or thosedisplaying enhanced capacity for CK signal transmis-sion suitable for enhancing agricultural productivity.This may be of particular importance in developing

countries, where conditions are frequently suboptimal[72].

Given the indeterminate nature of plant develop-ment, the powerful influence of the environment onplant growth and development, and the well estab-lished role of CKs in the regulation of growth and mor-phogenesis, it is not surprising that many developmen-tal and physiological responses to adverse environmen-tal stimuli are apparently mediated by alterations in CKaction. One explanation for regulation of stress-relatedgene expression by CK is that CK- and stress-relatedsignal transduction pathways may overlap in part oract in parallel [181, 196] in a manner resembling theapparent interaction of CK signalling in phototrans-duction [184]. However, more work is needed beforea clear role can be assigned to the role of CKs in theregulation of plant development under adverse condi-tions. Stress responses are likely to be the end-productsof complex interplay between a multitude of hormonaland non-hormonal signals, including intrinsic develop-mental control and cell specificity factors. Alterationsin CK action may not specify stress-responses directly,but simply facilitate their expression under adverseconditions. If this interpretation is correct, then bettercharacterised hormonal triggers of stress effects such asABA or ethylene may themselves initiate the appropri-ate responses, although an overall change in hormonalbalance, including alterations in CK action, might bean important determinant of response capacity. A mul-titude of secondary feedback “checks” is likely to beneeded to confirm that a stress of sufficient severityhas been encountered and that the necessary positiveor negative controls over genomic expression patternsmust be effected.

Since there can now be little doubt that the fine con-trol of cellular responses to environmental stimuli isnot accomplished by isolated signals but by a complexweb of interwoven signal transduction chains, futurework must focus firstly on assessment of the level ofvariety in signalling pathways that control plant growthand development before dissection of the intricacies ofcross-talk may be tackled. In this regard, the emergenceof at least a subset of COP/DET/FUS gene productsas central regulators, which appear to be involved inboth stress [119] and CK-related signalling [67, 184]is encouraging, since these may constitute a funda-mental signalling pathway which could provide a cen-tral framework from which to establish relationshipsto well characterised parts of more specific signallingpathways. Although the mechanism whereby an arrayof inputs is integrated remains unclear, it seems fea-

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sible that they may converge on a single downstreamtarget defined by DET/COP/FUS members and thenbranch further downstream.

To fully comprehend the role of CKs in plantstress responses at the molecular level, more will needto be understood concerning: (i) networks of inter-actions that control growth and development underoptimal and adverse conditions, (ii) the effects ofadverse environmental conditions on CK biosynthe-sis and metabolism in different plant parts, and (iii)alterations in the flux of CKs from roots to shootsduring stress/recovery cycles and clarification whetherthese correlate with stress-induced changes in shootgrowth and physiology. Discrimination between therelative importance of synthesis or mobilisation at thesite of action and transport from the roots is like-ly to be of importance in future attempts to enhancethe environmental tolerance of crops by altering CKaction. In this regard, examination of stress-responsesin plants exhibiting root-specific ipt expression suchas with a recently-developed Cu2+-inducible geneexpression system [125] may shed some light on theimportance of inter-organ transport. Exploitation ofstress-autoregulated ipt expression, as was recentlyachieved using a senescence-specific promoter [57],holds considerable potential in agricultural applica-tions, since transgenic crops would not exhibit devel-opmental abnormalities associated with ipt expressionin the absence of adverse environmental conditions.Theoretically, such a system could be fine-tuned toameliorate stress-effects in particular tissues duringsusceptible developmental periods.

Since unlike animal hormones, it is generallyaccepted that responses to plant hormones are achievednot only from a change in hormone concentration butalso from a change in sensitivity of the respondingtissue, CK activity in any tissue is likely to be re-stricted to environmentally and developmentally per-missive conditions. A likely explanation for the report-edly wide, and often conflicting, range of effects ofCKs on stress responses is a general failure to takeinto account the interactions between the effects ofdifferent growth regulators, the environment and thephysiological state of the plant material. At the cellu-lar level, it is likely that many physiological process-es respond to both internal developmental cues andenvironmental challenges [48]. Therefore, hormonalcontrol should ideally always be considered against abackground of other controlling influences. By theircooperative, independent or antagonistic participationin several signalling steps, the available evidence sug-

gests that CKs may assist in the complex but highly reg-ulated coordination of growth and development duringstress. It is hoped that the dissection of stress-relatedand CK-specific cellular signal transduction pathwayswill provide a clearer picture of the role that CKs playin plant responses to suboptimal growth conditions.

Acknowledgements

The financial assistance of the South African ProteinResearch Trust, Natal University Research Fund andFoundation for Research Development is gratefullyacknowledged. We thank R Zimmermann for prepa-ration of the figure.

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