Plant Growth Regulation 23: 33–40, 1997. 33c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Elucidating cytokinin response mechanisms using mutants

Jill DeikmanDepartment of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA

Received 23 April 1997; accepted in revised form 9 June 1997

Key words: Arabidopsis thaliana, cytokinin, mutant, Nicotiana plumbaginifolia

Abstract

Cytokinins (CKs) have powerful effects on many elements of plant development,but little is known about the cellularand molecular mechanisms of CK action.This review describes recent progress in identification and characterizationof mutants of flowering plants that may permit elucidation of CK response mechanisms. Several Nicotianaplumbaginifolia mutants that are resistant to high levels of exogenous CK have been isolated. Characterizationof these mutants has led to information about relationships between CKs and other hormones, and CKs andnutrient metabolism. Two Arabidopsis thaliana mutants that are specifically resistant to CKs in a root elongationassay, cyr1 and stp1, have been described, and may represent lesions in the CK signal transduction pathway. Amutant that produces elevated levels of CKs, amp1, has provided surprising information about the role of CKsin cotyledon formation. A set of tagged mutations that result in CK independent growth in culture has beenidentified, and the affected gene, CKI1, cloned. The possibility that this gene encodes a CK receptor is discussed.Continued molecular/genetic analysis of CK responses is predicted to result in rapid progress in the next few yearsin understanding how CKs act.

Abbreviations: ABA = abscisic acid; ACC = 1-aminocyclopropane-1-carboxylic acid; BA = 6-(benzylamino)purine; CK = cytokinin; GA = gibberellin; IAA = indole-3-acetic acid

1. Introduction

Cytokinins were named for their ability to promotecell division, and they are required for growth ofmost plant tissues in culture. Cytokinins also influenceprocesses such as nutrient mobilization, senescence,chloroplast development and lateral branching [23,33]. Although CKs have powerful and wide-rangingeffects on plant development, we understand very littleabout their mode of action. However, recent progressusing mutants defective in CK responses promises toremedy this deficiency in the near future.

The conventional paradigm for the mechanism ofCK action is derived from the well characterized ani-mal hormone models. It is expected that there will be ahormone receptor, that binding to the receptor will ini-tiate a signal transduction sequence, and that one targetof signal transduction will be gene expression [28]. Aswith other plant hormones, biochemical approaches

to identification of a receptor have proven to be diffi-cult. Although a number of CK-binding proteins havebeen identified, their characterization has not yet ledto evidence that they are involved in CK action [7].Several experiments have indicated that calcium playsa role in CK signal transduction in mosses [40, 41],and there is also evidence for calcium and calmod-ulin involvement in CK-induced betacyanin synthesisin Amaranthus seedlings [16, 17, 46]. A large num-ber of genes have been studied that are regulated byCK [12]. However, progress in studying the controlof these genes has been slowed by the fact that suchgenes are often regulated by signals in addition to CK,such as light or other hormones. Also, many of thesegenes are regulated at a post-transcriptional level, andanalysis of such regulation is not as straightforwardas is study of transcriptional regulation. Thus, at thispoint CK perception and signal transduction processesare still very much a black box.

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An effective approach to understanding plant hor-mone signal transduction is by analysis of mutantsdefective in response to the hormone [15]. One advan-tage of this approach is that it is not dependent on for-mation of an accurate hypothesis, but it does requireknowledge of the expected phenotype of the desiredmutant, and the availability of a specific screen. Thecomplexity of CK effects on development of floweringplants has made these conditions hard to satisfy. How-ever, some progress has been made in the last few yearsin finding CK response mutants in flowering plants.Mosses also have specific responses to CKs, and mossmutants that appear to be defective in regulating CKbiosynthesis, and in perception of CKs were identified.However, a recent review on CK mutants described themoss mutants thoroughly [48]. Therefore, this reviewwill discuss only CK mutants in flowering plants, andwill emphasize recent progress.

2. Cytokinin resistant mutants

Although CKs are thought of as promoters of growth,high levels of this class of hormone are toxic. Isola-tion of mutants that are resistant to elevated and toxiclevels of a plant hormone has been used to identifygenes involved in hormone perception and signal trans-duction. For example, a number of auxin resistantmutants identify genes thought to play a role in auxinsignal transduction [21]. CK resistant mutants havebeen identified in Nicotiana plumbaginifolia and inArabidopsis thaliana, and these mutants are describedbelow.

2.1 Mutants of N. plumbaginifolia

Several groups have conducted screens for mutants inN. plumbaginifolia that are capable of greater growththan wild type plants in the presence of toxic levels ofCKs, resulting in the identification of several mutationsthat affect sensitivity of plants to CKs [5, 25].

2.1a The aba1 locus (ckr1, iba1, esg)The first N. plumbaginifolia CK-resistant mutant wasidentified by incubating EMS-mutagenized seed on20 �M BA [5]. At this concentration, wild type seedsgerminate, but growth of both the root and shoot isinhibited. The CK-resistant mutant ckr1 developedleaves and had an elongated axis with a substantial rooton medium containing 20 �M BA. This single gene

mutation was recessive, and it had additional effects.In particular, it was noted that the mutant germinatedmore rapidly than wild type, and it had a wilty phe-notype. Wiltiness could be corrected by application ofABA, and the mutant was shown to be deficient in ABAbiosynthesis [35]. Specifically, the mutant appears tobe unable to carry out the last step in ABA biosynthe-sis, conversion of ABA-aldehyde to ABA.

A second group isolated a mutant based on earlyseed germination (esg). This mutant was shown to beallelic both to ckr1 and to a mutant identified on thebasis of auxin-resistant seed germination, iba1 [39].The reduction in ABA levels in these mutants was con-firmed. IAA levels were also measured in the mutants,and were normal in leaves, elevated in ckr1 in seeds,but normal in seeds of the other mutants [39]. Theseauthors concluded that the defect in ABA biosynthesisjustified renaming the locus aba1.

The specificity of the response of these mutants tohormones was investigated by testing their resistanceto ABA and auxin. The mutants responded normal-ly to ABA in terms of inhibition of seed germinationand stomatal closure [5, 39]. However, the mutantshad abnormal responses to auxin. Seed germinationand root formation of the mutants was more resistantthan wild type to inhibition by auxin [5, 39], but leafdevelopment was more sensitive to inhibition by aux-in [5]. In this regard it is interesting that the adultmutant plants had reduced apical dominance, suggest-ing greater production or sensitivity to CK, and/orreduced auxin sensitivity [39].

It is possible that ABA1 encodes a structural genein the ABA biosynthetic pathway, and that CK andauxin resistance is an artifact deriving from the morerapid germination conferred by reduced ABA levels[5]. However, it is also possible that this gene regu-lates the synthesis of ABA, as well as CK and auxinsensitivity, or that perturbations in CK sensitivity sec-ondarily affect ABA biosynthesis and auxin sensitivity.Additional investigation of this mutant should helpclarify the mechanism for interactions of CK, auxinand ABA on various processes of plant development.

2.1b zea mutantsIn a different screen, three complementation groupsof N. plumbaginifolia mutants were isolated that cangrow in the presence of high levels of zeatin, and werecalled zea1, 2, and 3 [25]. When grown on 100 �Mzeatin, expansion of wild type cotyledons and roots isarrested. However, the mutants exhibit hypertrophy of

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the cotyledons and hypocotyl under these conditions.The resistance of the mutants to zeatin is more clear-ly demonstrated by transfer of 4-leaf stage plants tomedium with different concentrations of zeatin. Forexample, at 0.1 �M zeatin, growth of the zea1 mutantis not appreciably affected compared to growth in theabsence of exogenous hormone. On the other hand,growth of wild type plants is severely inhibited atthis concentration of zeatin. However, at 1 �M zeatingrowth of the zea1 mutant is abnormal, and callusand ectopic shoots are produced. At this concentra-tion, development of wild type plants was arrested.Each of the zea mutations was nuclear and recessive.Interestingly, these mutations do not have significanteffects on growth or development of the adult plants.

Sensitivity of the zea1 mutant in response to variousCKs, including adenine and urea types, was moreextensively analyzed in a later report [34]. The cotyle-don hypertrophy response was specific to CKs knownto be active. However, the response of the zea mutantsto other hormones has not been demonstrated, leavingthe issue of specificity only partially resolved.

The zea3 mutant was isolated for a second timefrom the same lot of mutagenized seed in an unrelatedscreen for mutants defective in the ability to assim-ilate nitrate [19]. A mutant was isolated which hada jointed cotyledon phenotype reminiscent of zea3,and a complementation test confirmed that the muta-tions were identical. Cytokinins are known to influ-ence nitrate metabolism by promoting expression ofthe nitrate reductase gene [27, 32, 36]. Thus, it seemsconsistent that a mutant defective in response to CKwould also be deficient in nitrate metabolism. In addi-tion to a requirement for high nitrate for growth, themutant was also sensitive to high amounts of sugar inthe medium. Each aspect of the mutant phenotype wasconfined to seedlings, and it was concluded that themutation affects a set of cotyledon-specific functions.However, the exact relation between CK resistance andthe defect in carbon and nitrogen metabolism remainsto be elucidated.

2.2 Mutants of Arabidopsis

2.2a ckr1 of ArabidopsisThe search for Arabidopsis mutants defective in CKperception or signal transduction has been based onthe inhibitory effect of CK on root elongation [42].An extensive screen was conducted using low levels(2.5 �M) of BA, and multiple mutants with roots that

have reduced sensitivity to CKs were identified [42].These recessive mutants were all allelic, and the locuswas named ckr1. Despite the reduced sensitivity toCKs, the plants grew relatively normally in the absenceof exogenous hormone, except that the young rosetteleaves of the mutant were more cup-shaped and yellowthan those of the wild-type. The mutants had normalsensitivity to auxin, but it was subsequently learnedthat the mutant is also resistant to ethylene, and thatckr1 is allelic to the ethylene-insensitive mutant, ein2[8].

The mutant ein2 is one of several mutants isolatedbecause of the failure of etiolated seedlings to dis-play the characteristic ‘triple response’ in the presenceof ethylene [15]. This mutant has been well charac-terized, and it lacks all known ethylene responses.Double mutant analysis places it in a signal trans-duction pathway with the other ethylene responsemutants [38].

The discovery that ckr1 and ein2 are allelic led tofurther characterization of the role of ethylene in CKinhibition of root elongation in Arabidopsis [8]. It wasshown that the ethylene-insensitive mutants ein1 andein2 are both CK resistant. Furthermore, application ofchemical inhibitors of ethylene biosynthesis or actionto wild type plants can partially relieve CK inhibitionof root and hypocotyl elongation, while application ofethylene caused inhibition of root and hypocotyl elon-gation. Finally, the production of ethylene in responseto exogenous CK was demonstrated. The conclusionwas that CK induces the production of ethylene, whichinhibits root and hypocotyl elongation.

The finding that CK inhibition of root elongationacts by induction of ethylene does not invalidate theroot growth screen. That is, it should be possibleto obtain CK response mutants with this screen, butmutants of interest should not be insensitive to ethyl-ene as well.

2.2b cyr1 mutantThe cyr1 mutant was also isolated because it had anelongated root in the presence of a low concentration(4.4 �M) of BA [14]. However, in contrast to ckr1, thismutant has an extremely abnormal shoot phenotype,which has features consistent with a disruption in CKperception or signal transduction (described below).In addition, root elongation of the cyr1 mutant hasnormal sensitivity to auxin, and ACC, the precursor toethylene. It is somewhat more sensitive to ABA. Therecessive cyr1 mutation is not allelic with ckr1.

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The cyr1 shoot has abbreviated development [14].The cotyledons and leaves fail to expand. Only a fewleaves are produced, and then a single infertile flower ismade. In addition, the mutant has reduced chlorophyllaccumulation, and fails to accumulate anthocyanins inresponse to exogenous CKs. Most aspects of the shootphenotype are consistent with CK insensitivity. Forexample, cotyledon expansion and chlorophyll accu-mulation are well-studied CK responses [23], and theregulation of anthocyanin accumulation by CK has alsobeen characterized [13]. The cyr1 mutant also displaysCK insensitivity in culture. Normally, root explantsform shoots in culture when supplied with exogenousCK, but root explants of the cyr1 mutant fail to respondin this way to CK [14].

The shoot phenotype of cyr1 is very similar to thatof a mutant called embryonic flower (emf), which wasisolated because of its abnormal pattern of develop-ment [44]. Two EMF loci have been identified [50],and EMF2 maps to the same region of chromosome5 as CYR1 [14]. Several alleles were isolated of bothemf1 and emf2, and the screen should have result-ed in recovery of cyr1 mutants. Therefore, it is quitelikely that emf2 and cyr1 are allelic. However, a genet-ic cross to definitively determine allelism has not beencompleted. Yang et al. [50] proposed that the primaryfunction of both EMF genes is to maintain vegeta-tive growth. The finding that CKs negatively regu-late expression of genes important in flower formation[18] would support the idea that EMF genes could beinvolved in CK signal transduction. Double mutantanalysis suggested that EMF1 and EMF2 are not inthe same pathway [50], so the two genes may act bydifferent mechanisms. Analysis of CK responsivenessof both emf mutants would help clarify this issue.

2.2c stp1 mutantThe stunted plant 1 (stp1) mutant is recessive and wasisolated in a search for root development mutants [2].The stp1 mutation affects the rate of growth but notthe morphology of the plant. To determine whetherthe mutation influences hormone responses, the effectof exogenous hormone application on root elongationwas examined. Responses of stp1 to auxin, ABA, eth-ylene and GA3 were similar to wild type. However,the mutant had reduced sensitivity to zeatin and BA.Radial swelling of the root in response to ethylene andCK was also examined, and the mutant was also lesssensitive to CK in this assay, but had normal ethylenesensitivity.

There was no effect of the stp1 mutation on CK-dependent organogenesis in tissue culture. It was con-cluded that the role of the STP1 locus in CK responsesis specific to growth responses, and it was hypothe-sized that CK inhibits root growth by down regulatingSTP1 activity [2].

2.2d Auxin resistant mutants which are also resistantto cytokininTwo mutants that were identified because they areresistant to elevated levels of auxin, aux1 and axr1, arealso resistant to CK and ethylene [21]. Both mutantsare defective in gravity response of roots, and abun-dant evidence indicates that auxin plays a role in rootgravitropism. In addition, axr1 has a pleiotropic phe-notype that is consistent with a primary defect in auxinaction. Both the AUX1 and AXR1 genes have beencloned. The AUX1 predicted gene product has homol-ogy to amino acid permeases, and is expressed in rootapical tissues [3]. It was proposed that the AUX1 geneproduct is involved in auxin transport. The AXR1 geneencodes a protein with homology to ubiquitin activat-ing enzyme E1, and may play a role in regulation ofprotein turnover [30]. Work with auxin regulated geneshas also pointed to a regulatory role for protein degra-dation as a central part of the mechanism of auxinaction [1].

Because of the phenotypes of the mutants, theAUX1 and AXR1 genes are thought to be involved pri-marily in auxin function. It has been proposed thatthe observed cross-resistance to multiple hormones ofthese mutants is a consequence of the known interac-tions among the hormones [21]. Cytokinin and auxininteract in control of numerous plant processes, such asorganogenesis and apical dominance. However, cross-resistance to CK and ethylene in the Arabidopsis ckr1mutant was explained simply by CK-induced ethylenebiosynthesis (see above). Therefore, it may be neces-sary to explain only cross-resistance of aux1 and axr1mutants to auxin and ethylene. Future investigationswith the cloned AXR1 and AUX1 genes may provideinformation about the mechanisms of these hormonalinteractions.

3. Amp1 – a cytokinin overproducing mutant

Hormone biosynthesis mutants have been very usefulin elucidating the pathways for GA and ABA biosyn-thesis [37]. Unfortunately, mutants of flowering plants

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that are perturbed in CK biosynthesis have been elu-sive. However, one such mutant has been identified,altered meristem program 1 (amp1) [9]. Amp1, a reces-sive nuclear mutation, was identified in a screen forseedlings with multiple cotyledons, but was subse-quently found to have increased levels of CKs. amp1light-grown seedlings had six times more zeatin ribo-side than wild type, and similarly elevated zeatin anddihydrozeatin. Cytokinin levels were also elevated indark-grown amp1 seedlings [10]. The product of theAMP1 gene may be required for CK degradation, or itmay be a negative regulator of CK biosynthesis.

Several aspects of the phenotype of amp1 had sug-gested that the mutant could have increased CK levels.In particular, the mutants have decreased apical dom-inance, an elevated rate of leaf initiation, and a longerthan normal life span. In tissue culture, amp1 mutantshad greater regeneration frequency than wild type [9].

Other aspects of the phenotype could not have beenpredicted from current knowledge of CK effects. Forexample, CKs were not known to affect cotyledonnumber. The multiple cotyledon phenotype has onlypartial penetrance, with about 20% of the seedlingshaving an abnormal number of cotyledons [9].

Cytokinins have been implicated in control of flow-ering [4]. Therefore, it is interesting that amp1 mutantshave abnormalities related to flowering. Floweringoccurred after a shorter time in the mutant comparedto wild type (12 vs. 16 days), but the number of rosetteleaves at the time of flowering was greater for themutant (an average of 20 leaves for amp1 and 7 forwild type). There was also an elevated rate of flowermorphological abnormalities, as well as a reduction inboth male and female fertility.

The amp1 mutant has been useful for explor-ing the relation between light and CKs, since amp1plants have a partially de-etiolated phenotype whengrown in the dark [9]. In fact, one of the constitutivephotomorphogenesis mutants, cop2, is allelic to amp1[24, 29]. When grown in the dark, wild type plantshave elongated hypocotyls, and unexpanded cotyle-dons folded in an apical hook. The amp1 and cop2mutants have open and enlarged cotyledons in the dark,but have nearly normal hypocotyl length [9, 24]. Inter-estingly, application of CK to wild type dark-grownplants can quite convincingly mimic the dark-grownphenotype of amp1 [10]. The effects of the amp1 andcop2 mutations on photomorphogenesis are limited,unlike those of several other cop and de-etiolated(det) mutants, such as det1, cop1, and cop8-15 [49].In contrast to those mutants, cop2 displayed normal

light-regulated gene expression and plastid differenti-ation [24], although amp1 had small but discernibleeffects on these processes [10]. It was proposed thatthe CK signal transduction pathway operates in paral-lel with the phytochrome-mediated light-signal trans-duction pathway, and influences a subset of the de-etiolation responses [10]. This hypothesis is in agree-ment with one reached from studying the growth ofArabidopsis seedlings in response to CK and light [43].It was found that CKs and light have independent andadditive effects on inhibition of hypocotyl elongation.

4. Cytokinin response genes identified byactivation tagging

A novel approach has been used to identify genesimportant in CK activity, called activation tagging [47].For this approach, protoplasts are transformed witha T-DNA containing multiple copies of the enhancerfrom the 35S gene of CaMV which are situated adja-cent to the right border of the T-DNA. Cells are thengrown on medium lacking CK. Wild type cell cultureswill not grow under these conditions, but if a gene isactivated that can substitute for the requirement for CKin the medium, growth can occur. Mutants can thus beselected that have CK-independent growth. A furtheradvantage of this method is that the genes affected inthe mutants are tagged by the T-DNA insertion, so thatthe gene can be readily isolated. Furthermore, the dom-inant nature of the mutations allows confirmation thatthe correct gene was cloned by transformation of wildtype protoplasts with the rescued gene, and selectionfor growth in the absence of CK.

Two groups have reported success with this tech-nique in identifying mutants that have CK indepen-dent growth in culture. Walden et al. [47] have studiedfive lines selected for growth in the absence of CKs,but have not yet published detailed analysis of thesemutants.

Kakimoto [26], using the same T-DNA vector, iso-lated three different kinds of mutants. Cytokinin inde-pendent 1 (cki1) callus proliferated rapidly, and pro-duced shoots in the absence of CK. Four such calliwere selected, but the regenerated shoots were unableto produce roots and normal flowers, and all weresterile. A second type of mutant, cki2 had similarCK-independent growth in culture, but was able toregenerate fertile plants. The CK-independent growthin culture was heritable. A third mutant, called manyshoots (msh) formed many shoots in the absence of CK,

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but rapid proliferation did not occur. This plant wasfertile, and its progeny sometimes produced adventi-tious shoots on cotyledons and petioles when grownon hormone-free medium.

The gene responsible for the cki1 phenotype wasidentified, and transformation of wild type plants withthe tagged gene conferred CK-independent growth.A coding sequence was identified flanking the rightT-DNA border, and homologous transcripts were abun-dant in the cki1-1 line, but were not detectable intissues of wild type plants. Analysis of genomic DNAindicated that the T-DNA was inserted upstream ofthe CKI1 gene in all four of the callus-forming linesof the cki1 group. However, results of this analysisindicated that this gene was not affected in the cki2 ormsh mutants. Also, those mutants did not have elevatedexpression of the CKI1 transcript.

The predicted amino acid sequence of the CKI1gene indicated homology to the histidine kinase andreceiver domains of the bacterial two-component reg-ulators [26]. The sequence had greatest similarity tothe products of the bacterial LemA and BarA genes.Another plant gene in this family, ETR1 of Arabidop-sis, encodes an ethylene receptor [15]. ETR1 and CKI1do not share homology in the putative input domains.The intriguing suggestion was made that CKI1 couldencode a CK receptor, which when over-expressedallows cells to sense a low amount of endogenous CKs[26]. Further experiments will be needed to determinethe exact function of the CKI1 gene in CK action.

The activation tagging approach has the potentialto identify genes involved in CK signal transduction,or in CK biosynthesis or metabolism. So far, mole-cular characterization of only the cki1 mutants has beencarried out, but analysis of the cki2 and msh mutantsshould also contribute useful information about CKresponse mechanisms.

5. Conclusions

Several CK response mutants have been identified, andtheir phenotypes have provided information about theinteractions of CKs with other hormones and environ-mental signals such as light or nutrient availability.Several of the mutants, including amp1 and cyr1, havedevelopmental abnormalities that may lend insight intothe roles of CKs in plant development, if the primarylesions are shown to be specifically in CK functions.

There is now the potential to gain more infor-mation about the nature of these mutants and about

CK responses by carrying out classical genetic doublemutant analysis. For example, it would be interestingto know whether cyr1 and stp1 act in the same or inde-pendent pathways. Ultimately, cloning the genes iden-tified by the mutants described in this review shouldprovide critical information about mechanisms of CKfunction.

Discovery of CK response mutants has been hap-hazard, and at times serendipitous. Cytokinins have notbeen easy to study. Compared to other plant hormones,mutants defective in CK response or accumulationhave been very difficult to obtain. Cytokinins may playsuch an intrinsic role in plant development that manymutations in CK functions might be lethal. Alterna-tively, there may be overlapping, redundant gene sys-tems in plants for CK biosynthesis, metabolism andperception, thereby masking mutations. In this case,though, dominant mutants could have been obtained.A separate characteristic of CKs that has probably hin-dered research is that many CK effects are not rapid,since many involve effects in tissue culture or in com-plex developmental processes. Therefore, in studies ofCKs it has been difficult to identify a simple, specificresponse which could form the basis of an effectivemutant screen.

Finding new mutants will greatly assist elucidationof the pathways for CK signal transduction and CKbiosynthesis, and should be a priority. The experimentsdescribed in this review provide clues for improvedapproaches for screening for CK response mutants. Forexample, the infertility of the cyr1 and cki1 mutantssuggest that other CK mutants might be infertile, anda screen designed to recover heterozygotes should becarried out. Care must be taken to ensure early in anyscreen that mutants are altered specifically in responseto CKs, thus avoiding confusion with mutants that areprimarily affected in response to ethylene, auxin, orABA. At the same time, it should be recognized thatcross-resistance to other hormones may be a featureof some CK mutants. In such a case, additional fea-tures of the phenotype must be used to judge whetherthe mutation may affect CK response directly, or as asecondary effect.

Examining insensitivity of roots to toxic levels ofhormone may allow identification of only a subset ofpotentially interesting mutants, and additional types ofscreens should be carried out. Several ideas for newscreens are evident from considering recent work withCK effects on Arabidopsis plants. For example, CKscause a de-etiolated phenotype when supplied in highamounts to dark-grown plants [11], and one could look

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for mutants that remain etiolated in the dark in thepresence of CKs. Recently, an albino mutant calledpale cress 2 (pac-2) was shown to undergo greening inresponse to CK [20]. Perhaps this effect could serve asa good screen. That is, pac-2 mutants could be muta-genized and a second site mutation that prevents CK-induced greening could be sought. Another responsethat could form the basis of a screen is the CK induc-tion of anthocyanin accumulation [13]. One could lookfor mutants that fail to accumulate anthocyanins inresponse to CKs, but which have normal anthocyaninaccumulation in response to high light. An advantageof this approach is that it targets a downstream responsewhich is not essential for viability. Also, as pigmentsobservable with the unaided eye, anthocyanins haveproven to be powerful genetic tools. Finally, it seemslikely that the approach of screening for mutants unableto express a reporter gene or selectable marker underthe control of a CK-responsive promoter may be pro-ductive. Such mutant selection strategies have resultedin identification of mutants defective in regulation ofgene expression coupled to chloroplast development[45], regulation of gene expression by light [31], andregulation by the systemic acquired resistance pathway[6]. The advantage of this approach is that it could leadto identification of genes involved in the terminal stepsof CK signal transduction, and such mutations mayhave limited effects on development which would notinterfere with viability or fertility.

Considerable progress has been made since the lastreviews of CK mutants in 1994 [22, 48]. The nextcouple of years should produce exciting new advancesin understanding of the mode of action of this critical,but recalcitrant hormone.

Acknowledgements

I would like to thank Drs. Melanie Trull and BenjaminMoll for helpful comments on the manuscript.

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