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Plant Molecular Biology 26: 1483-1497, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1483

Cytokinin metabolism: implications for regulation of plant growth and development

B~etislav Brzobohat~, t,2,,, Ian Moore 2, 3 and Klaus Palme 2

1 Institute for Biophysics, AS CR, Krdlovopolsk6 135, CZ-61265 Brno, Czech Republic (* author for correspondence); 2 Max-Planck-Institut fiir Zfichtungsforschung, Carl-von-Linn6 Weg 10, D-50829 K~ln, Germany; 3Department of Plant Sciences, University of Oxford, South Parks Rd., Oxford, OX1 3RB, UK

Received and accepted 11 October 1994

Key words: plant hormone, cytokinin, metabolism, conjugation, fl-glucosidase, transgenic plants

In troduct ion

This review describes recent advances in the study of cytokinin metabolism. It highlights how plant development is influenced by cytokinin synthesis, conjugation and conjugate hydrolysis, and what has been learned of the enzymes that regulate these processes. Although cytokinin metabolism and physiology are complex issues, some of the key enzymatic players are now being identified. This holds out the prospect of rapid progress in the near future. Just as much of what we know about the control of animal cell proliferation was learned by studying the cellular counterparts of viral oncogenes, so important information about the control of plant development by phytohormones has come from studying the genes of bacterial pathogens that subvert host phytohormone metabolism to their own advantage. We will focus on what has been learned from the use of such genes, and describe progress in identifying their functional counterparts in plants.

C y t o k i n i n s

Plant growth and differentiation relies on two fun- damental cell activities - cell elongation and division. As early as in the end of the past century the concept of two separate and specific factors

controlling cell enlargement and division was for- mulated. The first experimental evidence of chemical control of cell division was provided by Haberlandt in 1913 [32]. He demonstrated that phloem diffusates could stimulate parenchyma- tous potato tuber cells to convert to mitotic cells. More than 40 years later, Skoog and co-workers were able to show that while auxin induced cell enlargement in tobacco pith tissue cells, a simul- taneous addition of the cytokinin kinetin (6- furfurylamino purine) was necessary to induce cell division [56, 57]. The first naturally occurring compound able to induce plant cell divisions was purified by Letham in 1963 and identified as 6-(4- hydroxy-3-methylbut-trans-2-enylamino) purine, commonly known as zeatin [46]. Since that time a number of compounds which stimulate plant cell division in combination with auxin have been isolated and named cytokinins (Fig. 1).

Skoog's classical experiments led not only to the discovery of the first cytokinin, but simulta- neously demonstrated a general feature of phytohormone action, namely involvement of more than one phytohormone in promoting a biological response. Later, it was established that phytohormones can interact both synergistically and an- tagonistically (e.g. cytokinins act together with auxin to stimulate callus cell division but oppose auxin with regard to stimulation of lateral bud formation).

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R1

/=(

R~

R2 R3

H H

H ribosyl

H riboside H

R1 '

*

N, H2

L:g:L987 . :~" " ~ N ~ ~N

I

I R3

R4

glucosyl

Trivial name

N6(A2-isopentenyl) adenine

N6(A2-isopentenyl) adenosin

N6(A2-isopentenyl) adenosine- 5 "-monophosphate

N6(A2-isopentenyl) adenine-7-glucoside

OH

H H H ribosyl H glucosyl H H alanyl H ribotide

glucosyl

trans- zeafin t-zeatin riboside t- zeatin-9-glucoside t-zeatin-7-glucoside lupinic acid t-zeatin riboside-5"-monophosphate

C~, H H H ribosyl

zeatin-O-glucoside zeatin riboside-O-glucoside

OH /,-%

H H H ribosyl H glucosyl H H alanyl H ribotide

glucosyl

dihydrozeatin dihydrozeatin riboside dihydrozeatin-9-glucoside dihydrozeatin-7-glucoside dihydrolupinic acid dihydrozeatin riboside-5"-monophosphate

OG H H / - % H ribosyl

H H ~ _ H ribosyl

H glucosyl H

Fig,. 1. Cytokinin structure and nomenclature. fl-D-ribofuranosyl group.

glucosyl

Glucosyl (G)

dihydrozeatin-O-glucoside dihydrozeatin riboside-O-glucoside

N6(benzyl) adenine N~(benzyl) adenosine N~(benzyl) adenine-9-glucoside NO(benzyl) adenine-7-glucoside

and ribosyl groups refer to the fl-D-glucopyranosyl and

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Although cytokinins were first discovered by their ability to induce cell division, they are now known to act in combination with other phytohormones to regulate diverse responses in plants, including seed germination, de novo bud formation, release of buds from apical dominance, leaf expansion, reproductive development, and senescence.

Significant effort has been dedicated to identification of molecular mechanisms of cytokinin signal transduction. More than twenty years ago a search for cytokinin receptors was initiated [29]. However, despite the isolation of a number of cytokinin binding proteins from different plant sources [3, 23, 34, 43, 62, 65, 75, 77, 79, 80, 99, 100] no functional cytokinin receptor has been reported yet. The ability ofcytokinins to stimulate transcription has been demonstrated in several experimental systems and cytokinin regulated promoters have been identified [ 1, 12, 15, 82, 98]. Although it is possible that upon cytokinin binding some soluble cytokinin binding proteins may regulate the activity of these promoters in a man- ner similar to animal steroid receptors, the involvement of receptor linked signalling cascades cannot be excluded. Membrane-bound receptor- like proteins have recently been cloned from plants and were shown to belong to the group of receptor kinases or the two-component signal receptors (see Theologis and Walker, this volume). However, the involvement of G-protein-linked receptors or other so far unknown receptor systems in phytohormone signalling cannot be excluded. An indication suggesting that phosphorylation is involved in cytokinin signalling pathway came from the demonstration that transcription of a cytokinin-inducible gene could be suppressed by a protein kinase inhibitor [17]. Further support for a role of protein phosphorylation in cytokinin response was provided recently by the demonstration that a wheat protein kinase homologue was transcriptionally regulated by cytokinins [81 ]. This finding may further support the notion of a link between protein phosphorylation and other players in cytokinin signal transduction, i.e. changes in calmodulin and calcium levels [37].

Understanding molecular mechanisms of cyto-

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kinin action will require both precise knowledge ofcytokinin signal transduction pathways and an understanding of how active cytokinin levels are modulated. An intriguing feature of cytokinins, as well as other phytohormones, is the complexity of metabolic transformations to which they can be subjected in the plant cell. It is conceivable that metabolic modifications may be important in modulating cytokinin activity, compartmentation and transportability. Thus the level of active cytokinin at a particular site of action may be influenced by a large number of factors: de novo

synthesis; oxidative degradation; formation and hydrolysis of inactive conjugates, transport into and out of particular cells; and subcellular com- partmentalisation to or away from sites of action. On top of this, there is also the possibility that physiological responses may be modulated by variations in the ability of cells to respond to a particular concentration of free cytokinin. In this paper we will concentrate principally on de novo

biosynthesis, degradation, conjugate formation, and conjugate hydrolysis, which will be dealt with in that order.

De novo synthesis o f cytokinins

Cytokinin biosynthesis in developed plants takes place mainly in roots [ 19, 36, 94, 107], although smaller amounts can be synthesized by the shoot apex and some other plant tissues. Two biosynthetic pathways have been reported for cytokinins: the de novo biosynthetic pathway [11, 101] and the tRNA pathway [33, 88]. It is very likely that the de novo biosynthetic pathway accounts for the majority of newly synthesized cytokinin. The key step in de novo cytokinin biosynthesis is the formation of N6-(AZ-isopentenyl) adenosine- 5 ' -monophosphate from A2-isopentenyl pyro- phosphate and adenosine-5 '-monophosphate catalysed by isopentenyltransferase (IPT). Although IPT activity has been detected in plants, attempts to purify the enzyme to homogeneity have not yet been successful [10, 76]. It may be that a gene- tagging approach such as that of Walden et al. (this volume) will provide a faster route to the

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identification of cytokinin biosynthetic enzymes. Recently, an Arabidopsis mutant, amp1, demon- strating various abnormalities reminiscent of cytokinin action, was shown to have a six-fold elevated cytokinin content. The elevated cytokinin content was proposed to be the primary effect of amp1 mutation, and one of the possible roles for wild-type AMP1 is as a negative regulator of cytokinin biosynthesis [9].

Bacterial isopentenyltransferase

The soil phytophatogen Agrobacterium tumefaciens causes crown gall disease on several plant species by transforming the host with a bacterial D N A sequence (T-DNA) that leads to increased phytohormone biosynthesis by the transformed cells, which in turn promotes cell proliferation. Molecular analysis of the genes on the T-DNA allowed the identification of one (tmr or ipt) coding for an isopentenyltransferase. Several groups have used the gene to study the effects of enhanced cytokinin biosynthesis on plant growth and development. Tobacco calli overexpressing the ipt gene from a strong promoter (CaMV 35S) initiated shoots more frequently, rapidly and pro- fusely than did calli containing the ipt gene expressed from its own promoter [91]. Similarly, the chimaeric CaMV 35S-ipt construct was able to induce shoot proliferation on undifferentiated cucumber tissues, whereas the ipt gene expressed from its own promoter was not. These changes correlated well with the measured increases in endogenous cytokinin level in these tissues [91], and mimiked the classic effects of cytokinin on plant tissue cultures described by Skoog and Miller [87].

Constitutive overproduction of cytokinin leads to almost complete suppression of root formation and, consequently, prevents plant regeneration. Controlled ipt expression in transgenic plants was first achieved by placing ipt under the control of heat-shock-inducible promoters (hsp) [54, 84, 89, 90]. Experiments with these transgenic plants led to a number of important conclusions about cytokinin action. Several morphological conse-

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quences of introducing an hsp-ipt gene into tobacco were observed. The transgenic plants displayed reduced height (to ca. 50 ~o of the control). This reduction was due to a decrease in internode length rather than node number. Fur- ther, the transgenic plants also have smaller leaves, which show delayed senescence [89], a greatly reduced root system, a less developed vascular system, and reduced apical dominance.

Apical dominance has also been modified through the use of bacterial genes that alter free auxin levels. When the free IAA content was increased 10-fold by expression of the A. tumefaciens T-DNA auxin biosynthetic genes iaaM and iaaH, the transformed plants are almost com- pletely apically dominant [42]. In addition, a gene encoding an IAA-lysine synthetase of Pseudomo- nas savastanoi has been used to reduce the free IAA content of transgenic plant material. When free IAA content was reduced by 10- to 20-fold, lateral buds are also released from apical dominance [78, 96]. Thus, it appeared that the apical dominance is not controlled by auxin or cytokinin alone, but by the ratio of the two growth factors. A high auxin/cytokinin ratio will result in the suppression of growth of lateral shoots, while a low ratio results in growth.

Interestingly, the morphological changes induced in plants containing the hsp-ipt construct were observed even in non-heat-treated plants. Although no ipt mRNA could be detected in such plants, the cytokinin levels were increased between twofold for zeatin riboside and up to sev- enfold for zeatin riboside 5'-monophosphate [54]. Whilst a single heat treatment dramatically increased the abundance of ipt mRNA and the cytokinin content (63-fold for zeatin, 24-fold for zeatin riboside and 3-fold for zeatin riboside 5'- monophosphate), no further phenotypic alterations were observed [ 54]. Smart and co-workers [89] described similar differences in appearance between transformed and untransformed plants at the control temperature, the transformed plants being shorter and having larger axillary shoots than untransformed plants. In addition, after multiple heat shocks the hsp-ipt transformed plants exhibited an abundance of tiny shoots at

the apex and a release of lateral buds, which was not found in the untransformed heat-shocked plant.

These studies parallel those of Kares and co- workers [41] who found that low to moderate increases in endogenous levels of IAA were sufficient to induce physiological responses in planta. This study used a heat-shock inducible system for increasing the endogenous IAA levels consisting of the auxin biosynthetic genes iaaM under the control of a heat shock promoter and iaaH tran- scribed from its own promoter. Interestingly, endogenous IAA in uninduced transformed plant material reached levels up to ten times higher than untransformed plants. This increase was able to promote root induction in three different plant systems. Heat-shock treatment caused a further increase in IAA levels in the range from 11-20 times compared to untransformed controls and increased dramatically the extent of root formation.

Hence, relatively minor changes in cytokinin and auxin levels are sufficient to cause the observed developmental alterations. Indeed, these are just two of several examples with transgenic plants where relatively small changes of 3- to 10- fold in measured hormone concentration (at least in whole tissue homogenates) are sufficient to sig- nificantly alter quantitative aspects of plant development [e.g. 26, 42, 96]. Trewavas [103] ar- gued that a true understanding of the control exerted by plant hormones over a given process in planta can come only from studying the effects of small fluctuations in hormone concentration around endogenous levels - dramatic increases or reductions in hormone levels can show only that a hormone is in some way able to promote a particular process, or that it is necessary for that process, without revealing anything about the control exerted by the hormone over that process in non-experimental material. In view of disagree- ments in the past about the relevance of some responses that are elicited with broad dose- response curves by application of exogenous phytohormone, it is gratifying that much of our classical understanding of phytohormone action is supported by small fluctuations in measured

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phytohormone concentration generated from within.

One other point that emerges from these studies is the lack of homeotic, or qualitative developmental changes in transgenic plants in response to changes in phytohormone content of up to 80- fold. The changes observed in hsp-ipt plants for example are essentially quantitative in nature. Tissue culture experiments clearly indicate the potential of phytohormones provided in the medium to greatly alter the developmental fate of cells [87]. Results obtained with exogenous cytokinin application have been extended by increasing endogenous cytokinin levels with help of ipt gene in transgenic calli. As low as a six-fold increase in endogenous cytokinin level (compared to a leaf cytokinin content) in N. plumbaginifolia led to induction of shoot formation. An approxi- mately threefold increase in endogenous cytokinin level has been associated with callus formation upon transformation in cucumber hypo- cotyles, whereas a 172-fold increase in endogenous cytokinins (compared to normal hypo- cotyl levels) was necessary to induce shoots on cucumber calli [91]. These levels of endogenous cytokinins are in the range achieved in hsp-ipt plants or only slightly exceed them. Thus, these results indicate the importance of the tissue in determining the nature of the response to a particular hormone content.

This type of morphogenic response has only been observed once in regenerated transgenic plants. In this case plants were regenerated which contained the ipt gene separated from the 35S CaMV promoter by the transposable element Ac ('35S-Ac-ipt' [ 27 ]). As a consequence of transposition in certain cells of the transgenic plants, the ipt gene was activated, and expressed to high levels producing sectors of high cytokinin concentration. This led to vivipary, that is, the formation of adventitious shoots derived from vascular parenchymal cells in the leaf. Thus, endogenous cytokinin can alter the developmental fate of certain cells in ex vitro cultured plants. However, adventitious shoots were observed to emerge only from the vascular parenchyma, whilst the activation of ipt gene by transposition occurs

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in numerous cell types, so the response of cells to cytokinin appears to be determined by cell type as much as by cytokinin concentration.

The local cytokinin concentrations induced by transposition in these 35S-Ac-ipt plants were much higher than in the non-heat-treated hsp-ipt which may account for the different morphogenic responses in these two systems. However, the highest cytokinin levels were detected in the ab- errant shoots of 35S-Ac-ipt plants, and, surpris- ingly, they were not much higher than in heat- shocked leaves containing the hsp-ipt gene fusion. As several of the adventitious shoots emerged from sites on a leaf quite distant from the site of transposition (and consequently of cytokinin biosynthesis) it may be the gradient of cytokinin which is important, or as suggested by Estruch et al. [27], transport to the vascular parenchyma cells may cause a local accumulation of cytokinin to high levels. Later in development, 35S-Ac-ipt plants formed epiphylous flower buds on leaf tips [28]. Some of these buds were morphologically normal while a fraction of them showed abnormalities such as organ transitions and fusion between organs located in different whorls. Inter- estingly, free cytokinin levels in the normal epiphyllous buds were only moderately (not more than ten times) higher compared to normal leaves or flower buds. In contrast, 100-1000-fold higher cytokinin levels were found in the abnormal epiphyllous flower buds. The increase in cytokinin levels was accompanied by a strongly reduced expression of tobacco homologues of floral homeotic genes DEFA, GLO and PLENA of Anti- rrhinum majus [28].

Thus, despite the lack of a well characterized plant isopentenyltransferase, the availability of a bacterial ipt has allowed some analysis of the consequences of deregulated cytokinin synthesis in plants. The results obtained in experiments in- volving small (5-10-fold) alterations in cytokinin content have confirmed much of the classical view of cytokinin action in plants, while the significance of developmental alterations associated with considerably larger changes in cytokinin content remains to be properly clarified.

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Cytokinin deactivation

Attenuation of hormone-induced cellular responses requires an efficient way of hormone inactivation. Active cytokinins can be" inactivated by degradation or conjugation to different low- molecular-weight metabolites, such as sugars and amino acids.

Degradation

At present the only plant enzymes known to ca- talyse the degradation of cytokinins to inactive products that lack the N6-side-chain are cytokinin oxidases. An enzyme activity converting N 6- (A2-isopentenyl) adenosine to adenosine was demonstrated in a cell-free system for the first time by Paces et al. [66]. Whitty and Hall [111] were the first to use the term 'cytokinin oxidase' for a similar enzyme activity detected in maize kernels. Cytokinin oxidase activities have now been isolated from a number of higher-plant sources [5, 8, 40, 44, 51, 63, 66, 67, 111]. N6-(A 2- isopentenyl) adenosine is the preferred substrate for all cytokinin oxidase activities studied. Zeatin and zeatin riboside were also substrates in all cases studied. However, enzymes from certain sources exhibit clear preference for N6-(A 2- isopentenyl) adenosine compared to zeatin [ 8, 40, 63, 67]. The presence and position of the double bond in the isoprenoid side-chain appears to be important to substrate activity. Thus, dihydrozeatin is resistant to cytokinin oxidase action in vitro [8, 40, 51, 63, 111] and appears to be a major cytokinin in tissues with a high cytokinin oxidase activity [64]. Similarly, O-glucosylation of the side chain protects cytokinins from the in vitro attack of cytokinin oxidase [51]. On the other hand, N-glucosylation or other ring substi- tutions may decrease the affinity of cytokinin oxidase for a given cytokinin, but do not necessarily eliminate substrate activity [51]. Cytokinin oxidase cannot accept cytokinin nucleotides as substrates [44]. Plant cytokinin oxidases exert very low or no activity against N6-benzyladenin and kinetin in vitro [8, 40, 44, 51, 63, 111] and

these cytokinins are degraded only very slowly/n vivo [18, 108].

Cytokinin oxidase activity and cytokinin degradation can be regulated on several levels. Tis- sue culture experiments have demonstrated that cytokinin oxidase activity can be induced by transient increases in the cytokinin supply available in the culture medium [8, 40, 63, 69, 102]. A possible role of cytokinin oxidase compartmentalization in modulation of cytokinin degradation by P. vulgaris and P. lunatus calli has been suggested by Kaminek and Armstrong [40]. Phaseolus vulgaris calli can degrade exogenously supplied cytokinins much faster than P. lunatus calli. The cytokinin oxidase isolated from P. lunatus was apparently not glycosylated and exhibited a pH optimum of 8.4, features typical of intracellular enzymes. In contrast, the cytokinin oxidase from P. vulgaris was glycosylated and exhibited a pH optimum of 6.5, features often detected in secreted proteins. Thus, the faster degradation of cytokinins in tissue culture media by P. vulgaris calli could be explained if the P. vulgaris cytokinin oxidase is secreted into the cell exterior and consequently has a better access to exogenously supplied cytokinins than the P. lunatus cytokinin oxidase se- questered in an internal cell compartment. Cyto- kinin degradation in vivo can be influenced by other phytohormones. Thus, auxins stimulated cytokinin degradation in several experimental systems [35, 53, 70, 74, 112, 113], although not necessarily by increasing the level of cytokinin oxidase protein [63]. On the other hand, abscisic acid caused suppression of kinetin degradation in lettuce seeds [ 55 ] and the conversion of zeatin to dihydrozeatin in P. vulgar& [93]. Limited atten- tion has been paid to the role of cytokinin degradation in the regulation of plant growth and development. However, from the data available it appears that cytokinin degradation varies in different tissues and in the same tissue during its development [39, 86, 104, 111] suggesting that cytokinin oxidase may have a significant role in the control of cytokinin levels during plant development.

Significant progress in understanding the role of cytokinin oxidase in the regulation of plant

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development can be expected from the application of molecular techniques. Therefore, the recent success in obtaining antibodies against maize cytokinin oxidase and their use in the isolation of a cDNA clone [6] are of particular importance.

Conjugation

A common feature of plant hormones is that they frequently occur in the cell as conjugates to various sugars and amino acids. These conjugates usually possess low intrinsic physiological activity, and what activity they do show correlates with their rate of hydrolysis in plant tissues. Con- jugation is often seen as a way of removing free, active hormones from a tissue, however the conjugation process is often reversible, and, as conjugates can frequently accumulate in great excess over free forms of phytohormone, the conjugate pools must also be considered as sources of free hormone and may represent storage or inactive transportable forms of the hormone. This section considers the biochemistry and physiology of conjugate formation after which we discuss the physiological role of conjugate formation and hydrolysis.

Cytokinins occur frequently as N-glucosides and O-glucosides. They can be N-glucosylated on the purine ring and O-glucosylated on the N 6- substituted side-chain. N-glucosylation at the 3, 7, or 9 position of zeatin and dihydrozeatin have been identified in tobacco, radish, and lupin [14, 31, 52]. The radish cytokinin-7-glucosyltransferase was studied in more detail [20, 21 ]. The enzyme catalysed the formation of both 7- and 9-glucosides, but favoured the 7 position. Both UDP-glucose and TDP-glucose served as glucosyl donor. The enzyme can use trans-zeatin (Km 0.15 mM) as well as other natural and synthetic cytokinins as substrates.

N-glucosides of cytokinins are biologically inactive and very stable in vivo where there do not appear to be hydrolases that can release the free phytohormone from the conjugate. Thus it is sus- pected that N-glucosylation may be important in regulating levels of active cytokinin through inac-

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tivation [47]. Indeed, increased cytokinin production upon heat shock treatment in hsp-ipt plants (see above) was accompanied by accumulation of zeatin-N7-fl-glucoside at high levels [ 54]. Similarly, after external cytokinin application a significant portion of the internalized cytokinin was converted to N-glucosides [52, 71, 72, 73]. Nevertheless, the precise function of these compounds in normal plant development is not known, but it has been proposed that they play a role in Agrobacterium rhizogenes pathogenicity (see below).

Cytokinins such as zeatin and dihydrozeatin with hydroxylated N6-side-chain are often glucosylated to form the O-glucosides. Cytokinin-O- glucosides have been identified in many plant species [see 47]. The enzyme mediating O- glucosylation, O-glucosyltransferase, was isolated from P. lunatus [16]. The O-glucosyltransferase has a distinct substrate specificity. The enzyme recognizes trans-zeatin, but not dihydrozeatin, cis-zeatin, or ribosylzeatin. Both UDP-glucose and UDP-xylose serve as glycosyl donors for the enzyme in vitro, although UDP-glucose was the favoured substrate. Immunologically related proteins can be detected in both dicots and mono- cots [59, 60]. It is possible that the amp1 mutant of Arabidopsis thaliana described above is defec- tive in some aspect of cytokinin glycosylation, leading to increased accumulation of the free forms [9].

The function of O-glucosyl derivatives ofzeatin is not clear. However, their accumulation when cytokinin accumulates in tissues and their decrease during phases of active growth have been taken as an evidence in favour of a storage role [47].

Other types of conjugates

While cytokinin glucosides appear to be ubiqui- tous cytokinin conjugates, cytokinin-O-xylosides seem to be specific for Phaseolus vulgaris [45, 105]. Interestingly, although O-xylosylzeatin has been found to be even more active than zeatin in some biossays, it was converted to zeatin only slowly

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in the same tissue. Therefore, it is possible that O-xylosylzeatin represents a conjugate with intrinsic cytokinin activity [58]. The O-xylosyltransferase has been purified and characterized [105 ]. The enzyme utilizes trans-zeatin as the preferred substrate and UDP-xylose as the donor of the xylosyl moiety. The enzyme also accepts dihydrozeatin as a substrate, but not cis-zeatin or ribosylzeatin. The enzyme is specifically expressed in endosperm [49, 50, 60]. Interestingly, the zeatin-O-xylosyltransferase was found to be associated with both the nucleus and cytoplasm [50]. The biological significance of this finding is unclear, but the authors suggested possible role(s) for the enzyme in transport or targeting of cytokinins or cytokinin-related molecules between the nucleus and cytoplasm [50]. Interestingly, cytokinins have recently been localized to both nucleus and cytoplasm in developing somatic embryos of Dactylis glomerata L. [38]. These find- ings will certainly increase interest in studying the role of subcellular compartmentalization in phytohormone action.

In addition to glycosylated forms of cytokinin, conjugates of cytokinins with amino acids have been identified. Alanine can be conjugated to the nitrogen 9 of the cytokinin purine ring. An enzyme catalysing this reaction, fl-(9-cytokinin) alanine synthase, was purified from immature Lupi- nus luteus seeds. The product derived from zeatin was identified as lupinic acid [22]. As with N-glucosylation of cytokinins, alanyl conjugation probably represents a process of cytokinin inactivation since the alanine conjugates are also biologically inactive and stable.

Conjugate hydrolysis - bacterial enzymes

RolC

The study of phytohormone conjugates was given a boost by the discovery a few years ago that the plant pathogenic soil bacterium Agrobacterium rhizogenes may exploit conjugate pools during pathogenesis. A. rhizogenes induces the hairy root disease in infected plants, resulting in neoplastic

root proliferation. These neoplasias result from activities of the rol genes (mainly rolA, B and C) which are transferred to the plant genome by the T-DNA of A. rhizogenes RJ plasmids. In an at- tempt to decipher the role of individual rol genes and to get an insight into the molecular basis of the hairy root disease, individual rol genes have been transformed into various plant species. Dis- tinct pleiotropic morphological changes in transgenic plants expressing each of the genes demonstrated that the products of each rol gene can interfere with normal plant developmental processes independently and in distinct ways.

Recently, the gene product of rolC has been shown to release cytokinins from their N- glucosides [26]. Transgenic tobacco plants expressing the rolC gene from its own promoter resulted in reduced apical dominance and inter- nodal distance, altered leaf morphology, small flowers and reduced fertility [83]. More severe phenotypes were observed in transgenic plants expressing the rolC gene under the control of the strong 35S CaMV promoter. These plants were very small, the apical dominance and internode length were drastically reduced, leaves were pale green and lanceolate, and flowers were male- sterile [ 30, 83 ]. An interesting aspect of rolC gene action is its cell-autonomy both in root induction on leaf discs [83] and in the reduction of leaf pigment content [95]. This observation has been interpreted as evidence that the rolC biological effect is not due to the rolC-mediated synthesis of a growth factor that is transported in leaf tissue [95].

The glycoconjugate hydrolysing activity of ROLC protein may cause increased levels of cytokinins in rolC transgenics [26, 85]. However, only some of the morphological and physiological changes associated with the rolC phenotype, namely reduction of apical dominance and height, and enhanced tuber formation in potatoes, can be explained by simply postulating increased cytokinin levels. Stimulation of root initiating and growth, and reduction in chlorophyll content which are typical of rolC transgenic plants are not observed either upon application of exogenous cytokinin or in plants expressing the ipt gene. In

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addition, the cell-autonomous behaviour of rolC contrasts with the ability of cytokinins generated by IPT to act at a distance [27]. It may be that some process such as intra- and intercellular transport of cytokinin is altered when their glycosylation is disturbed, leading to accumulation of free cytokinin within the cells. However, an unambiguous interpretation of data from such transgenics can be complicated by the fact that free hormone levels are influenced by numerous input and output pathways and these can be altered during tissue development. Only when these are better understood will we be able to interpret effects such as those of rolC with confidence.

Conjugate hydrolysis - plant enzymes

The stability of cytokinin-O-glucosides in different tissues varies markedly. While they accumulate to relatively high levels in tissues such as endosperm many plant tissues contain glycosidase activities that can release active cytokinin. The ability of plant tissues to hydrolyse the cytokinin-O-glucosidic bond contrasts with their apparent inability to hydrolyse cytokinin-N- glucosides to free cytokinin. Consequently, cytokinin-O-glucosides are frequently active in bioas- says, and they have been suggested to be a storage or transport form of cytokinins. The biological activity exhibited by cytokinin-O-glucosides is associated with the release of free cytokinins, so cytokinin-O-glucosides are considered to be in- trinsically inactive forms of cytokinins which can be easily converted to free active cytokinins by action of fl-glucosidases. Cytokinin-O-glucoside levels have been observed to decrease rapidly during some phases of plant development, for example during germination in maize seeds [92], lateral bud development in bean plants [68], and the breaking of dormancy and apical bud growth in potato tubers [ 106]. Regulated release of free cytokinins from their glucosides may require the action of specific fl-glucosidases. Although glycosidase activities were initially thought to have broad substrate specificities, it has appeared that when single glycosidase species are investigated

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they often show considerable selectivity for the sugar and aglycone portion of their substrates [131.

Recently, a novel 60 kDa/~-glucosidase (p60) has been identified by photoaffinity labelling with azido-IAA in maize coleoptiles [7]. The purified enzyme exhibited activity towards general /%glucosidase substrates (e.g. p-nitrophenyl-/?-D- glucopyranoside or 6-bromo-2-naphthyl-/~-D- glucopyranoside), and the activity of p60 on various auxin and cytokinin conjugates was investigated, p60 was able to hydrolyse cytokinin-O- and -N3-glucosides but not cytokinin-N7- or -N9-glucosides. It also hydrolysed indoxyl- glucoside but not the auxin conjugates IAA- aspartate, IAA-glycine or IAA-myo-inositol, or IAA glucose ester, consistent with the specificity of the enzyme for the glucosidic bond. It had no or very low activity on several other naturally occurring glucosides (e.g. salicin, cellobiose and laminiaribose) which were cleaved efficiently by a broad-specificity/~-glucosidase from Caldocellum saccharolyticum [7]. Interestingly, expression of this enzyme is tightly regulated during the maize life cycle with the highest levels being found in the developing seedling. One possible role for a /%glucosidase specific for this developmental stage might be to release free phytohormones from their conjugates which are transported from the endosperm to the growing regions.

The activity of p60 in vitro on cytokinin- glucosides indicated that it has the potential to modulate the intracellular concentration of active cytokinin. However, as mentioned previously, the kinetics of cytokinin accumulation are likely to be highly complex, and assays of activity in vitro do not clarify what impact a particular enzyme will have on the equilibria that control the metabolism and transport of cytokinin in the highly compart- mentalized environment of the cell. The physiological relevance of p60 in cytokinin metabolism has been investigated in a biological assay. Firstly, amino acid sequence analysis of p60 allowed isolation of a cDNA, Zm-p60.1, coding for a protein closely related to p60 and with the same enzymatic properties. It was found that tobacco pro- toplasts that expressed Zm-p60.1 either tran-

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siently or constitutively could use inactive cytokinin glucosides such as zeatin-O-glucoside and kinetin-N3-glucoside to initiate cell division in the absence of any other exogenous cytokinin source [4].

In the roots of maize seedlings Zm-p60.1 was found to be located in the meristematic cells. It was suggested that in young maize root meristems Zm-p60.1 liberates free cytokinins from the exogenous supply of cytokinin-O-glucosides arriving from the endosperm and consequently helps to maintain meristem activity [4]. Thus, Zm-p60.1 may be one of the key enzymes involved in regulation of equilibria between free phytohormones and their conjugates, and a valuable tool to study and manipulate these equilibria.

It is noteworthy that the endogenous cytokinin glucosidase, Zm-p60, and the bacterial enzyme, ROLC, have different substrate specificities, each of which appears tailored to its biological role. Thus Zm-p60 hydrolyses O-glucosides which are thought to be transient storage forms of cytokinin, but it does not hydrolyse N7- or N9- glucosides which appear to be terminally inactivated forms of cytokinin. The ROLC protein, however, is able to hydrolyse these N7- and N9- glucosides in addition to O-glucosides. Conse- quently, during pathogenesis, the bacterium is able to exploit the normally inactive N7- and N9- glucoside pools (which can accumulate to considerable levels in tissues of certain species [47]) to increase cytokinin levels, and by reversing de novo glycosylation on N7 and N9, it simulta- neously antagonizes one of the pathways available to plant cells to compensate for the increased accumulation of free cytokinin.

Interestingly, IAA glucose ester was found to be a particularly efficient inhibitor of cytokinin glucoside hydrolysis by Zm-p60.1. This may ex- plain why this protein was labelled by IAA azido derivative. The inhibitory effect of IAA glucose ester on cytokinin glucoside hydrolysis raises the possibility of interaction between auxin and cytokinin conjugate metabolism. Evidence in favour of mutual interactions between phytohormones at the metabolic level has been reported; IAA and its derivatives were found to be competitive in-

hibitors of cytokinin-alanine synthase in vitro [74], cytokinins inhibited 2,4-D conjugation to amino acids in tissue culture [61], ABA stimulated formation of cytokinin-O-glucosides from free cytokinins in tobacco plants [110] and ipt gene expression in N. glutinosa tumours resulted in both cytokinin and auxin autonomy accompanied by IAA accumulation [2]. Interestingly, transgenic plants expressing the ipt gene under the control of an auxin-inducible SA UR gene promoter exhibited increased tolerance to the toxic effects of exogenously applied auxins and auxin transport inhibitors [48].

Indirect evidence supporting an important role for Zm-p60.1 in maize development comes from genetic studies. In maize, a single locus (Glul) coding for fl-glucosidase activity has been identified up to now, and a large number of alleles have been described at this locus [97]. As the Glul locus was characterized on the basis of fl-glucosidase activity found in coleoptiles and since only one fl-glucosidase has been identified in this material [25] it is likely that Glul contains the Zm-p60 gene family. Lines originally reported to carry null alleles have now been shown to contain the enzyme in amounts similar to that of normal genotypes, but in a poorly soluble complex that did not lend itself to the original zymo- gram assay method. Thus, of the numerous alleles that are known at the Glul locus, no null allele has yet been identified [24], supporting the proposition that the G L U 1 glucosidase activity is critical for maize development.

Conclusions

The study of bacterial enzymes that modify cytokinin metabolism has allowed progress to be made in elucidating the physiological roles of cytokinins in numerous plant systems, and has highlighted the potential importance of the conjugate pools. There has also been recent progress in identifying some of the enzymatic activities that are likely to prove central to the control of cytokinin synthesis, compartmentalization, and inactivation. These initial studies must now be fol-

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lowed through with purification of the enzymes themselves, and isolation of the corresponding cDNA clones, through genetics, T-DNA tagging, or classical biochemistry. With these tools in hand, perhaps the most urgent questions to ad- dress will be the tissue and subcellular localization of each activity, and the consequences of overexpression, ectopic expression or antisense expression of the cDNAs. In this way a picture should begin to emerge of the spatial and tempo- ral control of cytokinin levels in plants. This will provide a valuable basis for understanding the other important aspects of cytokinin action, namely its perception and signal transduction, two processes that are currently poorly understood.

Acknowledgements

B.B. is supported by a grant from the Grant Agency of the Czech Republic (204/93/0350) and K.P. by a grant from the DFG. We wish to thank Dr Csaba Koncz and Dr Christopher Redhead for critically reading the manuscript.

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