Control of cytokinin biosynthesis and metabolism

P.J.J. Hooykaas, M.A. Hall, K.R. Libbenga (Eds.), Biochemistry and Molecular Biology of Plant Hormones

0 1999 Elsevier Science B.V. A11 rights reserved

CHAPTER 6

Control of cytokinin biosynthesis and metabolism Eva Zaiimalovii and Miroslav Kaminek De Montjort University Norman Borlaug Centrefor Plant Science, Institute of Experimental Botany ASCR, Rozvojova 135, Prague 6, CZ 165 02, Czech Republic

Alena BEezinovii and Viiclav Motyka Institute of Experimental Botany ASCR, Rozvojova 135, Prague 6, CZ 165 02, Czech Republic

1. Introduction Together with auxins. cytokinins are key substances in hormonal regulation of plant development. The existence of cell division promoting substances was proven experimentally at the beginning of this century by Haberlandt [l] and later, in the fifties, the auxin:cytokinin model was proposed by Skoog and Miller [2] for regulation of morphogenesis in plants. Individual compounds exhibiting cytokinin-like biological activity were identified first in a non-plant source [3] and later in the milky endosperm of Zea mays [4,5]. In spite of the first native cytokinins being known for more than thirty years, the knowledge about their biosynthetic and metabolic pathways is still limited. This is particularly true of the biosynthesis of cytokinins in “normal”, i.e. non-transformed higher plant cells. This situation might partially reflect the existence of many (currently more than 40) native substances with more or less pronounced cytokinin activity. All native cytokinins are derivatives of adenine with at least one substituent (at N 6 position). According to this N 6 substituent, these compounds may be classed into ( I ) isoprenoid (zeatin, N 6-A2-isopentenyladenine and their derivatives), (2) isoprenoidderived (dihydrozeatin and its derivatives) and ( 3 ) aromatic cytokinins. Native cytokinins and their derivatives are summarised in Fig. 1 together with abbreviations used here.

2. Cytokinin biosynthesis In general, biosynthetic pathways are an integral part of the overall metabolism. Moreover, in some cases it is very difficult to distinguish exactly and unambiguously between “biosynthetic” and other “metabolic” reactions. In view of the hypothesis that free cytokinin bases are the true biologically active forms [6], the reactions resulting in the formation of key cytokinin bases (i.e. iPA, Z , DHZ and BA) are summarised in the part “Cytokinin biosynthesis”. All other processes leading to modifications and/or degradation of these compounds are included in the part “Cytokinin metabolism”. 141

142 Isoprenoid and isoprenoid-derived cytokhins: XI

CH3 -C e H 3

Xz

Abbreviation

X, X, X, Name H H H H

H

H H H G H H G H H H H

H

H H H H CH,S H

H H

H G H H

H H CHZOH H

H H H

Nh-(A2-isopenteny1)adenine N6-(A’4sopentenyl)adenosine R 2-methylthio-N6-(A2-isopentenyl)adenosine RP N6-(A2-isopenteny1)adenosine-5’-monophosphate H H N6-(A2-isopentenyl)adenine-3-glucoside G H N6-(A2-isopentenyl)adenine-7-glucoside H G N6-(A2-isopentenyl)adenine-9-glucoside

R

iPA iPAR MTiPAR iPARMP 1pa3g 1pa7g 1pa9g

~.

-G

C H 3

H H H H

trans-zeatin R rrans-zeatin riboside RP trans-zeatin-riboside-5‘-monophosphate H trans-zeatin-3-glucoside H trans-zeatin-7-glucoside G trans-zeatin-9-glucoside Ala lupinic acid

Z ZR ZRMP Z3G Z7G Z9G z9a1a

-&a2,+,H

‘“3

H

H H

H H H G

cis-zeatin cis-zeatin-9-glucoside

cis-z cis-Z9G

‘“2%

H H

H H

H H

H

R

trans-zeatin-0-glucoside rrans-zeatin-riboside-0-glucoside

ZOG ZROG

H H

H H

H H

H R

trans-zeatin-0-xyloside trans-zeatin-riboside-0-xyloside

ZROX

H H CH20H H H -‘OH3 H H H

H H H G H H H

H H H

dihydrozeatin dihydrozeatin riboside RP dihydrozeatin-riboside-5’-monophosphate

CH20G H

H H

CH20Xy H

H H

-cQcH3

“‘2y ‘’

-cQCH3 _,($CH.,

H

H

H

H

R

zox

DHZ DHZR DHZRMP DHZ3G DHZ7G DHZ9G DHZ9Ala

H

dihydrozeatin-3-glucoside

H

H

dihydrozeatin-0-glucoside

dihydrozeatin-riboside-0-glucoside

DHZOG DHZROG

H H

H

dihydrozeatin-0-xyloside dihydrozeatin-tiboside-0-xyloside

DHZOX DHZROX

G H dihydrozeatin-7-glucoside H G dihydrozeatin-9-glucoside H Ala dihydrolupinic acid

H

R

R

Fig 1 Cytokinins identified and confirmed in plants, scheme of structure, names and abbreviat~onsused in this chapter, data compiled from [74,7S,Sl,l00,179-lS4] R = P-D-nbofuranosyl group, RP = P-D-nbofuranosyl-5’-monophoaphate group, G = P-D-glucopyranosyl group, Xy = P-D-xylopyranosyl group and Ala=alanyl group Scheme of structure (XI-X,= substituents) NHXI N,

ti

511

y4

$J

143 Aromatic cytokinins: I _ _

___

-

XI

X,

X, X, X,

Name

Abbreviation

H

H G H H H

N6-benzyladenine N6-benzyladenine-3-glucoside N6-benzyladenine-7-glucoside N6-benzyladenine-9-glucoside

cH2-o / \

_____

OH

H H G H

H

H H R

Nb-benzyladenosine

BA BA3G BA7G BA9G BA9Ala BAR

H H H

H H H

H

H R G

N6-(ortho-hydroxybenzy1)adenine N‘-(ortho-hydroxybenzyl)adenosine N6-(ortho-hydroxybenzyl)adenine-9-glucoside

oOHBA oOHBAR oOHBA9G

H

H H H

H H H R H G

N6-(rneru-hydroxybenzyl)adenine N6-(metu-hydroxybenzy1)adenosine N6-(metu-hydroxybenzyl)adenine-9-glucoside

mOHBA mOHBAR mOHBA9G

H

H H

H

H H

H H G Ala

N6-benzyladenine-9-alanine

Fig. 1. Continued

2.1. De novo formation of isoprenoid and isoprenoid-derived cytokinins Two compounds common in plant metabolism are believed to be precursors of isoprenoid cytokinins in plants: adenosine-5 ’-monophosphate (AMP) and A’-isopentenylpyrophosphate (iPP). As a final product of the mevalonate pathway, the latter substance serves also as a precursor for a wide spectrum of metabolites including some other plant hormones, as abscisic acid, gibberellins and brassinosteroids. The hypothetical scheme of reactions resulting in the formation of iPA, Z and DHZ is given in Fig. 2. The “enzyme of entry” into isoprenoid cytokinin formation is A2-isopentenylpyrophosphate: 5 ’-AMP-A2-isopentenyltransferase (EC 2.5.1.8, trivially named “cytokinin synthetase”). This enzyme activity was first detected in a cell-free preparation from the slime mould Dictyostelium discoideum [7.8]. Later the enzyme from higher plants (cytokinin-independent tobacco callus [9,10] and immature Zea mays kernels [ l l ] ) was described and the data were recently summarised in 1121. The enzyme is very specific as far as the substrate is concerned 113,141: only the nucleotide AMP can be converted and only iPP (with a double bond in A2 position) may function as a side chain donor. 5’-Nucleotidase [15] followed by adenosine nucleosidase [16] are expected to be the enzymes responsible for the step-by-step conversion of the cytokinin nucleotide to the base iPA. Both of these reactions may proceed also in the opposite direction, and in this case they are catalysed by adenosine phosphorylase (ribosylation of iPA, [17]) and adenosine kinase (phosphorylation of iPAR, [ 18-20]). These enzymes are common in the mutual conversions of adenine and purine metabolites (reviewed in 1211) and their properties have been summarised by [22]. These enzyme activities seem to be the key for understanding the fate of ‘‘C-labelled adenine (Ade) and adenosine (Ado) in feeding experiments [summarised by 231. Z may be formed from iPA (and also ZR from iPAR) by hydroxylation of one of

144

terminal side chain methyl groups (enzyme trans-hydroxylase ?). In cauliflower microsomes this reaction is fully inhibited by CO and metyrapone, which indicates the involvement of cytochrome P-450 in the regulation of cytokinin metabolism [24]. Further studies on transgenic Nicotiana tabacum calli expressing the ipt gene indicated that in this system trans-hydroxylation may preferentially proceed at the nucleotide level 1251. The enzyme converting Z to DHZ, zeatin reductase, was characterised in Phaseolus vulgaris embryos [26]. The reduction proceeds only in the presence of NADPH and the enzyme is very specific, with the highest affinity for Z (cZ, ZR, iPA, iPAR are not substrates, see also part 3.1 .l. of this article). Taking into account that bases of isoprenoid cytokinins may represent the physiologically active forms of cytokinins [6], the conversion of Z to DHZ by zeatin reductase may prevent the loss of cytokinin activity caused by degradation of Z (but not that of DHZ, cf. part 3.1.5. of this article) by cytokinin oxidase. Significant in this context are feeding experiments with labelled Ade and Ado [23 and references therein]: after 3H-Ade(Ado) application to plant tissues, Z and ZR were

Fig. 2. Hypothetical scheme of de novo formation of isoprenoid and isoprenoid-derived cytokinins in plants; modified according to [I 151. Numbers refer to the individual enzymes andor enzyme activities: 1 = A’-isopentenylpyrophosphate:S’-AMP-A%sopentenyltransferase (“cytokinin synthetase”) 2 =5 ’-nucleotidase activity

3 =adenosine kinase 4 =adenosine nucleosidase 5= adenosine phosphorylase 6=trans-hydroxylase activity (7) 7=zeatin reductase

145

preferentially accumulated, while almost no label was incorporated into iPA and iPAR [recently in 27,281. This indicates that in plants cytokinins may be also formed in another way, as e.g. by the attachment of already hydroxylated iPP to AMP. There are Arabidopsis thaliana (ampl, [29]) and Physcomitrella patens (ove, [30]) mutants showing an altered cytokinin accumulation, perhaps due to changes in the biosynthetic pathway. In Arabidopsis ampl the product of the amp1 gene, AMP1, is suggested to regulate the isopentenyltransferase-like enzyme and maybe also the hydrolysis of cytokinins from their conjugates. The use of plant hormone mutants in phytohormone research is discussed elsewhere in this issue.

2.1 . I . Micro-organisms Paradoxically, the enzyme involved in the first step in the biosynthesis of isoprenoid cytokinins is known in detail not from plants but from bacteria. The Agrobacterium tumefaciens Ti plasmid contains genes for the biosynthesis of both auxin (genes 1 and 2) and cytokinins (gene 4, ipt). The ipt gene product, isopentenyltransferase, catalyses the formation of iPARMP from iPP and AMP [31-331, i.e. it possesses the same activity as A‘4sopentenylpyrophosphate : 5‘-AMP-A’-isopentenyltransferase (EC 2.5.1.8) described earlier in slime mould and tobacco (see above). The ipt gene was sequenced [34-36] and the properties of the product, the IPT enzyme, were described [37]. By free-living A. tumefaciens an isopentenyltransferase is expressed, which is encoded by the tzs gene of the virulence region of nopaline-type Ti plasmids [ 131 and which is probably responsible for high secretion of Z in response to certain plant phenolics released after wounding. In fact, “cytolunin-producing” genes are relatively frequent also in other prokaryotes (e.g. Agrobacterium rhizogenes, Pseudomonas syringae pv. savastanoi, Pseudomonas solanacearum, Azotobacter chroococcurn, Erwinia herbicola pv. gypsophilae, Rhodococcus fascians, etc., reviewed in [38,39]) and so these micro-organisms often produce cytokinins, sometimes of rather unusual structures (e.g. 1‘-methyheatin and its riboside with a methyl group in the 1’-position of the isoprenoid side chain, and zeatin 2’-deoxyriboside with a hydrogen atom instead of OH-group in the 2’-position of the p-Dribofuranosyl group, detected in Pseudomonas syringae pv. savastanoi, summarised in 1401). The investigation of these non-plant genes cannot directly contribute to the understanding of cytokinin biosynthesis in plants, but it may provide (and now indeed provides) a useful tool for manipulation of the plant genome and consequently of cytokinin biosynthesis in transgenic plants. 2.1.2. Transgenic plants Remarkably increased endogenous levels of cytokinins (mainly Z-derivatives) were reported in crown gall tissues having the T-DNA from the Ti plasmid of Agrobacterium tumefaciens incorporated in the genome [41-47]. The ipt gene from Agrobacterium tumefaciens was also introduced into the plant genome under its own or alternative promoter control in many laboratories [reviewed in 48-5 I]. Predominantly Z-type cytokinins were usually accumulated in transformed plants; this may reflect the rapid stereospecific hydroxylation of IPARMP, iPAR andor iPA to ZRMP, ZR and Z, respectively. This corresponds to the results of some I4C-Adefeeding experiments in Vinca

146

rosea crown-gall tissue [52,53], where only labelled Z-type compounds were found. However, in some cases also iPA and iPAR levels [54-561 increased significantly. When dealing with transgenic plants one should take into account the potential existence of a “plant” cytokinin-biosynthetic pathway different from the “bacterial” ipt encoded pathway. This hypothetical pathway may participate in the accumulation of cytokinins in transformed plants; however, this is generally ignored. 2.1.3. tRNA as a possible source of free cytokinins In addition to free cytokinins, cytokinin moieties also occur as constituents of some tRNA species of a wide range of organisms including plants [57]. They are located at the strategic 37 position adjacent to the 3’-end of the anticodon [58]. In contrast to the formation of free cytokinins the biosynthetic pathways of tRNA cytokinins are well understood. The first step in their formation is post-transcriptional isopentenylation of Ade37using iPP and unmodified tRNA as substrates. This reaction is catalysed by A2isopentenylpyrophosphate : tRNA-A*-isopentenyltransferase (EC 2.5.1 .8) which was partially purified from yeast [59], E. coli [60] and corn [61]. This enzyme is encoded by E. coli miaA and yeast MOD5 genes which were sequenced and show significant homology with Agrobacterium tumefaciens miaA gene [62,63]. The isopentenylated Ade37 may be further modified by hydroxylation of one of the side chain methyl groups. In plant tRNAs the cis-methyl group is preferentially hydroxylated to yield the cis-isomer of Z ([23] and references therein). tRNA cytokinins have two different functions, viz. (1) as regulators in tRNA operation during protein synthesis and (2) as potential precursors of free cytokinins. The proposed regulatory role of tRNA cytokinins in protein synthesis was supported by experiments with bacterial and yeast mutants lacking the cytokinin moiety at Ade37,resulting in the suggestion that cytokinins in tRNA enhance tRNA translational efficiency [64,65]. As far as the cytokinin donor function is concerned there are indications that tRNA cytokinins may contribute to the pool of free cytokinins. Based on pulse-chase experiments with labelled cytokinin precursors it was estimated that 40-50% of the free cytokinins in plant cells may be of tRNA origin [66,67]. However, there are serious limitations to tRNA as a possible source of free cytokinins: (1) As compared with bacteria, plant tRNAs contain very limited amounts of cytokinins, ( 2 ) “cytokinin” moieties in some plant tRNAs (tRNAphe) consist of hypermodified nucleosides which support the operation of tRNA in protein synthesis but axe not active as cytokinins, (3) there are some cytokinins (BA-type) in plants which are not constituents of tRNA and cannot be derived from isoprenoid tRNA cytokinins 168, 691, (4) plant tRNAs contain cis-Z as a predominant “cytokinin” moiety which almost lacks cytokinin activity.

However, the existence of a cis-trans-isomerase [22] (see part 3.1.2. of this article) catalysing the interconversion between the cis- and trans-isomers of Z may support the function of tRNA as a supplementary source of free cytokinins in plants.

147

2.2. Formation of aromatic cytokinins BA and its derivatives, originally considered only as synthetic substances exhibiting cytokinin activity, were later found as native cytokinins in plants, namely in Populus robusta leaves, first in the early seventies [70,71], later also in other plant species in the eighties (Zuntedeschia aethiopica fruits [72,73], Pimpinella anisum cell culture [74], primary tomato crown gall tumour [75]), and in the nineties (Populus x canadensis Moench cv. Robusta [76,77], Elaeis guinernsis [78]). BA-derivatives were frequently detected in plants as products of exogenous BA metabolism and/or uptake from culture medium (recently e.g. [79,80], reviewed in [Sl]). To date there is no report about the biosynthesis of aromatic cytokinins. In view of the dissimilarity between the aromatic and the isoprenoid(-derived) N 6 side chains it is likely that their biosynthetic pathways are quite different. Phenylalanine may be considered as a starting compound and benzaldehyde and/or hydroxylated benzaldehydes as immediate side chain precursors. However, the existence of some “crossing-points” between aromatic and isoprenoid side chain formation cannot be completely excluded. There is also the possibility that the enzymes of adenine and/or purine metabolism, which are not strictly specific, may catalyse some mutual conversions among BA-bases, nucleosides and nucleotides [81].

3. Cytokinin metabolism Cytokinin metabolism is very complex and reflects the existence of many different native compounds, sometimes not very close in their structure, but possessing a varying degree of cytokinin-like biological activity. In terms of reaction type, cytokinin metabolism includes mainly mutual conversions among cytokinin bases, ribosides and ribotides (i.e. riboside-5’-monophosphates), conjugation and conjugate-hydrolysing reactions and degradative (i.e. oxidation) reactions. All these reactions and their regulations are very important in view of the very different relative biological activity of individual cytokinin derivatives (structure-activity relationships are discussed elsewhere in this Book). Fig. 3 lists cytokinin-metabolising reactions in relation to the part of cytokinin molecule affected. 3.1. Reactions resulting in Nbside chain modijication

N b side chain substitution (i.e. introduction of an X , substituent into the molecule of Ade, cf. Fig. 1) is what converts the precursor compounds into true cytokinins. Thus, reactions leading to changes in this part of the molecule are more or less specific for cytokinins and are of remarkable physiological significance. 3.1.1. Side chain reduction The enzyme zeatin reductase, responsible for the reduction of the side chain double bond in zeatin and subsequent formation of dihydrozeatin, was partially purified from Phaseolus vulgaris embryos and characterised [26]. As already mentioned in part 2.1. of

148

Scheme of cytokinin structure

Part of molecule affected

Type of reaction

N6-sidechain

0-glucosylation 0-xylosylation 0-acetylation

NHX, I

Substituent affected

reduction cis-trans isomerisation

degradation

I x5

x3

purinering

ribos ylation phosphorylation phosphoribosylation N-glucosylation N-alanine-conjugation

~~

Fig. 3 .

Summary of cytokinin-metabolising reactions in plants

this chapter, the enzyme is very specific for Z, which implies that the reaction may proceed only at the free base level. This seems to be in contrast with the earlier observations [82] that both ZRMP and DHZRMP levels increased rapidly after 3H-ZR application on soybean explants. The question is whether these different results are due to the different specificity of the respective enzymes in Glycine and Phaseolus, or whether there is another reductase not so strictly specific.

3.1.2. Cis-trans isomerisation The existence of the enzyme catalysing the conversion between cis- and trans-isomers of zeatin is the prerequisite for possible involvement of tRNA as a source of free cytokinins (cf. part 2.1.3. in this chapter). Indeed, the cis-trans-isomerase was isolated and partially purified from the endosperm of immature Phaseolus vulgaris seeds. The reaction may proceed in the presence of FAD or FMN cofactors and light in both directions, but the conversion of the cis- to the trans-isomer is preferred. The enzyme seems to be a glycoprotein and is specific for both free bases (Z, cis-Z) and their ribosides [83,84]. 3.1.3. Side chain conjugation and hydrolysis of the side chain substituents Side chain conjugations comprise the formation of 0-glycosides (glucosides and xylosides) and 0-acetyl-derivatives. It is evident that these conjugates may be formed only from cytokinin derivatives bearing a hydroxyl-group in the side chain, i.e. from Z, DHZ, and OH-derivatives of BA. The 0-acetyl-conjugates were encountered only infrequently in plants, they were detected as 0-acetyl-ZRMP and 0-acetyl-DHZRMP in Lupinus angustifolius after

149

application of radiolabelled ZR and DHZ [85] and later as naturally occurring substances in plant tumours [86]. In contrast, the 0-glycosyl-conjugates are common if not prevailing forms of cytokinins in plants irrespective of species, plant organs and phase of development. These compounds were first identified as products of feeding experiments and, almost in parallel, as native compounds in the seventies and early eighties (e.g. [87-911, first review in [92], recent ones in [22,81,931). In the 0-glycosyl-derivatives of cytokinins two saccharide moieties are known to be bound to the aglycone: the hexose glucose and the pentose xylose, both in P-D-pyranoside forms. The enzymes catalysing 0-glycosylation were characterised in Phaseolus species: UDP-xylose :zeatin 0-xylosyltransferase (EC 2.4.2.-) from Phaseolus vulgaris [94] and 0-glucosyltransferase from Phaseolus lunatus seeds [95]. Both enzymes were isolated and studied in detail. They possess similar physico-chemical properties but they differ in substrate specificity: the former recognises Z and DHZ as substrates and only UDPX may serve as the donor of the saccharide moiety while the latter requires only Z as substrate and both UDPG and UDPX as the source of the saccharide substituent (reviewed in [22,93]). In view of the strict substrate specificity of the 0-glucosyltransferases there is an open question how the recently predicted [81] and very recently detected [80] O-glucosylderivatives of BA (namely m-0-glucosylBA and its riboside) are formed. The conjugation of cytokinins via 0-glucosylation is a reverse process; ubiquitous enzymes possessing a P-glucosidase-like activity are responsible for the cleavage of these cytokinin conjugates. The hydrolysis of cytokinin-0-glucosides was suggested andor detected in various plants, e.g. Glycine max, Lupinus luteus, Phaseolus vulgaris, Knca rosea and Zea mays ([96-991, summarised [in 501). It is still not clear whether cytokinin-0-glucosides do possess high physiological activity per se [83,100] or due to the immediate (e.g. reference [loll) P-glucosidasecontrolled cleavage resulting in free cytokinin bases and/or ribosides. At any rate, the metabolic system of 0-glucosyltransferase/P-glucosidase seems to be very significant in the regulation of physiological activity of cytolunins during plant development, and cytokinin-0-glycosides are candidates for cytokinin transport and storage forms. 3.1.4. Methylation The unusual cytokinins 1'-methylzeatin and its riboside, both with a methyl group instead of hydrogen atom in C'-position of the side chain, were identified in the plant pathogenic bacteria Pseudomonas syringae subsp. savastanoi and P. amygdali ([40] and references therein, [102]). These compounds have not yet been detected in plants and nothing is known about the path(s) of their formation. 3.1.5. Degradation Cytokinin degradation via N 6 side chain cleavage is another process regulating levels of biologically active cytokinins in plant cells. Unlike other metabolic steps the cleavage of the N 6 side chain from the cytokinin molecule results in an irreversible destruction of cytokinin structure. which is of course associated with a complete loss of biological activity.

150

4-

Fig. 4. The scheme of cytokinin oxidase reaction.

The existence of an enzyme activity catalysing cytokinin degradation in plants was first demonstrated in crude homogenates from cultured tobacco cells [ 1031. Subsequently, the enzyme was characterised in a number of higher plants (reviewed in [104,105]) and named cytokinin oxidase [106]. The presence of cytokinin oxidase activity was also reported in moss protonema [107], cellular slime moulds [lo81 and yeast [109]. Cytokinin oxidase seems to be a copper-containing amine oxidase (EC 1.4.3.6, [lOS]) catalysing specifically the N side chain cleavage of isoprenoid cytokinins, releasing Ade or its derivatives and the corresponding side chain aldehyde in the presence of molecular oxygen (Fig. 4). Naturally occurring substrates of cytokinin oxidase are iPA, Z and their ribosides, N-glucosides and N-alanyl conjugates. Cytokinins bearing saturated N 6 side chains (DHZ-type cytokinins), bulky substituents on the side chain (0-glucosides, aromatic cytokinins and kinetin, with two reported exceptions [107,1lo]) and cytokinin nucleotides are not degraded by the enzyme (e.g. [110-1141). In spite of very similar substrate specificities, cytokinin oxidases from various plant species differ markedly in their molecular weight, pH optima and kinetic constants (reviewed in [104,115]). These differences may be caused in part by a various degree of protein glycosylation [113,116], which may also affect compartmentation and excretion of the enzyme in plant cells and, subsequently, the access of the substrate to the enzyme. Isolation and sequencing of the cytokinin oxidase gene has not been successful so far although antisera have been raised against the purified maize enzyme [ 1171 and used to isolate a hgtll clone carrying a part of the cytokinin oxidase gene [118]. With the exception of two other preliminary notes [119,120] no further progress in cloning of the cytokinin oxidase gene has as yet been reported. 3.2. Reactions resulting in the modijication of the purine ring 3.2.1. Mutual conversions among cytokinin buses, ribosides and ribotides

These interconversion reactions, i.e. (de-)ribosylation, (de-)phosphorylation and phosphoribosylation in position N 9 on the purine ring are analogous to those known from the basic metabolism of adenine and purine. The enzymes were isolated from plant sources and partially characterised (reviewed recently in [22,81,93]). Some of them take part also in biosynthetic reactions and were already mentioned in part 2.1. of this chapter.

151

Generally, all conversions in the “biosynthetic” direction, i.e. iPARMP+ iPAR +iPA (catalysed by 5‘-nucleotidase, (EC 3.1.33, and adenosine nucleosidase, (EC 3.2.2.7), respectively, cf. Fig. 2) may also proceed in the opposite direction, i.e. basenucleoside nucleotide (catalysed by adenosine phosphorylase and adenosine kinase, respectively). All these enzymes require both Ade and iPA or Ado and iPAR, respectively, as substrates. They were characterised in wheat germ [15-18] and lupin seeds [19]. Interestingly, no K,-constants were reported for Z-type cytokinins (see summary in 1221). However, as seen in 3H-labelIed Z-derivatives feeding experiments, Z-type cytokinins are also interconverted in a similar way [82,121,122]. Moreover, the specificity of these enzymes is not too strict with respect to the N 6 side chain configuration and one may speculate that this complex may function for most if not all native cytokinins [21,81]. One more enzyme belongs to this system, converting a free base directly into a riboside5’-monophosphate (adenine phosphoribosyltransferase, EC 2.4.2.7). The enzyme partially purified from wheat germ [I231 converted iPA into iPARMP; moreover, the crude enzymes extracted from Arubidopsis thaliana and Lycopersicon esculentum plants were able to convert also BA into BARMP [124,125, respectively].

-

-

3.2.2. Conjugation on purine ring and hydrolysis of purine ring substituents These types of cytokinin-modifying reactions consist of (de-)ribosylation in position N9, glucosylation and conjugate hydrolysis in positions N3, N 7 and N9, and formation of alanyl-conjugates and their hydrolysis in position N9 of the purine ring. (De-)ribosylation reactions are briefly summarised in paragraphs 2.1. and 3.2.1. 3-, 7- and 9-Glucosides of both isoprenoid(-derived) and aromatic cytokinins are ubiquitous in many plants and were detected also as products of various feeding experiments. Unlike the cytokinin-0-glycosides, these compounds feature an N-glycosidic bond. The formation of 7- and 9-glucosides of BA was studied in detail in radish cotyledons [126,1271. Two proteins possessing glucosyltransferase activity were detected and the more abundant one, named cytokinin-7-glucosyltransferase, was further characterised. The enzyme is specific for highly active cytokinins (Z, BA), but also for DHZ, cis-Z and, in spite of its name, it catalyses to a lesser extent also formation of 9-glucosides. UDPG and also TDPG may function as donors of the glucosyl moiety. The array of N-glucosylation derivatives depends on the type of assay (in vivo vs. in vitro), plant material, type of labelled cytokinin applied, and other factors. The presence in plants of another enzyme(s) possessing this type of activity cannot be excluded. Cytokinin 7- and 9-glucosides are biologically relatively inactive [ 128-1301 and they are not substrates for plant P-glucosidases [130,131]. Thus they are proposed to be detoxification or simply inactivation products [92]. In contrast, 3-glucosyl-derivatives of Z and BA can be cleaved by these enzymes to corresponding biologically active cytokinin bases [130--132] and also possess some biological activity per se [130]. Nothing is known about the enzyme responsible for N3glucoside formation in plants. With respect to their possible turnover in plants these cytokinin conjugates may be considered as cytokinin storage forms [ 1301. N9-Alanyl derivatives of Z and DHZ (lupinic and dihydrolupinic acids, Z9Ala and DHZ9Ala, respectively) were identified as minor native cytokinins in Lupinus luteus [ 1331 and, together with BA9Ala, as metabolic products of Z and BA, respectively, in legumes

152

[92,97]. Formation of these amino acid conjugates is catalysed by p-(9-cytokinin)-alanine synthase, classified as C-N ligase, and characterised in lupin seeds [134]. The enzyme requires 0-acetylserine as donor of the alanine moiety and recognises all main cytokinin bases (including BA) and many other purine derivatives as substrates. Similarly to cytokinin 7- and 9-glucosides, also cytokinin 9-alanyl derivatives are biologically inactive and metabolically stable, and are therefore candidates to be cytokinin-inactivation and detoxification products.

4. Mechanisms of regulation of cytokinin metabolism in plants There is no doubt that the endogenous cytokinin levels are precisely regulated in plants with respect to such developmental events as cell and growth cycles of cell cultures [ 135-1371, morphogenic response in tissue culture [ 1381, somatic embryogenesis [ 139-1411 and organ development (e.g. [ 142-1481) including floral induction [ 149-1531, Also environmental factors such as light (e.g. [144]), various stresses (e.g. [154]) and nutrition conditions affect the endogenous cytokinin content. It should be mentioned that only the momentary contents of cytokinins can be experimentally monitored (as total sum or as levels of individual compounds) and these data result from a number of contributions of several, sometimes actually antagonistic, metabolic processes. 4.1. Control of cytokinin metabolism in plant cell There are several regulatory elements taking part in the control of cytokinin metabolism (including biosynthesis) in plant cells and consequently affecting the momentary ratio between active cytokinins and their metabolites exhibiting low or no cytokinin activity. The cytokinins themselves and other phytohormones (auxins in particular) belong amongst the most frequently investigated regulatory factors. It is obvious that their action(s) are enzyme-mediated. A scheme of mutual regulations among exogenous cytokinins and auxins, “pool” of intracellular cytokinins and consequent physiological responses is proposed in Fig. 5. A model describing the regulation of the dynamics of cytokinin levels and its function in control of physiological processes in plant cells is described elsewhere [ 1551. The complete mosaic of processes leading to ultimate “cytokinin homeostasis” in plant cells should be supplemented with data on the compartmentation of both cytokinins themselves (reviewed in [ 1551) and the enzymes and hypothetical carriers responsible for cytokinin modifications and uptakeitransport, respectively. 4.1.I . Regulation of individual enzymes in cytokinin metabolism Cytokinin degradation seems to be a very important tool for regulation of the active cytokinin “pool” in plant cells. Cytokinin oxidase activity in plant cells is subject to multiple control (reviewed in [ 104,1551). Most of the control mechanisms depend directly on the concentration and/or compartmentation of the cytokinins in the cell. Cytokinin degradation in plant cells is significantly enhanced in vivo after their exposure to exogenous cytolunins [ 156,1571. This phenomenon is probably mediated via

153

the promotion of cytokinin oxidase activity in response to both substrate and non-substrate exogenous cytokinins [ 112,113,1581. Recent studies revealed that cytokinin oxidase activity may be enhanced also by endogenous cytokinins over-produced in the cells transformed by the cytokinin biosynthetic ipt gene expressed from its native [25] or conditionally-induced promoter [159,160]. These data suggest a substrate induction of cytokinin oxidase activity which may contribute to hormone homeostasis in plant cells. Differences in glycosylation of the enzyme and consequent differences in both subcellular compartmentation and excretion of the protein may represent additional mechanisms controlling cytokinin degradation. Two molecular forms of cytokinin oxidase differing in their pH optima and glycosylation patterns were identified in cultured tissues of two Phaseolus species [113] and tobacco cultivars [116] indicating a different intracellular localisation of the individual cytokinin oxidase iso-forms. Genotypic

\ \ \

I

// /

/

+?

INTRACELLULAR CYTOKININS

?

I

I

AUXINS

v

I'

b'

'.

/*

\

\,? \

\

AUXINS

Physiological response(s)

Fig. 5. Hypothetical model of regulations in cytokinin metabolism.

The way of contribution of individual metabolic processes and/or cytokinin derivatives to the "pool" of intracellular cytokinins is indicated by ___ b, the regulatory action (positive or negative, + and -, respectively) is represented by ----b,the action of cytokinins on physiological processes is figured as

b.

154

variation in the enzyme properties was correlated with different ability to degrade cytokinins in two Phaseolus lines [161]. That is why the substrate specificity of cytokinin oxidase and the access of substrate to the enzyme should be considered as other aspects of the control of cytokinin degradation. However, many naturally occurring and synthetic cytokinins that are not recognised as substrates by cytokinin oxidase (e.g. DHZ-type and aromatic cytokinins) are degraded in vivo by the N6 side chain cleavage in a number of plant tissues (reviewed in [81,92,93,104]). Degradation of such cytokinin-oxidase-non-substrate cytokinins may be attributed to an as yet unknown separate enzyme system which makes the control of cytokinin catabolism even more complex. The “0-conjugationhydrolysis” system together with N-conjugation represent different ways how to regulate endogenous cytokinin levels ([22,50,115], cf parts 3.1.3. and 3.2.2. of this chapter). In addition to cytokinin degradation the formation of both cytokinin 0and N-conjugates is perhaps the common way how to balance the overproduction of cytokinins in transgenic plants [46,162,163].

4.1.2. Regulation of cytokinin accumulation by cytokinins themselves A significant increase of endogenous isoprenoid and isoprenoid-derived cytokinin levels after treatment with both native-like aromatic (BA-type) and synthetic heterocyclic (kinetin) and urea-type (e.g. thidiazuron) cytokinins was observed in different plant species (Nicotiana sp. [ 160,164-166) and Beta vulgaris 11671). Because ( I ) the applied cytokinins could not be converted into isoprenoid(-derived) cytokinins due to their quite different structure, and because ( 2 ) the response was very fast, one might speculate that the isoprenoid(-derived) cytokinin increase was partially due to their de novo synthesis. The enhancement of ipt gene expression in transformed tobacco callus after BA application [25] supports such opinion. These findings are in agreement with the hypothesis that the cell competence for cytokinin autonomy is associated with increased endogenous cytokinin levels and that the maintenance of this autonomy is based on a positive feedback when cytokinins either induce their own accumulation or inhibit their own degradation [ 1681. 4.1.3. Regulation of cytokinin accumulation by other phytohormones

Endogenous cytokinin levels in plant tissues are undoubtedly regulated by other plant hormones; in particular, the role of auxin(s) in the control of cytokinin metabolism has been summarised in several recent reviews [104,155,169]. In contrast to the effect of exogenous cytokinins (see 4.1.2) an increase of the auxin concentration either exogenously applied [25,54,170] or resulting from expression of auxin biosynthetic genes in transgenic plant tissues [171] resulted in a significant decrease of endogenous cytokinin levels. On the other hand, induced or enhanced free cytokinin accumulation has been reported after partial or total auxin deprivation 1137,1651 or inactivation of auxin synthesising genes in transformed plants [46,172]. The regulatory effect of auxin(s) on cytokinin metabolism seems to be transient and its duration corresponds to the period required for an induction of certain developmental process(es) [ 1731. Down-regulation of cytokinin concentration by auxin(s) in plant cells is supposed to function either directly at the level of cytokinin biosynthesis [ 174,1751 andor indirectly

155

as promoted metabolic cytokinin inactivation, either by N-glucosylation or through oxidative degradation [25,170,1711. Although the stimulation of cytokinin catabolism by auxin(s) in vivo has been reported for several plant systems, data concerning auxin effects on cytokinin oxidase activity in vitro are highly contradictory and depend on assay conditions [25,158,170,17 11. Regulatory links between cytokinin metabolism and other plant hormones (abscisic acid, ethylene) include both synergistic and antagonistic interactions and have been described in a number of plant tissues [176-1781. In spite of the rather scant present knowledge, it is evident that the balance between such synergistic and antagonistic relationships is the dominating principle of integral hormone action in plants.

5. Conclusion The current knowledge of the biosynthesis and metabolism of cytokinins derives from the level of the methods employed for cytokinin extraction, determination and identification, and methods for isolation and characterisation of appropriate enzymes. Further development of the methods (miniaturisation, enhanced detection sensitivity and specificity) is expected to bring improvements of our knowledge, and to offer new possibilities (e.g., LC-MS or GC-MS combinations are highly promising for metabolic studies). The genetic approach to the study of metabolism, above all the use of transformants and mutants, opens new vistas for research, in particular for deciphering the regulatory mechanisms of metabolism and its dynamics. The use of mutants for the study of cytokinin metabolism is somewhat limited because, owing to the indispensable role of cytokinins in the regulation of key processes of plant development, many mutations in genes encoding enzymes of cytokinin metabolism may be lethal. Ideally, the study of cytokinin metabolism should bring to the fore an association of analytical and biochemical techniques with state-of-the-art cytological approaches, especially with in situ immunolocalisation of cytokinins and molecules reacting with them, as well as of enzymes of cytokinin metabolism. In the future, this approach should facilitate the elucidation of biochemical and physiological processes not only under static conditions (as is the case for most current studies), but dynamically as a real-time biomolecular interaction analysis.

Acknowledgements The authors appreciate the support of their research work by the Grant Agency of the Academy of Sciences of the Czech Republic (project No.: A6038706), by the Grant Agency of the Czech Republic (projects No.: 206/96/K188 and 522/96/K186) and by the Volkswagen Stiftung (project U72076.).

References [l] Haberlandt. G. (1913) Sitzungsber. K. Preuss M a d . Wiss.. 318-345. [2] Skoog, F. and Miller, C.O. (1957) Symp. SOC.Exptl. Biol. No XI, 118-140

156 [3] Miller, C.O., Skoog, F., Okomura, F.S., von Saltza, M.H. and Strong, F.M. (1956) J. Am. Chem. Soc. 78, 1345-1350. [4] Letham, D.S. (1963) Life Sci. 8, 569-573. [5] Miller, C.O. and Witham F.H. (1963) Regul. Nat. Croissance Vegetale, Colloques Intemationaux du Centre National de la Recherche Scientifique, p. 123. [6] Laloue, M. and Pethe, M. (1982) In: P.F. Wareing, (Ed.), Plant Growth Substances 1982. Academic Press, London, pp. 185-195. [7] Taya, Y., Tanaka,Y. and Nishimura, S. (1978) Nature 271, 545-547. [XI Ihara, M., Taya, Y., Nishimura, S. and Tanaka, Y. (1984) Arch. Biochem. Biophys. 230, 6 5 2 4 6 0 . [9] Chen, C.M. and Melitz, C.K. (1979) FEBS Lett. 107, 15-20. [lo] Chen, C.M. (1982) In: P.F. Wareing (Ed.), Plant Growth Substances 1982. Academic Press, London, pp. 155-164. 11 11 Blackwell, J.R. and Horgan, R. (1994) Phytochemistry 35,339-342. 1121 Chen, C.M. and Ertl, J.R. (1994) In: D.W.S. Mok and M.C. Mok (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, London, Tokyo, pp. 81-85. [13] Moms, R.O.. Blevins, D.G., Dietrich, J.T., Durley, R.C., Gelvin, S.B., Gray, J., Hommes, N.G., Kaminek, M., Mathews, L.J., Meilan, R., Reinbott, T.M. and Sayavedra-Soto, L. (1993) Aust. J. Plant Physiol. 20, 62 1-637. [14] Koshimizu, K. and Iwamura, H. (1986) In: N. Takahashi (Ed.), Chemistry of Plant Hormones. CRC Press, Boca Raton. pp. 153-200. [l51 Chen, C.M. and Kristopeit, S.M. (1981) Plant Physiol. 6 7 ,4 9 4 4 9 8 . [I61 Chen, C.M. and Kristopeit, S.M. (1981) Plant Physiol. 68, 1020-1023. [171 Chen, C.M. and Petschow, B. (1978) Plant Physiol. 62, 871-874. [ 181 Chen, C.M. and Eckert, R.L. (1977) Plant Physiol. 5 9 ,4 4 3 4 4 7 . [19] Guranowski, A. (1979) Arch. Biochem. Biophys. 196,220-226. [201 Faye, F. and Floc’h, F. (1997) Plant Physiol. Biochem. 35, 15-22. [21] Burch, L.R. and Stuchbury, T. (1987) Physiol Plant. 69, 283-288. [22] Mok, D.W.S. and Martin, R.C. (1994) In: D.W.S. Mok and M.C. Mok (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, London, Tokyo, pp. 129-137. 1231 Prinsen, E.. Kaminek, M. and Van Onckelen, H. (1997) Plant Growth Regul. 23, 3-15. [24] Chen, C.M. and Leisner, S.M. (1984) Plant Physiol. 7 5,4 4 2 4 4 6 . 1251 Zhang, R., Zhang, X., Wang, J., Letham, D.S., McKinney, S.A. and Higgins, T.J.V. (1995) Planta 196, 84-94. 1261 Martin, R.C., Mok, M.C., Shaw, G. And Mok, D.W.S. (1989) Plant Physiol. 90, 1630-1635. 1271 Hocart, C.H. and Letham, D.S. (1990) J. Exp. Bat. 41, 1525-1528. 1281 Van Staden, J. and Drewes, F.E. (1993) J. Exp. Bot. 44, 1411-1414. [291 Chin-Atkins, A.N., Craig, S., Hocart, C.H., Dennis, E.S. and Chaudhury, A.M. (1996) Planta 198, 549-556. 1301 Wang, T.L. (1994) In: P.J. Davies (Ed.), Plant Hormones. Kluwer Academic Publishers, The Netherlands, pp. 255-268. [31] Akiyoshi, D.E., Klee, H., Amasino, R.M., Nester, E. and Gordon, M.P. (1984) Proc. Natl. Acad. Sci. USA 81,5994-5998. [321 Barry, G.F., Rogers, S.G., Fraley, R.T. and Brand, L. (1984) Proc. Natl. Acad. Sci. USA 81, 4776-4780. 1331 Buchman, I., Marner, F.J., Schroder, G., Waffenschmidt, S. and Schroder, J. (1985) EMBO J. 4, 853-859 [341 Heidekamp, F., Dirkse, W.G., Hille, J. and Van Ormondt, H. (1983) Nucl. Acids Res. 1I , 621 1-6223. [351 Goldberg, S.B., Flick, J.S. and Rogers, S.G. (1984) Nucl. Acids Res. 12,46654677. [361 Lichtenstein, C., Klee, H.J., Montoya, A., Garfinkel, D.J., Fuller, S., Flores, C., Nester, E.W. and Gordon, M.P. (1984) J. Mol. Appl. Genet. 2, 354-362. [371 Blackwell, J.R. and Horgan, R. (1993) Phytochemistry 34, 1477-1481. [381 Frankenberger, W.T. Jr. and Arshad, M. (1995) Phytohormones in Soil: Microbial Production and Function. pp. 503. Marcel Dekker, Inc., New York, Basel, Hong Kong. (391 Moms R.O. (1995) In: P.J. Davies (Ed.), Plant Hormones. Kluwer Academic Publishers, The Netherlands, pp. 318-339.

157 [401 Evidente. A., Fujii, T., lacobellis, N.S., Riva, S., Sisto, A. and suic0, G. (1991) phytochemistry 30, 35053510. [411 Miller, C.O. (1974) Proc. Natl. Acad. Sci. USA 71, 334-338. [421 Einset, J.W. (1980) Biochem. Biophys. Res. Commun. 93, 510-515. [43] Scott, I.M., Browning, G. and Eagles, I. (1980) Planta 147, 269-273. 1441 Weiler, E.W. and Spanier, K. (1981) Planta 153, 326337. [451 Scott, I.M. and Horgan, R. (1984) Planta 161, 345-354. [46] McGaw, B.A., Horgan, R., Heald, J.K., Wullems, G.J. and Schilperoort, R.A. (1988) Planta 176, 230-234. [47] Estruch, J.J., Prinsen, E., Van Onckelen, H, Schell, J. and Spena, A. (1991) Science 254, 13641367. [481 smigocki, A.C. (1991) Plant Mol. Biol. 16, 105-1 15. [49] Hamill, J.D. (1993) Aust. J. Plant Physiol. 20, 405423. 1501 BrzobohatL, B., Moore, I. and Palme, K. (1994) Plant Mol. Biol. 26, 1483-1497. [511 Klee. H.J. and Lanahan, M.B. (1995) In: P.J. Davies (Ed.), Plant Hormones. Kluwer Academic Publishers, The Netherlands, pp. 340-353. [52] Stuchbury, T.L., Palni, L.M.S., Horgan, R. and Wareing, P.F. (1979) Planta 147, 97-102. [531 Palni, L.M.S., Horgan, R., Darral, N.M., Stuchhury, T. and Wareing, P.F. (1983) Planta 159, 50-59. [54] Beinsherger, S.E.I.,Valcke, R.L.M., Deblaere. R.Y., Clijsters, H.M.M., De Greef, J.A. andVan Onckelen, H.A. (1991) Plant Cell Physiol. 32, 489496. [5Sl Catskji J. PospiSilovL, J., MachiEkova, I., Synkova, H., Wilhelmovi, N. and Sestik, Z. (1993) Biol. Plant. 35, 191-198. [56] Von Schwartzenberg, K., Doumas, P., Jouanin, L. and Pilate, G. (1994) Tree Physiol. 14, 27-35. [S7] Taller, B.J. (1994) In: D.W.S. Mok and M.C. Mok (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, London, Tokyo, pp. 101-112. [58] Sprinzl, M., Dank, N., Nock, S. and Schon, A. (1991) Nucleic Acid Res. 19. 2127. 1591 Kline, L., Fittler, F. and Hall, R. (1969) Biochemistry 8,43614371. [60] Barz, L. and Soll, D. (1972) Biochimie 54, 31-39. [61] Holtz, J. and Klambt, D. (1978) Hoppe Seyler’s Z. Physiol. Chem. 359, 89-101. [62] Connoly, D.M. and Winkler, M.E. (1991) J. Bacteriol. 173, 1711-1721. [63] Gray, J., Wang, I. and Gelvin, S.B. (1992) J. Bacteriol. 174, 1086-1098. [64] Laten, H., Gorman, J. and Bock, R.M. (1978) Nucleic Acid Res. 5,43294342. [65] Landick, R., Yanofski, C., Choo, K. and Phung, L. (1990) J. Mol. Biol. 216, 25-37. [66] Barnes, M.F., Tien, C.L. and Gray, J.S. (1980) Phytochemistry 19,409412. 1671 Klambt, D., Holtz, J., Helbach, M. and Maass, H. (1984) Ber. Deutsch. Bot. Ges. 97, 57-65. [68] Kaminek, M. (1974) J. Theor. Biol. 48,489492. 1691 Kam’nek, M. (1982) In: P.F. Wareing (Ed.), Plant Growth Substances 1982. Academic Press, London, pp. 21 5-224. [70] Hogan, R.. Hewett, E.W., Purse, J. and Wareing, P.F. (1973) Tetrahedron Lett. 30, 2827- 2828. 1711 Horgan, R., Hewett, E.W., Horgan, J.M., Purse, J. and Wareing, P.F. (1975) Phytochemistry 14, 1005-1008. [72] Das Neves. H.J.C. and Pais, M.S.S. (1980) Biochem. Biophys. Res. Commun. 95, 1387-1392. [731 Das Neves. H.J.C. and Pais, M.S.S. (1980) Tetrahedron Lett. 21,43874390. [74] Emst, D., Schafer, W. and Oesterhelt, D. (1983) Planta 159, 222-225. [75] Nandi, S.K., Letham, D.S., Palni, L.M.S., Wong, O.C. and Summons, R.E. (1989) Plant Sci. 61, 189-196. 1761 Stmad, M., Peters, W., Beck, E. and Kaminek, M. (1992) Plant Physiol. 99,74-80. [77] Stmad, M., Peters, W., HanuS, J. and Beck, E. (1994) Phytochemistry 37, 1059-1062. [78] Jones, L.H., Martinkovi, H., Stmad, M. and Hanke, D.E. (1996) J. Plant Growth Regul. 15, 3 9 4 9 . 1791 Vahala, T., Eriksson, T., Tillberg, E. and Nicander, B. (1993) Physiol. Plant. 88, 439-445. [80] Werbrouck. S.P.O., Stmad, M., Van Onckelen, H.A. and Debergh, P. (1996) Physiol. Plant. 98,291-297. [Sl] Van Staden, J. and Crouch, N.R. (1996) Plant Growth Regul. 19, 153-170. [82] Singh, S., Letham, D.S., Jameson, P.E., Zhang, R., Parker, C.W., Badenoch-Jones, J. and NoodCn, L.D. (1988) Plant Physiol. 88, 788-794. [83] Mok, M.C., Martin, R.C., Mok, D.W.S. and Shaw, G. (1992) In: M. Kam’nek, D.W.S. Mok and E.

158 Zaiimalovi (Eds.), Physiology and Biochemistry of Cytokinins in Plants. SPB Academic Publishing, The Hague, pp. 4 1 4 6 . 1841 Bassil, N.V., Mok, D.W.S. and Mok, M.C. (1993) Plant Physiol. 102,867-872. [85] Jameson, P.E., Letham, D.S., Zhang, R., Parker, C.W. and Badenoch-Jones, J. (1987) Aust. J. Plant Physiol. 14, 695-7 18. [86] Laloue, M. and Pethe, M. (1988) In: R.P. Pharis and S.B. Rood (Eds.), Abstr. 13th Int. Conf. Plant Growth Substances. Int. Plant Growth Substances Association, Springer-Verlag, Canada. p. 93. [87] Parker, C.W., Wilson, M.M., Letham, D.S., Cowley, D.E., and MacLeod, J.K. (1973) Biochem. Biophys. Res. Commun. 55, 137&1376. [88] Parker, C.W., Letham, D.S., Wilson, M.M., Jenkins, J.D., MacLeod, J.K. and Summons, R.E. (1975) Ann. Bot. 39, 375-376. [89] Letham, D.S., Parker, C.W., Duke, C.C., Summons, R.E., and MacLeod, J.K. (1976) Ann. Bot. 41. 261-263. [90] Palmer, M.V., Horgan, R. and Wareing, P.F. (1981) J. Exp. Bot. 32, 1231-1241. [91] Lee, Y.H., Mok, M.C., Mok, D.W.S., Griffin, D.A. and Shaw, G. (1985) Plant Physiol. 77,635-641. 1921 Letham, D.S. and Palni, L.M.S. (1983) Ann. Rev. Plant Physiol. 34, 163-197. 1931 Jameson, P.E. (1994) In: D.W.S. Mok and M.C. Mok (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, London, Tokyo, pp. 113-128. [941 Turner, J.E., Mok, D.W.S., Mok, M.C. and Shaw, G. (1987) Proc. Natl. Acad. Sci. USA 84,37163717. [951 Dixon, S.C., Martin, R.C., Mok, M.C., Shaw, G. and Mok, D.W.S. (1989) Plant Physiol. 90, 1316-1321. [96] Van Staden, J. and Papaphilippou, A.P. (1977) Plant Physiol. 60, 649-650. 1971 Parker, C.W., Letham, D.S., Gollnow, B.I., Summons, R.E., Duke, C.C. and MacLeod, J.K. (1978) Planta 142,239-251. 1981 Palmer, M.V., Scott, I.M. and Horgan, R. (1981) Plant Sci. Lett. 22, 187-195. [991 Horgan, R., Palni, L.M.S., Scott, I. and McGaw, B. (1981) In: J. Guem and C. PCaud-Lenoel (Eds.), Metabolism and Molecular Activities of Cytokinins. Springer-Verlag, Berlin, pp. 56-65. [I001 Mok, D.W.S and Mok, M.C. (1987) Plant Physiol. 84,596-599. [I011 McGaw, B., Horgan, R. and Heald, J.K. (1985) Phytochemistry 24, 9-13. 11021 MacDonald, E.M.S., Powell, G.K., Regier, D.A., Glass, L., Kosuge. T. and Morris, R.O. (1986) Plant Physiol. 82, 742-747. 11031 PaEes. V., Werstiuk, E. and Hall, R.H. (1971) Plant Physiol 48, 775-778. 11041 Amstrong, D.J. (1994) In: D.W.S. Mok and M.C. Mok (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, London, Tokyo, pp. 139-154. [I051 Hare, P.D. and Van Staden, J. (1994) Physiol. Plant. 91, 128-136. [I061 Whitty, C.D. and Hall, R.H. (1974) Can. .I. Biochem. 52,789-799. 11071 Gerhauser, D. and Bopp, M. (1990) J. Plant Physiol. 135, 714-718. IlOS] Armstrong, D.J. and Firtel, R.A. (1989) Dev. Biol. 136,491499. 11091 Van Kast, C.A. and Laten, H.M. (1987) Plant Physiol. 83, 726-727. [ I 101 Laloue, M. and Fox, J.E. (1989) Plant Physiol. 90, 899-906. [ l l l ] McGaw, B.A. and Horgan, R. (1983) Planta 159, 30-37. [ I 121 Chatfield, J.M. and Armstrong, D.J. (1986) Plant Physiol. 80, 493499. [I131 Kaminek, M., a n d h s t r o n g , D.J. (1990) Plant Physiol. 93, 153C1538. [ I 141 Motyka, V. and Kam’nek, M. (1992) In: M. Kaminek, D.W.S. Mok and E. ZaiimalovB (Eds.), Physiology and Biochemistry of Cytokinins in Plants. SPB Academic Publishing, The Hague, pp. 33-39. [I151 Kaminek, M. (1992) Trends Biotechnol. 10, 159-164. [ I 161 Motyka, V., Gomes, A.I.M. and Kaminek, M. (1994) Biol. Plant. 36, S-31. I1 171 Burch, L.R. and Horgan. R. (1989) Phytochemistry 28, 1313-1319. [1181 Burch, L.R. and Horgan, R. (1992) In: M. Kaminek, D.W.S. Mok and E. Zaiimalovi (Eds.), Physiology and Biochemistry of Cytokinins in Plants. SPB Academic Publishing, The Hague, pp. 29-32. 11191 Meilan, R. and Morris, R.O. (1994) Plant Physiol. (Suppl.) 105, p. 68. [I201 Schreiber, B.M.N., Roessler, J.A. and Jones, R.J. (1995) Plant Physiol. (Suppl.) 108, p.80. 11211 Knypl, J.S., Letham, D.S. and Palni, L.M.S. (1985) Biol. Plant. 27, 188-194. 11221 Letham, D.S. and Zhang, R. (1989) Plant Sci. 64, 161-165. L1231 Chen, C.M., Melitz, D.K. and Clough, EW. (1982) Arch. Biochem. Biophys. 214, 634441.

159 [I241 Burch, L.R. and Stuchbury, T. (1986) Phytochemistry 25, 244-2449. El251 Moffafi, B., Pethe, C. and Laloue, M. (1991) Plant Physiol. 95, 900-908. 11261 Entsch, B. and Letham, D.S. (1979) Plant Sci. Lett. 14, 205-212. 11271 Entsch, B., Letham, D.S.,Parker, C.W. and Summons, R.E. (1979) Biochem. Biophys. Acta 570, 124-139. [I281 Letham, D.S., Palni, L.M.S., Tao. G.-Q., Gollnow, B.I. and Bates, C.M. (1983) Plant Growth Regul. 2, 103-1 15. [1291 Van Staden, J. and Drewes, F.E. (1991) Plant Growth Regul. 10, 109-1 15. [I301 Van Staden, J. and Drewes, EE. (1992) J. Plant Physiol. 140, 92-95. [I311 Letham, D.S., Wilson, M.M., Parker, C.W., Jenkins, I.D., MacLeod, J.K. and Summons, R.E., (1975) Biochim. Biophys. Acta 399.61-70. 11321 Letham, D.S. and Gollnow, B.I. (1985) Plant Growth Regul. 4, 129-145. [I331 Summons, R.E., Letham, D.S., Gollnow, B.I., Parker, C.W., Entsch, B., Johnson, L.P., MacLeod, J.K. and Rolfe, B.G. (1981) In: J. Guern and C. Peaud-Lenoel (Eds.), Metabolism and Molecular Activities of Cytokinins. Springer-Verlag, Berlin, Heidelberg, New York, pp, 69-80. [I341 Entsch, B., Letham, D.S., Parker, C.W., Summons, R.E. and Gollnow, B.I. (1980) In: F. Skoog (Ed.), Plant Growth Substances 1979. Springer-Verlag, Berlin, Heidelberg, New York, pp. 109-1 15. [I351 Redig, P., Shaul, O., lnzC, D., Van Montagu, M. and Van Onckelen, H. (1996) FEBS Lett. 391, 175-180. [I361 Nishinari, N. and Syono, K. (1986) Plant Cell Physiol. 27, 147-153. [137] Zaiimalovi, E., Bkzinovl, A,, Holik, J., Opatm9, Z. (1996) Plant Cell Rep. 16, 76-79. [I381 Centeno, M.L., Rodriguez, A., Feito, I. and Femandez, B. (1996) Plant Cell Rep. 16, 58-62. [139] Emst, D. and Oesterhelt, D. (1985) Plant Cell Rep. 4, 140-143. [ 1401 Van Staden, J., Upfold, S.J., Altman, A. and Nadel, B.L. (1992) J. Plant Physiol. 140,466469. [141] Biezinovi, A,, Holik, J., Zaiimalova, E., Vlasakova, V. and Mali, J. (1996) Plant Physiol Biochem., Spec. Issue, SO3-18, p. 31. [142] Einset, J. and Silverstone, A. (1987) Plant Physiol. 84, 208-209. [143] Tagaki, M., Yokota, T., Murofushi, N., Saka, H. and Takahashi, N. (1989) Plant Growth Regul. 8, 349-3 64. [I441 Rossi, G., Marziani, G.P., Uneddu, P. and Longo, C.P. (1991) Physiol. Plant. 83,647-651. [I451 Niedenveiser, J.G., Van Staden, J., Upfold, S.J. and Drewes, F.E. (1992) S. Air. J. Bot. 58, 236-238. [ 1461 Auer, C. and Cohen, J.D. (1993) Plant Physiol. 102,541-545. [I471 Zhu, Y., Qui, R., Shan, X. and Chen, Z. (1995) Plant Growth Regul. 17, 1-5. [I481 Bollmark, M., Chen, H.-J., Moritz, T. and Eliasson, L. (1995) Physiol. Plant. 95, 563-568. [ 1491 Hansen, C.E., Kopperud, C. and Heide, O.M. (1988) Physiol. Plant. 73,387-391. [lSO] MachBEkova, I., Krekule, J., Eder, J., Seidlova, F. and Stmad, M. (1993) Physiol. Plant. 87, 160-166. [I511 MachaEkova, I., Eder, J., Motyka, V., HanuS, J., and Krekule, J. (1996) Physiol. Plant. 98,564-570. [152] Lejeune, P., Bernier, D., Requier, M.-C. and Kinet, J.-M. (1994) Physiol. Plant. 90, 522-528. (1531 Kinet, J.-M., Houssa, P., Requier, M.-C. and Bernier, G. (1994) Plant Physiol. Biochem. 32, 379-383. [I541 Von Schwartzenberg, K. and Hahn, H. (1991) J. Plant Physiol. 139, 218-223. [155] Km’nek, M., Motyka, V. and Vaiikovd, R. (1997) Physiol. Plant 101, 689-700. [I561 Terrine, C. and Laloue, M. (1980) Plant Physiol. 65, 109CL1095. [I571 Palmer, M.V. and Palni, L.M.S. (1987) J. Plant Physiol. 126, 365-371. [I581 Motyka, V. and Kaminek, M. (1990) In: H.J.J. Nijkamp, L.H.W. Van der Plas and J. Van Aartrijk (Eds.), Progress in Plant Cellular and Molecular Biology. Kluwer Academic Publishers, Dordrecht, pp. 492497. [159] Motyka, V., Faiss, M., Stmad, M., Kam’nek, M. and Schmiilling, T. (1996) Plant Physiol. 112, 1035-1043. 11601 Redig, P., Motyka, V., Van Onckelen, H.A. and Kam’nek, M. (1997) Physiol. Plant. 99, 89-96. [I611 Mok, M.C., Mok, D.W.S., Dixon, S.C., Armstrong, D.J. and Shaw, G. (1982) Plant Physiol. 70, 173-178. [I621 Eklof, S.. Astot, C., Moritz, T., Blackwell, J., Olsson, 0. and Sandberg, G. (1996) Physiol. Plant. 98, 333-344. 11631 Redig, P., Schmiilling, T. and Van Onckelen, H. (1996) Plant Physiol. 112, 141-148. 11641 Thomas, J.C. and Katterman, F.R. (1986) Plant Physiol. 81, 681483.

160 [I651 Hansen, C.E., Meins, Jr. F. and Aebi, R. (1987) Planta 172,520-525. [I661 Vaiikova, R., Kaminek, M., Eder, J. and VanE-k,T. (1987) J. Plant Growth Regul. 6, 147-157. [I671 Vaiikovi, R., Hsiao, K.-C., Bornman, C.H. and Gaudinova, A. (1991) J. Plant Growth Regul. 10, 197-199. [168] Meins, Jr., F. (1989) Annu. Rev. Genet. 23, 395408. 11691 Coenen, C. and Lomax, T. (1997) Trends Plant Sci. 2,351-356. (1701 Palni, L.M.S., Burch, L. and Horgan, R. (1988) Planta 174, 231-234. [1711 Eklof, S., h o t , C., Blackwell, J., Moritz, T., Olsson, 0. and Sandberg, G. (1997) Plant Cell Physiol. 38. 225-235. 1172) Akiyoshi, D.E., Moms, R.O., Hinz, R., Mischke, B.S., Kosuge, T., Garfinkel, D.J., Gordon, M.P. and Nester, E.W. (1983) Proc. Natl. Acad. Sci. USA 8 0 , 4 0 7 4 1 1. [1731 Vaiikova, R., Gaudinova, A., Kaminek, M. and Eder, J. (1992) In: M. Kaminek, D.W.S. Mok and E. Zaiimalovi (Eds.), Physiology and Biochemistry of Cytokinins in Plants. SPB Academic Publishing, The Hague, pp. 47-5 1. [I741 Song, J.Y., Choi, E.Y., Lee, H.S., Choi, D.-W., Oh, M.-H. and Kim, S . 4 . (1995) J. Plant Physiol. 146, 148-1 54. [I751 Zhang, X.D., Letham, D.S., Zhang, R. and Higgins, T.J.V. (1996) Transgen. Res. 5,57-65. 11761 Sondheimer, E. and Tzou, D. (1971) Plant Physiol47. 516-520. [ 1771 Miemyk, J.A. (1979) Physiol. Plant. 45, 6 3 4 6 . [178j Bollmark, M. and Eliasson, L. (1990) Physiol. Plant. 80,534-540. [179j Tao, G.Q., Letham, D.S., Palni, L.M.S. and Summons, R.E. (1983) J. Plant Growth Regul. 2, 89-102. [I801 Sugiyama, T., Suye, S.-I. and Hashizume, T. (1983)Agric. Biol. Chem. 47, 315-318. 11811 McGaw, B.A., Heald, J.K. and Horgan, R. (1984) Phytochemistry 23, 1373-1377. [1821 McGaw, B.A. and Burch, L.R. (1995) In: P.J. Davies (Ed.), Plant Hormones. Kluwer Academic Publishers, The Netherlands, pp. 98-1 17. [1831 Nicander, B., Bjorkman, P.-O. and Tillberg, E. (1995) Plant Physiol. 109, 513-516 [1841 Strnad, M. (1997) Physiol. Plant. 101, 674-688.