CYTOKININS: BIOSYNTHESIS, METABOLISM AND PERCEPTION

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1 In Vitro Cell. Dev. Biol. Plant 36: , March April Society for In Vitro Biology /99 $ CYTOKININS: BIOSYNTHESIS, METABOLISM AND PERCEPTION MACHTELD C. MOK *, RUTH C. MARTIN, and DAVID W. S. MOK Department of Horticulture and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon (Accepted 30 November 1999; editor T. A. Thorpe) Summary Cytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins. Key words: plant hormone; phytohormone; growth regulator; zeatin; thidiazuron. Introduction Cytokinins were discovered as factors promoting cell division in tobacco tissue cultures (Miller et al., 1955). Since then, cytokinins have been shown to regulate a host of additional developmental events, such as de novo bud formation, release of buds from apical dominance, leaf expansion, delay of senescence, promotion of seed germination and chloroplast formation (Mok, 1994). Naturally occurring cytokinins are derivatives of N 6 -(D 2 -isopentenyl)adenine (i 6 Ade) and cytokinins with a hydroxylated side-chain, such as zeatin (trans-zeatin), are major constituents in plants. Synthetic cytokinins include adenine derivatives, such as kinetin, as well as compounds structurally unrelated to natural cytokinins, such as certain phenylureas (Shaw, 1994; Shudo, 1994). Although cytokinins are known to have pronounced effects on plant development and there is a wealth of knowledge on cytokinin chemistry, little is known about their mode of action. It is possible that severe impairment of cytokinin action is lethal and therefore strategies of isolating mutants, which have been essential in elucidating signal transduction pathways of other plant hormones, notably ethylene, have not been successful. The rarity of cytokinin mutants may also explain the slow progress in our understanding of cytokinin biosynthesis in plants, as plant genes controlling cytokinin biosynthesis have yet to be identified. Nevertheless, genes involved in cytokinin perception and genes encoding cytokinin metabolic enzymes have recently been cloned, providing useful tools for further molecular analyses. This review summarizes key findings on cytokinin biosynthesis, metabolism and perception, with an emphasis on newly discovered genes. Cytokinin Biosynthesis Two biosynthetic pathways have been proposed for cytokinin *Author to whom correspondence should be addressed; mokm@ bcc.orst.edu biosynthesis. The first is the direct pathway, involving formation of N 6 -isopentenyladenosine monophosphate (i 6 AMP) from AMP and dimethylallyl pyrophosphate (DMAPP), followed by hydroxylation of the side-chain to form zeatin-type compounds. The second pathway is the indirect pathway, in which cytokinins are released by turnover of trna containing cis-zeatin. In spite of extensive effort having been focused on these pathways (see review by Prinsen et al., 1997), information is still highly fragmented. Several studies using intact plant tissues have shown incorporation of radiolabel from adenine, adenosine or AMP into cytokinins; however, results of tissue incubations cannot be construed as evidence for a particular pathway. The main evidence in favor of direct biosynthesis comes from studies with enzyme preparations converting AMP and DMAPP to i 6 AMP. Isopentenyl transferases mediating this conversion have been isolated from the slime mold Dictyostelium discoideum and tobacco callus (reviewed by Chen and Ertl, 1994). The slime mold enzyme is the only enzyme that has been purified extensively, 6700-fold (Ihara et al., 1984). The tobacco enzyme was highly unstable and attempts to achieve over 40-fold purification led to a complete loss of activity (Chen and Melitz, 1979). There are indications for a similar biosynthetic pathway in monocots. Crude protein extracts from immature maize kernels catalyzed the formation of i 6 AMP from AMP and DMAPP (Blackwell and Horgan, 1994). However, the enzyme involved has not been purified. Plant AMP isopentenyltransferase genes have remained elusive in spite of the identification of such genes from bacteria such as Agrobacterium tumefaciens (Akiyoshi et al., 1984; Barry et al., 1984; Beaty et al., 1986). Two homologous genes are located on the Ti plasmid of A. tumefaciens: one in the T-region (ipt) and one near the vir region (tzs), both encoding enzymes of about 27 kda. Other microorganisms, such as Pseudomonas savastanoi, also contain genes homologous to ipt and tzs (Powell and Morris, 1986). The only plant genes with considerable homology to these microbial genes encode trna isopentenyltransferases, which mediate the transfer of an isopentenyl group to adenine of trnas recognizing the codon 102

2 CYTOKININS 103 group starting with U (Skoog and Armstrong, 1970; Taller, 1994). It is possible that the evolution of plant and bacterial isopentenyltransferases diverged early and that the bacterial AMP isopentenyltransferase genes arose from trna isopentenyltransferase genes. Hydroxylation of the i 6 AMP side-chain, to give rise to zeatin nucleotide, was shown in a microsomal fraction of cauliflower (Chen and Leisner, 1984) and in feeding experiments of Actinidia callus using high levels of i 6 Ade (Einset, 1986). However, purification of the enzyme has not been reported, and no incorporation of radioactivity from labeled isopentenyl adenosine into zeatin-type cytokinins was observed in other incubation experiments (Mok et al., 1982b). As discussed by Einset (1986), hydroxylation may occur only in certain species; therefore, the possible existence of a different biosynthetic pathway for the direct synthesis of hydroxylated cytokinins, without i 6 AMP as intermediate, cannot be excluded. Cytokinins occur adjacent to the anticodon in certain trnas recognizing codons beginning with U (Skoog and Armstrong, 1970; Taller, 1994). The indirect pathway involves release of cytokinins from breakdown of such trna. Although the weakly active ciszeatin is the major cytokinin in plant trna, cis-zeatin can be converted to the highly active trans-zeatin by cis trans isomerization (Bassil et al., 1993). Based on the half-life of trna, it was estimated that trna turnover can account for a significant portion of the total cytokinins in plant tissues (Barnes et al., 1980; Maaß and Klämbt, 1981). However, a critical constraint of the indirect pathway as a major contributor to the cytokinin pool is the fact that trna degradation is not a regulated process, while the biosynthesis of cytokinins should be precisely controlled. Moreover, cytokinin synthesis seems to be localized, with centers of high biosynthetic activity in root tips, shoot meristems and developing seeds (Letham, 1994), while trna turnover is not tissue-specific. In summary, the direct pathway of cytokinin biosynthesis is probably the more important of the two, but the steps of the pathway remain hypothetical until genes encoding specific enzymes have been isolated. It is possible that enzymes and genes somewhat different from those found in the microbial systems occur in plants. Cytokinin Metabolism Plant tissues contain an array of cytokinin metabolites, primarily derivatives of zeatin. The presence of an adenine ring structure in zeatin allows for metabolic conversions similar to those for adenine and, therefore, zeatin riboside and zeatin nucleotide are ubiquitous metabolites. Interconversions between the three forms occur rapidly in plant tissues. A number of the enzymes involved in the conversion between base, nucleoside and nucleotide have been isolated from plants. These enzymes are not cytokinin-specific and generally have higher affinity for adenine, adenosine and AMP than for the corresponding cytokinins (reviewed by Chen, 1997). Mutants deficient in adenine phosphoribosyltransferase (APRTase) were isolated from Arabidopsis (Moffatt and Somerville, 1988) and two genes encoding this enzyme were cloned (Moffatt et al., 1992; Schnorr et al., 1996). One of the two recombinant enzymes (APT1) has a much higher affinity for adenine than benzyladenine; the other (APT2) has a slightly higher affinity for benzyladenine. However, affinities for the naturally occurring cytokinins were not reported. It is not known whether the male sterility associated with Apt1 mutations is due to changes in cytokinin composition or Fig. 1. Side-chain modifications of zeatin. The enzymes involved in these conversions are cytokinin oxidase (a), zeatin O-glucosyltransferase (b), zeatin O-xylosyltransferase (c) and zeatin reductase (d). Genes encoding (a), (b) and (c) have been isolated. adenine salvage (Gaillard et al., 1998). An adenosine kinase gene was isolated from the moss Physcomitrella, but its substrate specificity has not yet been determined (Von Schwartzenberg et al., 1998). Other modifications of the adenine ring include glucosylation at the 3-, 7- or 9-position and the formation of an alanine conjugate at the 9-position. A glucosyltransferase catalyzing the formation of cytokinin glucosides at the 7- and 9-positions, but with a preference for the 7-position, was isolated from radish (Entsch and Letham, 1979). The enzyme converting zeatin to its 9-alanyl derivative (lupinic acid) was partially purified from lupin seeds (Entsch et al., 1983). Both enzymes lack specificity, but show higher conversion rates of cytokinins than adenine (Entsch et al., 1979, 1983). The most significant metabolic changes affect the N 6 -side-chain, as even small substitutions have pronounced effects on cytokinin activity (Skoog and Armstrong, 1970). The most frequent modifications of the trans-zeatin side-chain include reduction to dihydrozeatin, conjugation to O-glycosides (O-glucosylzeatin and O-xylosylzeatin), and side-chain cleavage by cytokinin oxidases (Fig. 1). Other changes, such as O-acetylation, have been observed only in a few species (Jameson, 1994). Cytokinin oxidases selectively cleave the unsaturated N 6 - isoprenoid side-chain of cytokinins (e.g., zeatin, i 6 Ade and their ribosides), while compounds with a saturated isoprenoid side-chain (e.g., dihydrozeatin) are resistant to oxidase attack (Armstrong, 1994). The oxidation products of i 6 Ade were identified as adenine and the side-chain fragment as 3-methyl-2-butenal (Brownlee et al., 1975). A cytokinin oxidase was isolated as early as 1971 by Paces et al. from tobacco callus tissue. Since that time numerous cytokinin oxidases have been isolated, from maize, beans, poplar, wheat and Vinca rosea crown gall tissues. As summarized in reviews by Armstrong (1994) and Jones and Schreiber (1997), the cytokinin oxidases constitute a diverse class of enzymes, with ph optima from 6.0 to 9.0 and a molecular mass range from 25 to 94 kda; most, but not all, are glycosylated. Cytokinin oxidase activity was induced by

3 104 MOK ET AL. both adenine- and phenylurea-type cytokinins (Chatfield and Armstrong, 1986). Recently, maize cytokinin oxidase genes were isolated by two independent groups (Houba-Hérin et al., 1999; Morris et al., 1999). Houba-Hérin et al. purified a cytokinin oxidase from maize using a photo-affinity label to trace the cytokinin oxidase during purification. Based on internal peptide sequences of the purified enzyme, degenerate primers were synthesized for PCR, generating a partial gene sequence. The full-length gene was obtained and the open reading frame (ORF) expressed in moss protoplasts, resulting in increased cytokinin oxidase activity in the culture medium. Morris et al. (1999) also purified the maize oxidase and obtained internal peptide sequences. The product from PCR with degenerate primers was used to isolate a clone from a maize genomic library. The predicted ORF was expressed in Pichia and an active oxidase was secreted in the culture medium. The two groups seem to have isolated the same maize gene as the two clones differ only in seven nucleotides (three amino acids). The enzyme has a flavin-binding domain and a mass of 57 kda. BLAST searches revealed homology to an Arabidopsis accession as well as the fas5 gene of Rhodococcus fascians. The oxidase genes will be useful in investigating the mechanisms of down-regulating cytokinin levels. Cytokinin O-glycosides (O-glucoside or O-xyloside) are resistant to cytokinin oxidases (Armstrong, 1994). As O-glycosides can be converted back to the active aglycones by the corresponding glycosidase, O-glycosylation may be a regulated step to reversibly control the level of active cytokinins. Two zeatin O-glycosyltransferases have been isolated: an O-xylosyltransferase from Phaseolus vulgaris (Turner et al., 1987), and an O-glucosyltransferase from P. lunatus (Dixon et al., 1989). The two enzymes are similar in mass (about 50 kda), but differ in charge (Martin et al., 1990). The O- glucosyltransferase uses uridine 5 0 -diphosphoglucose (UDPG) and UDP-xylose (UDPX) as glycosyl donor but has a much higher affinity to UDPG, while the O-xylosyltransferase uses only UDPX. The substrate recognition of the enzymes is highly specific: besides zeatin, the only other substrate identified thus far is dihydrozeatin, while cis-zeatin and all corresponding ribosides do not serve as substrates for these enzymes. Monoclonal antibodies specific for these enzymes (Martin et al., 1990) were used to isolate the cdna of zeatin O-glucosyltransferase from an expression library of P. lunatus (Martin et al., 1999a). The ORF encodes a polypeptide of 459 amino acids with a mass of 51.4 kda. The recombinant protein catalyzes the formation of O-glucosylzeatin (OGZ) from zeatin and UDPG and displays properties identical to the native enzyme. The gene ZOG1 (for zeatin O-glucosyltransferase) contains no intron. A gene encoding the O-xylosyltransferase, ZOX1 (for zeatin O-xylosyltransferase), was cloned from P. vulgaris by inverse PCR and PCR with primers based on the sequence of ZOG1 (Martin et al., 1999b). The gene contains an ORF coding for a peptide of 51 kda and does not have any intron. The recombinant protein of ZOX1 is identical to the native enzyme in terms of substrate affinity and catalytic properties. The ZOG1 and ZOX1 genes exhibit 93 and 87% identity at the DNA and amino acid levels, respectively. Northern analyses indicated high expression of the genes in immature Phaseolus seeds (Martin et al., 1999a,b) but low expression in vegetative tissues. Southern hybridization detected the presence of two or three homologous genes in each species. Recently, additional homologs have been isolated from Phaseolus as well as Glycine max (Shan et al., unpublished results from our laboratory). The enzymatic properties of the gene products are being determined. With this family of genes, it is now possible to examine more precisely the role of cytokinin O-glycosylation in plant development. Conversion of glucosides to the corresponding aglycones is important in restoring cytokinin activity. A maize b-glucosidase was purified (Campos et al., 1992) and, subsequently, the gene encoding this enzyme was cloned (Brzobohatý et al., 1993). The recombinant enzyme cleaves cytokinin O-glucosides as well as kinetin-n3- glucoside, indicating low substrate specificity (Brzobohatý et al., 1993). A similar gene was isolated from Brassica napus (Falk and Rask, 1995). The recombinant enzyme converted zeatin-o-glucoside to zeatin; however, other substrates, such as related O- glucosides and cytokinin N-glucosides, were not tested. As dihydrozeatin is resistant to degradation by cytokinin oxidase, reduction of the zeatin side-chain may serve an important function, especially in tissues with high levels of oxidases. This conversion is mediated by zeatin reductase, which has been isolated from immature bean seeds (Martin et al., 1989). The enzyme is highly specific for zeatin; it does not reduce cis-zeatin, trans-zeatin riboside, i 6 Ade or zeatin-o-glycosides. The enzyme requires NADPH as a cofactor. There are two isoforms of the enzyme, 25 and 55 kda, with quantitative differences between Phaseolus species. Another enzyme isolated from Phaseolus is a zeatin cis trans-isomerase (Bassil et al., 1993). This enzyme is unlikely to be important in the metabolism of zeatin as the conversion from the cis to the trans form is favored, but may play a role in zeatin biosynthesis via the indirect pathway (see above). The approach of identifying metabolic enzymes followed by cloning of the corresponding genes will continue to provide the molecular tools to assess the significance of individual metabolites in plant growth and development. Analyses of gene expression and phenotypes of transgenic plants will provide important cues regarding the function of the gene products. Cytokinin Perception Early studies on cytokinin signaling involved isolating cytokininbinding proteins. However, as stated in a thorough review (Brinegar, 1994), none of the proteins possesses properties required of a true receptor. Since 1994, several additional cytokinin-binding proteins have been reported (Brinegar et al., 1996; Kulaeva et al., 1996, 1998; Mitsui et al., 1996; Nogué et al., 1996; Brault et al., 1999), but their biological function has not been elucidated. The first gene implicated in cytokinin signal transduction was reported by Kakimoto in The gene CKI1 (for cytokinin independence) was obtained by activation tagging of Arabidopsis thaliana. The insertion of an enhanced 35S promoter upstream of the gene led to recovery of a mutant with traits indicative of overproduction of cytokinins. CKI1 encodes a 125 kda protein with homology to two-component systems that are prevalent in bacterial signal transduction (Parkinson and Kofoid, 1992). The CKI1 protein contains a receptor domain with two membrane-spanning regions, a histidine kinase region and a response regulator domain, and has similarity to ETR1, a known ethylene receptor (Chang et al., 1993; Schaller and Bleecker, 1995). When Arabidopsis callus was transformed with a vector containing the CKI1 gene driven by a 35S promoter, the callus regenerated into shoots in the absence of cytokinin (Kakimoto, 1996).

4 CYTOKININS 105 A very different putative receptor gene was identified based on sequence analyses. Plakidou-Dymock et al. (1998) noticed a partial sequence in the Arabidopsis EST (expressed sequence tag) database as a potential receptor, due to its homology to known bacterial receptors, and subsequently isolated the full-length gene GCR1 (for G-protein cytokinin receptor). The deduced amino acid sequence contains seven predicted membrane-spanning domains and has homology to known 7TM G-protein coupled receptors. When the GCR1 gene in antisense under the control of a 35S promoter was transferred to Arabidopsis, the transformants had reduced sensitivity to cytokinins. The effects of the transgene are suggestive of GCR1 encoding a cytokinin receptor, but cytokinin-binding properties are yet to be determined. Genes with rapid response to cytokinins may be involved in early steps of the signal transduction chain. Brandstatter and Kieber (1998) identified the IBC6 and IBC7 (for induced by cytokinin) genes in a differential display analysis of Arabidopsis seedlings treated with or without benzyladenine. Compounds exhibiting cytokinin activity, including thidiazuron, were effective inducers while auxins had no effect. Most importantly, induction occurred very shortly after exposure to cytokinin (within 10 min) and was independent of protein synthesis. Analyses of the gene sequence revealed the presence of a response regulator domain comparable to that of the bacterial two-component signal transduction systems (Parkinson and Kofoid, 1992). Iwamura et al. (1998) isolated the ARR (for Arabidopsis response regulator) genes by searching the EST database for similarity to bacterial receptors. Subsequently, five genes of this family, ARR3 ARR7, were shown to be induced by cytokinin (Tanaguchi et al., 1998) and some were found to be identical to IBC genes described by Brandstatter and Kieber (1998). A cytokinin-responsive gene isolated from maize, ZmCip1 (for Zea mays cytokinin inducible protein), also has homology to these genes (Sakakibara et al., 1998). Using the yeast two-hybrid system, Yamada et al. (1998) isolated an Arabidopsis cdna clone encoding a protein able to bind ARR4. The gene was isolated previously by Alliotte et al. (1988) and shown to encode a lysinerich DNA-binding protein. The significance of this protein is not known. Other genes induced by cytokinins have been identified (see Crowell and Amasino, 1994), but the timeframe of induction indicates that they act further downstream. The discovery of the candidate genes described above portends a more rapid dissection of the cytokinin-signaling pathways in the near future. found to be O-glucosides (Mok and Mok, 1985). However, it is not known whether the enzymes involved are similar to ZOG1. The fact that the chemical structures of the adenine- and phenylurea-type cytokinins differ greatly raises the question whether the two groups can have common sites of action. Alternatively, the phenylureas may act indirectly, by regulating endogenous cytokinin biosynthesis or metabolism. Studies of structure activity relationships and steric modeling suggest that phenylurea cytokinins have characteristics similar to those of adenine cytokinins (Iwamura et al., 1980; Fox, 1992) and thus may act through a common receptor. Other findings supporting this view include induction of the early response genes, IBC6 and IBC7, by TDZ (Brandstatter and Kieber, 1998) and the discovery of proteins capable of binding to both adenine and phenyurea cytokinins (Brinegar, 1994; Shudo, 1994; Nogué et al., 1996). Indirect effects of phenylureas are manifested in cytokinin autonomy in callus cultures (Mok et al., 1979; Capelle et al., 1983) and inhibition of cytokinin nucleotide formation (Capelle et al., 1983). Phenylureas also modulate cytokinin oxidase activity (Armstrong, 1994); in fact, TDZ is an inhibitor of various cytokinin oxidases (Chatfield and Armstrong, 1986; Armstrong, 1994). Thus it is possible that compounds like TDZ exert direct effects at the site of cytokinin action. However, they also modify endogenous cytokinin metabolism and their activity is enhanced by their high stability in plant tissues. Concluding Remarks Tissue culture and biotechnology of plants have relied on judicious manipulation of plant growth regulators, primarily cytokinins and auxins. Conversely, tissue cultures have been essential in defining cytokinin activities of new compounds and delineating structure activity relationships. In recent years, significant advances have been made in identifying genes involved in cytokinin metabolism and perception. To study the function of these genes and the proteins they encode, it will be necessary to alter their expression in plant tissues through transgenic techniques. Moreover, as more cytokinin genes become available, specific steps in cytokinin biosynthesis, metabolism and perception will be targets for genetic modification, in order to enhance crop performance and yield. Thus, advances in cytokinin research and in vitro manipulations will continue to be mutually supportive. Metabolism and Mode of Action of Phenylurea Cytokinins In 1955, Shantz and Steward showed the cytokinin activity of diphenylurea (DPU). Subsequently, much more potent urea derivatives were discovered, such as N-phenyl-N 0 -(2-chloro-4- pyridyl)urea and TDZ, with cytokinin activities exceeding that of zeatin (Takahashi et al., 1978; Mok et al., 1982a; Shudo, 1994). There is no evidence that these types of compounds occur naturally in plant tissues, although DPU was initially reported as a component of coconut milk (Shantz and Steward, 1955). The high cytokinin activity of some phenylureas may be due partly to their extreme stability in plant tissues. While zeatin is completely metabolized by plant tissues within hours of application, the metabolism of TDZ is extremely slow and the main metabolites were Acknowledgements Our research on cytokinins was supported by grants from USDA/NRI ( ), NSF (IBN ) and Pioneer Hi-Bred International Inc. This is paper no of the Oregon Agricultural Experiment Station. References Akiyoshi, D. E.; Klee, H.; Amasino, R. M.; Nester, E. W.; Gordon, M. P. T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81: ; Alliotte, T.; Engler, T. G.; Peleman, J.; Caplan, A.; van Montagu, M.; Inze, D. 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