SummaryCytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the N 6 -(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1 -(2-azido-6-chloropyrid-4-yl)-3-(4-[ 3 H])phenylurea ([ 3 H]-azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme.Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD-dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.

Formation of the riboside-5'-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N6-benzyladenine (BA). Formation of Nl-benzyladenosine-5'-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and pnmanly converted to BAMP and to BA 7-and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N6-benzyladenosine was not affected in the mutant plantlets.In vitro conversion of BA to its nucleoside-5'-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5'-monophosphate in extracts of wildtype plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRTdeficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets. lism, but few of the enzyme activities effecting these steps in vivo have been identified. Furthermore, which modified form(s) constitutes the "active" cytokinin remains an open question. Elucidation of the enzymes involved in the synthesis and metabolism of cytokinins may provide valuable insight into the function of the various cytokinin metabolites, how their level in particular tissues is regulated and how these levels relate to physiology of plants.The interconversion of cytokinin bases, ribosides and nucleotides is a major feature of the metabolism of cytokinins (5,17,20,26), and it may be catalyzed by the enzyme system that metabolizes adenine. Several of these enzymes have been purified and shown to act on N-substituted substrates in vitro, albeit at significantly lower rates than those measured on the corresponding adenine substrates (3-6). Results of in vivo feeding experiments also support the notion that the purine metabolizing enzymes may be the main route by which cytokinins are interconverted (7).

Cytokinin oxidases (CKOs) play a major role in the regulation of hormone levels in plants by irreversibly degrading cytokinins. Two new cDNAs from maize (CKO2 and CKO3) were cloned and CKO activity of a recombinant CKO3 enzyme was demonstrated. CKO2 and CKO3 encode flavoproteins with 93% identity among each other compared with 45% identity with CKO1. The respective genes were mapped to BIN 3.05/06 and BIN 8.06 which belong to duplicated regions of the maize genome. For a better understanding of the role of CKO2 and CKO3 in maize development, their expression profiles were analysed in different organs and during kernel development via semi-quantitative RT-PCR. Different spatial and temporal expression patterns were observed for the two genes, as well as for CKO1 and two additional genes CKO4 and CKO5. CKO2 to CKO5 genes were mainly expressed in vegetative tissues, with unique expression patterns. CKO1 was most strongly expressed in the kernel. All five genes were expressed at early stages of kernel development, a period when a peak in cytokinin levels and a high cell division rate in the endosperm have been described. However, each gene had its own expression profile with a major difference concerning the onset of expression.

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The behaviour of the naturally occurring cytokinin zeatin 1 in aqueous acidic conditions has been studied, after it was observed that this compound was relatively more stable than another related cytokinin, 6-(3-methylbut-2-eny1amino)purine 2. Zeatin 1 reacted readily in 1 rnol dm-3 aqueous HCI at 100 "C. Three products were characterized, a diol resulting from the hydration of the double bond of the aliphatic chain, and two cyclized products, a hydroxypyrrolidine and a dihydropyrrole. A scheme is proposed to explain the formation of these products. However, zeatin was found to be stable in 0.1 rnol dm-3 aqueous HCI at 100 "C and in 1 rnol dm-3 aqueous HCI at 50 "C, conditions which allow the hydrolysis of zeatin riboside.The plant hormones zeatin 1 and 6-(3-methylbut-2-enyIamino)purine 2, of the cytokinin family, are present in plantsnot only as the free bases, but also as the 9-P-~-ribofuranosides, as the corresponding 5'-nucleotides and as various N-glucosides. In the case of 1 and of its riboside, 0-glycosyl conjugates are also found.' It has been shown that 2 is particularly unstable in HCI solution2*3 and the products resulting from the reactivity of the chain have been fully characterized (Scheme 1). Acidic treatment of zeatin 1 with HCl(1 rnol dm-3) Scheme 1 at 100 0C,4.5 gave rise to two products, one of them being a hydration product of the chain double bond, the second being unknown. The question of the stability of the allylic chain of cytokinins in acidic media has to be considered, since acidic treatments are commonly performed during their isolation and purification from plant tissues, in particular the hydrolysis of the N-glucosyl conjugates when a quantitative determination of the hormone pool is required.We describe here the study of the transformation of zeatin 1 in aqueous HCI which allowed us to characterize additional products not seen in the previous ~tudies,~,' and to define acidic conditions under which zeatin is stable and which can therefore be used to hydrolyse zeatin N-glycosides, especially zeatin riboside.