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Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized, O-glucosyltransferase and O-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatin O-glucosyltansferase) was isolated from P. lunatis (Martin et al., 1999). Based on the ZOG1 sequence, the ZOX1 gene (zeatin O-xylosyltransferase) was cloned from P. vulgaris. ZOX1 contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 and ZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules.

The effects of a highly cytokinin-active urea derivative, N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N(6)-(Delta(2)-isopentenyl)[8-(14)C]adenosine ([(14)C]i(6) Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [(14)C]i(6) Ado was similar for the two genotypes, but differed with the medium used. [(14)C]i(6) Ado was rapidly converted to N(6)-(Delta(2)-isopentenyl)[8-(14)C]adenosine 5'-P ([(14)C]i(6) AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [(14)C] i(6) AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [(14)C]i(6) Ado, while [(14)C]i(6) AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related.

The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N(6)-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern x Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N(6)-isopentyladenine, N(6)-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N(6)-[Delta(2)-isopentenyl]adenine, N(6)-[Delta(2)-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of (14)C-N(6)-(Delta(2)-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of (14)C-N(6)-(Delta(2)-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites.

The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5'-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.