Cytolocalization of zeatin O-xylosyltransferase in Phaseolus.

Abstract

Zeatin O-xylosylbansferase (EC 2.4.2.-) mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose in immature seeds of Phaseolus vulgaris. Tissue printing with a monoclonal antibody specific for the enzyme and a cDNA probe demonstrated that the enzyme was primarily localized and synthesized in the endosperm. Immunolocalization performed on monolayer endosperm at the free-nuclei stage and on EM sections demonstrated that the enzyme was associated with the nucleus as well as with the cytoplasm. Immunoanalysis of nuclear fractions revealed that the enzyme was retained in the nuclear pellet. Western analysis also showed that the enzyme was present in the nuclei of cotyledons and endosperm callus. The findings suggest that the enzyme may be involved in the nuclear-cytoplasmic transport of cytokinins and related molecules or, possibly, with chromatin of rapidly dividing cells.Cytokinins are plant hormones mediating cell division and differentiation (1). Zeatin is a naturally occurring cytokinin highly active in most plant systems (2). Genetic differences in cytokinin metabolism between species of Phaseolus were investigated by incubating immature seeds with radiolabeled zeatin (3). Exogenously supplied zeatin was rapidly converted to O-xylosylzeatin by immature seeds of P. vulgaris, while O-glucosylzeatin was formed in P. lunatus. The species-specific formation of zeatin metabolites was related to the occurrence ofa single, distinct enzyme in immature seeds of each of the two species; zeatin O-xylosyltransferase in P. vulgaris and O-glucosyltransferase in P. lunatus. Both enzymes are highly specific for trans-zeatin but differ in specificity for the donor ofthe glycosyl moiety (4, 5). UDP-xylose (UDPX) serves as the donor for O-xylosyltransferase from P. vulgaris, whereas the O-glucosyltransferase ofP. lunatus can utilize both UDP-glucose (UDPG) and UDPX to mediate the formation of O-glucosylzeatin and O-xylosylzeatin, respectively, but has 10 times higher affinity for UDPG. The two glycosyltransferases have a similar molecular size (Mr 50,000) but can be separated by ion exchange chromatography (4).Two additional zeatin-metabolizing enzymes, a reductase and a cis-trans isomerase, have also been isolated from seeds of Phaseolus (6, 7). Interestingly, the activities of all four enzymes are particularly pronounced in immature seeds. We are interested in characterizing the developmental regulation of these enzymes. Part of this study is focused on localizing enzymes in the developing seed.Zeatin O-xylosyltransferase was chosen as the first enzyme for localization studies because monoclonal antibodies (mAbs) specific for the enzyme have been generated (8) and immunopositive cDNA clones have been isolated from Agtll expression libraries (9). In this paper we describe results obtained from tissue printing using a mAb (XZT-1) and a cDNA (pXZT82) as probes, immunolocalization performed on monolayer endosperm and EM sections, and Western analyses ofcellular and nuclear fractions. The results indicate t...

Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L

“…Antibodies specific to two of these enzymes have been obtained (Martin et al, 1990) and used to isolate genes involved in cytokinin metabolism (Mok et al, 1992a) and to study their spatial expression (Martin et al, 1993). We intend to use similar strategies for further analyses of the isomerase.…”

The Five "Classical" Plant Hormones.

“…Zeatin O-xylosyl transferase has been isolated from bean embryos, and antibodies have been prepared. The enzyme is predominantly localized in the endosperm (Martin et al, 1993).…”

Reactivation of Meristem Activity and Sprout Growth in Potato Tubers Require Both Cytokinin and Gibberellin

“…CDC, Cell division cycle; DP, docking protein. thought to be involved in CK homeostasis (Martin et al, 1993).…”