Zeatin O-xylosyltransferase in Phaseolus vulgaris : immunological and molecular analyses Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/r494vn959

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  • Cytokinins are a group of plant hormones mediating cell division and differentiation. Zeatin is a naturally occurring and highly active cytokinin that is rapidly metabolized in Phaseolus seeds. The metabolites and enzymes mediating the conversion have been studied extensively. Zeatin metabolic enzymes may be utilized in studying the regulation of cytokinin metabolism. This thesis describes such an approach using zeatin O-xylosyltransferase as the candidate. In immature seeds of P. vulgaris, zeatin is rapidly converted to O-xylosylzeatin; while in P. lunatus, O-glucosylzeatin is formed. The enzymes that mediate both of these conversions have been isolated and characterized. Samples containing the O-xylosyltransferase, purified over 2500-fold, were used as antigen to produce monoclonal antibodies (MAbs) which recognized both O-glycosyltransferases. The antibody was used for localization and gene cloning experiments. Tissue printing and immunolocalization studies revealed that the enzyme occurred predominantly in the endosperm, and is associated with both the cytoplasm and the nucleus. An expression cDNA library derived from mRNAs of immature P. vulgaris seeds was screened with the antibodies and two full length cDNAs were chosen for further analyses. The clones were highly homologous. The ORFs encode proteins of approximately 70 kD and the amino acid sequences were 90% identical. Recombinant proteins generated in prokaryotic (E. coli) and eukaryotic (insect cells via baculovirus) expression systems were insoluble in aqueous solutions and no enzyme activity was detected. Proteins obtained from in vitro transcription/translation were soluble and could be processed to smaller proteins with the addition of bean endosperm extracts. Processing of preproteins and the effects of the cDNA on plant development were examined by generating transgenic tobacco containing the ORF under the control of a constitutive promoter. Plants harboring the transgene were sensitive to auxin in comparison to no-insert control plants. Auxin also induced processing of the transgenic protein. These results suggest that the cDNAs encode preproteins that require precise processing to yield active enzymes and that hormonal interactions such as cytokinin/auxin balance may be mediated via auxin stimulation of cytokinin metabolic enzymes.
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