Abstract:
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.
