Identification of cytokinin reductase in Phaseolus embryos Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vm40xt692

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  • A critical balance of hormones is necessary for normal plant development. Therefore, precise mechanisms must exist to maintain proper levels of cytokinins, a group of hormones regulating cell division and differentiation. Structural modifications of the naturally occurring cytokinin, zeatin; including the oxidation, O-glycosylation and reduction of the N⁶-side chain; may represent such control mechanisms to regulate active cytokinin levels. This study concerns the identification and partial purification of the enzyme responsible for the reduction of zeatin in Phaseolus embryos. The presence of zeatin reductase was indicated by the occurrence of dihydrozeatin derivatives and O-xylosyldihydrozeatin in vegetative and embryonic tissues, respectively, in this genus. A cytokinin reductase catalyzing the conversion of zeatin to dihydrozeatin was initially detected in soluble fractions of immature embryos of Phaseolus. Subsequently, the enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. NADPH was required for enzyme activity, but was found to be inhibitory at high (0.5-1.0 mM) concentrations. The reaction had a pH optimum of 7.5-8.0 and was linear up to one hour. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cis-zeatin. O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The high molecular weight isozyme (M [subscript] r 55,000 +/- 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M [subscript] r 25,000 +/-5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level and Phaseolus embryos are useful for the detection of zeatin specific metabolic enzymes. Preliminary studies also indicate that quantitative differences in reductase exist in Phaseolus species. As dihydrozeatin (and possibly its derivatives) is more active than zeatin in most of the Phaseolus bioassays, the zeatin reductase identified in this study may be representative of enzymatic processes converting zeatin to a more active compound in selected tissues. The significance of qualitative and quantitative variations of this enzyme between species and tissues will be examined further to study this hypothesis.
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