Abstract:
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.
