Abstract
Hydroxylation of isopentenyladenine (iP)-type cytokinins to trans-zeatin (tZ)-type cytokinins is thought to be catalyzed by cytochromes P450. To isolate the gene(s) involving the hydroxylation step, we searched P450 genes from Arabidopsis thaliana using yeast expression system. Yeast strain expressing isopentenyltransferase (IPT), an enzyme catalyzes the initial step of cytokinin biosynthesis, secretes iP-type cytokinins into the culture medium. We assumed that if IPT and the aimed P450 genes are co-expressed in a yeast cell, the yeast secretes tZ-type cytokinins into the culture medium. Using this strategy, we screened P450 genes from A. thaliana and found two positive candidates. To identify the hydroxylase activity against iP-type cytokinins, we prepared microsome fractions from the yeast expressing these candidate genes, and measured the activity in vitro. We will present data of kinetic properties of the enzymes and expression profile of the genes.
