Selective interaction of triazole derivatives with DWF4, a cytochrome P450monooxygenase of the brassinosteroid biosynthetic pathway, correlates withbrassinosteroid deficiency in Planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazo le-type brassinosteroid biosynthesis inhibitor that induces dwarfism in var ious plant species. The target sites of brassinazole were investigated by c hemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treat ed Catharanthus roseus cells. The levels of castasterone and brassinolide i n brassinazole-treated plant cells were less than 6% of the levels in untre ated cells. In contrast, campestanol and 6-oxocampestanol levels were incre ased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabid opsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylas e, was expressed in Escherichia coli, and the binding affinity of brassinaz ole and its derivatives to the recombinant DWF4 were analyzed. Among severa l triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity an d the activity for inhibiting hypocotyl growth were well correlated among t he derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs, These results indica te that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this h ydroxylation reaction.