CHARACTERIZATION OF FLAVONOID 3',5'-HYDROXYLASE IN MICROSOMAL MEMBRANE-FRACTION OF PETUNIA-HYBRIDA FLOWERS

We have detected a flavonoid 3',5'-hydroxylase (F3',5'H) in the micros omal fraction of Petunia hybrida flowers. Activity varied with the dev elopment of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3',5'H activity in flower extracts from genetically defined floral color mutants correlated strictly wit h the genotypes Hf1 and Hf2. No activity was detected in flowers from mutants homozygous recessive for both alleles. F3',5'H activity was de pendent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4'-trihydroxy flavonone at the 3' and 5' positions, and of 5,7,3',4'-tetrahydroxyfla vonone and dihydroquercetin at the 5' position. Hydroxylase activity w as inhibited by plant growth regulators (1-aminobenzotriazole and tetc yclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cyto chrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or p henylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPH-Cyt P450 reducta se inhibited the F3',5'H. The data are consistent with the suggestion that the P. hybrida F3',5'H is a monooxygenase consisting of a Cyt P45 0 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsom al membranes and in solubilized detergent extracts of these membranes. F3',5'H activity was sensitive to low concentrations of all detergent s tested, and therefore solubilization of the active enzyme was not ac hieved. Reaction products other than flavanones were observed in F3',5 'H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has no t been described in P. hybrida is discussed.