A cytochrome b 5 is required for full activity of flavonoid 3′,5′-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors

The substitution pattern of anthocyanin pigments is a main determinant of flower color. Flavonoid 3',5'-hydroxylase (F3'5'H) is a cytochrome P450 enzyme (Cyt P450) that catalyzes the 3', 5'-hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. Species such as rose and carnation lack F3'5'H activity and are, therefore, unable to generate purple or blue flowers. Petunia, on the other hand, contains two loci, termed hf1 and hf2, that encode a Cyt P450 with F3'5'H activity. Here we report the identification of an additional petunia gene that is required for 3',5' substitution of anthocyanins and purple flower colors. It encodes a cytochrome b5 and is expressed exclusively in the flower. Inactivation of the gene by targeted transposon mutagenesis reduced F3'5'H enzyme activity and the accumulation of 5'-substituted anthocyanins, resulting in an altered flower color. However, no phenotypic effect on the activity of other Cyt P450s, involved in the synthesis of hormones or general phenylpropanoids, was observed. These data provide in vivo evidence for the regulation of the activity of specific Cyt P450s by a cytochrome b5.