Molecular and biochemical characterization of torenia flavonoid 3′-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of these genes

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Cytochrome P450 (P450) enzymes play important roles in the biosynthesis of flavonoids that determine flower color. Three P450s, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′,5′H) and flavone synthase II (FNSII), are involved in torenia flavonoid biosynthesis. In this study, we isolated a full-length cDNA of F3′H from a torenia petal cDNA library. The deduced amino acid sequence of torenia F3′H has 82 and 80% identity to those of Arabidopsis and petunia F3′Hs, respectively. Phylogenetic analysis showed that F3′H and F3′,5′H genes diverted before speciation of higher plants during evolution. Expression of torenia F3′H cDNA in yeast demonstrated that torenia F3′H catalyzed hydroxylation at the 3′ position of naringenin, dihydrokaempferol, kaempferol and apigenin. Km values for these compounds were 0.83, 3.95, 2.96 and 21.5 μM, respectively. Northern analysis showed that the accumulation of anthocyanins and flavones was transcriptionally regulated and that the transcription of the FNSII gene was differently regulated from F3′H and F3′,5′H genes. The torenia, whose F3′,5′H expression had been suppressed, was further transformed with the F3′H gene driven by a constitutive promoter. Some of the transgenic torenia plants had an elevated amount of cyanidin-type anthocyanins and thus redder flower color. Co-suppression of the FNSII gene in the torenia successfully decreased the amount of flavones and increased the amount of flavanones, and yielded paler flower color.