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Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.

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Nierembergia sp., a popular floricultural species, only has violet and white flower color and lacks pink to red. To elucidate the reason, we analyzed its flavonoids. Its major anthocyanin was determined to be delphinidin 3-O-(6-O-(4-O-(4-O-(6-O-caffeoyl-b-D-glucopyranosyl)-p-coumaroyl)-a-L-rhamnosyl)-b-D-glucopyranosyl)-5-O-b-D-glucopyranoside. The petals rarely contained cyanidin and pelargonidin, and they contained more flavonols than anthocyanins. We also characterized the biosynthetic pathway by cloning the cDNAs encoding enzymes involved in the flavonoid biosynthesis pathway: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3Ј-hydroxylase (F3ЈH), flavonoid 3Ј,5Јhydroxylase (F3Ј5ЈH), dihydroflavonol 4-reductase (DFR), flavonol synthase (FLS), and UDP-rhamnose: anthocyanidin 3glucoside rhamnosyltransferase (3RT). Northern blot analysis revealed that the expressions of CHS, F3Ј5ЈH, DFR, and 3RT genes were coordinately regulated in parallel with anthocyanin accumulation in the petals, indicating that anthocyanin biosynthesis is transcriptionally regulated; on the other hand, the transcripts of the F3ЈH gene were rarely detected. Antisense suppression of the F3Ј5ЈH gene decreased the amount of F3Ј5ЈH transcripts and that of delphinidin. It was noteworthy that the color of the transgenic flower changed from violet to white rather than to reddish, which was the expectation.

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