Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.

The gene Hf1 plays a key role in the expression of floral color in petunias. Hf1 encodes a flavonoid-3',5'-hydroxylase (F3'5'H). The recessive allele (hf1) in an inbred line of petunia is known to be generated by the insertion of a transposable element (Psl). We isolated a novel Mutator-like transposable element (named dTph9) from the hf1 allele of a commercial petunia with red flowers. Another novel transposable element (named rTph1) was found in hf1 of another red petunia. rTph1 shared features with the copia-like retrotransposable element family. These novel elements were inserted independently in the third exon of the Hf1 gene, at different positions. The hf1 allele harboring dTph9 or rTph1 cannot produce an active F3'5'H enzyme because there are stop codons in the dTph9 and rTph1 sequences. Southern analysis showed that these elements were present in relatively low copy numbers and that mutation of the Hf1 locus was associated with the transposition of both elements. We conclude that a loss-of-function mutation of the petunia Hf1 gene is caused by the insertion of at least two different transposable elements, other than Psl, within the Hf1 gene.