ABSTRACTThe cDNAs encoding dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) that are involved in flavonoid biosynthesis determining flower colour were cloned from Rosa hybrida petal cDNA library. Leucoanthocyanidins and flavonols are synthesized from dihydroflavonols, the same substrates, through the action of DFR and FLS enzymes, respectively. Sequence comparison of the rose DFR with the reported DFR genes revealed that they were homologous each other. The amount of DFR mRNA in rose petals was developmentally regulated and paralleled anthocyanin production in petals. Sepals, thorns, styles and stamens also contained anthocyanins and DFR mRNA. No DFR mRNA was observed in mature leaves and a small amount of the transcript was detected in young leaves. A petunia cultivar, whose colour was pale pink due to a deficiency in flavonoid 3′-hydroxylase and 3′5′-hydroxylase, was transformed with binary vector containing a rose DFR cDNA cloned behind a constitutive promoter. Petals and anthers of the resultant transgenic petunia plants were salmon pink and contained pelargonidin, an anthocyanidin rarely found in petunia. FLS transcripts peak earlier than chalcone synthase and DFR transcripts in the petal development, which is consistent with the findings of preceding accumulation of flavonols to anthocyanidins in rose petals. The results indicate that flavonol biosynthesis is transcriptionally and differently regulated from anthocyanin biosynthesis. A phylogenetic analysis of 2-oxoglutarate dependent dioxygenases including FLS, flavanone 3-hydroxylase and anthocyanidin synthase indicates that these genes had diverted before flowering plants speciated and that FLS is closely related to anthocyanidin synthase. Rose and petunia FLS expressed in yeast required 2-oxoglutarate, ferrous ion and ascorbate for the full activity. Rose FLS catalyzed flavonol synthesis from dihydrokaempferol, dihydroquercetin and dihydromyricetin while petunia FLS only utilized dihydrokaempferol and dihydroquercetin. Flavonols contribute to bluing of flower colour by forming complex with anthocyanins. Cosuppression of FLS gene in transgenic petunias by petunia FLS gene yielded redder flower colour due to decrease of flavonols and increase of anthocyanins. The flowers of transgenic petunia expressing rose FLS cDNA had paler flower due to decrease of anthocyanidins.