Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley11Nucleotide and polypeptide sequence data reported here will appear in the EMBL, GenBank and DDBJ databases under the accession numbers AY230247 (FNS I), AY230248 (FHT) and AY230249 (FLS).

AbstractFlavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2S)-naringenin. While many plants express a microsomal-type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants. FNS I belongs to the FeII/2-oxoglutarate-dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation. Using a homology-based reverse transcription polymerase chain reaction approach, two additional flavonoid-specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3β-hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells. Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS. Analogous to FNS I, FLS oxidizes the flavonoid γ-pyrone by introducing a C2, C3 double bond, and (2R,3S)-dihydrokaempferol (cis-dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis. Incubation of either FNS I or FLS with cis-dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation. However, the lack of apigenin in these incubations ruled out cis-dihydrokaempferol as a free intermediate in FNS I catalysis. Furthermore, neither (+)-trans-dihydrokaempferol nor unnatural (−)-trans-dihydrokaempferol and 2-hydroxynaringenin served as a substrate for FNS I. Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions