The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3'-hydroxylase on leucoanthocyanidins.

MAIN CONCLUSION: In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3' position by flavonoid 3'-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3' or 3' and 5', respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3'H and F3'5'H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3'H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3'-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3',5'-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3'Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.

Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background.