Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

Abstract

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (K cat/K m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa.

Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca

“…vesca and F . × ananassa was reflected in the expression of F3’H and DFR1, and F3’H deficient lines displayed white or pale pigmentation phenotype [ 34 ]. In this study, a set of flavonoid pathway genes were down-regulated in yellow woodland strawberry fruits, including C4H, CHS, CHI, F3H, DFR and ANS, which indicated the transcript abundance of these genes were positively related to the accumulation of anthocyanin.…”

The rare orange-red colored Euphorbia pulcherrima cultivar ‘Harvest Orange’ shows a nonsense mutation in a flavonoid 3’-hydroxylase allele expressed in the bracts

“…(2- 14 C)-Malonyl-coenzyme A (55 mCi/mmol) was purchased from New England Nuclear Corp. GmbH (Vienna, Austria). ( 14 C)-Labeled flavonoids naringenin, DHK, DHQ, and DHM were synthesized as previously described [ 16 , 17 ] using recombinant F3’5’H from Sollya heterophylla and recombinant F3′H from Arabidopsis thaliana .…”

Automatic Identification of Players in the Flavonoid Biosynthesis with Application on the Biomedicinal Plant Croton tiglium

“…N at position 3 of the substrate-determining domain was associated with recognition of all three dihydroflavonols [ 55 ]. D at position 3 prevented the acceptance of dihydrokaempferols [ 55 ], while L or A led to a preference for dihydrokaempferol and substantially reduced the processing of dihydromyricetin [ 55 , 56 ]. Although this position is central for the substrate specificity, other positions contribute to the substrate specificity [ 57 ].…”