Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula

Abstract

Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.

The elephant grass (Cenchrus purpureus) genome provides insights into anthocyanidin accumulation and fast growth

“…Flavanone 3‐hydroxylase (F3H), flavonoid 3′‐hydroxylase (F3′H) and flavonoid 3′,5′‐hydroxylase (F3′5′H) can catalyse the production of dihydroflavonols, which are precursors of leucoanthocyanins (Cao et al, 2018). Dihydroflavonol 4‐reductase (DFR) was the first enzyme shown to produce leucoanthocyanins (Zhu et al, 2018), and DFR genes have been expanded and positively selected in Cp (Figure 5a,b). Anthocyanidins are converted from leucoanthocyanins by anthocyanidin synthase (ANS) and further glycosylated by anthocyanidin 3‐ O ‐glucosyltransferase (3GT; Zhang et al, 2016).…”

A R2R3-MYB Transcription Factor, VvMYBC2L2, Functions as a Transcriptional Repressor of Anthocyanin Biosynthesis in Grapevine (Vitis vinifera L.)

“…DFR participates in anthocyanin biosynthesis by catalyzing the conversion of dihydroflavonol to leucoanthocyanidins. Constitutive expression of the Vitis bellula gene VbDFR increased anthocyanin accumulation in tobacco [38], while, over-expression of the grapevine transcription factor genes carrying the C2-motif repressor, VvMYBC2L1 and VvMYBC2L3 , significantly decreased the transcript level of DFR in Petunia hybrida [25]. UFGT is involved in glycosylation of anthocyanidins to form anthocyanins, and was specific induced in red cultivars of V .…”

Ectopic Expression of Grapevine Gene VaRGA1 in Arabidopsis Improves Resistance to Downy Mildew and Pseudomonas syringae pv. tomato DC3000 But Increases Susceptibility to Botrytis cinerea

“…Grape is an economically important crop cultivated worldwide. However, its yield and quality are restricted by various pathogens [1,2]. Repeated use of fungicides not only increases production costs and causes environmental pollution, but also leads to the drug resistance of pathogens, which may eventually cause sudden pathogen outbreaks in the future [3,4].…”