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.
Highlights
Dihydroflavonol 4-reductase (DFR) is a key late enzyme in the plant flavonoid pathway toward both anthocyanins and proanthocyanidins (Figure 1)
We have demonstrated that the formation of proanthocyanidins and flavan-3-ols in V. bellula is via two pathways, the anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) pathways [28,30]
This amplification produced an approximately 1 kb cDNA fragment (Figure 2a). Sequencing confirmed that this fragment was composed of 1014 nucleotides including the full length of open reading frame (ORF) from the start codon to the stop codon, which was deduced to translate 337 amino acids
Summary
Dihydroflavonol 4-reductase (DFR) is a key late enzyme in the plant flavonoid pathway toward both anthocyanins and proanthocyanidins (Figure 1). It catalyzes the key step from dihydroflavonols, such as dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM), to leucoanthocyanidins, such as leucopelargonidin, leucocyanidin, and leucodelphinidin [1]. DFR genes have been cloned from multiple plants and its mutation has been demonstrated to cause the loss of anthocyanins and proanthocyanidins in plants [2,3,4,5]. DFR is an economically important plant gene, given that anthocyanins and proanthocyanidins are two groups of antioxidants relating to high nutritional values of crop, food, and beverage (such as wine and green tea) products [6,7,8,9]. A common metabolic phenotype is that the activation or Molecules 2018, 23, 861; doi:10.3390/molecules23040861 www.mdpi.com/journal/molecules
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