Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin

Abstract

Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in Ophiorrhiza japonica is still unknown. Here, three putative DFR-like genes were firstly isolated from O. japonica. Phylogenetic analysis indicated that OjDFR1 was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that OjDFR1 transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of OjDFR1 in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in Escherichia coli, but confirming that only OjDFR1 encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing OjDFR1 in Arabidopsis tt3-1 mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of OjDFR1 in transgenic tobacco contributed to darker flower color via up-regulating the expressions of endogenous NtANS and NtUFGT, which suggested that OjDFR1 was involved in flower color development. In summary, this study validates the functions of OjDFR1 and expands our understanding of anthocyanin biosynthesis in O. japonica.