Functional and correlation analyses of dihydroflavonol-4-reductase genes indicate their roles in regulating anthocyanin changes in Ginkgo biloba

Anthocyanins, a class of flavonoids, are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4-reductase (DFR) catalyzes the first reaction leading to anthocyanins, and it is considered a key regulatory enzyme of anthocyanin biosynthesis in plants. In this research, we isolated three new DFR genes by searching the whole-genomic sequence data of Ginkgo biloba. The genes have similar structures and are clustered in the same scaffold. Sequence analyses of GbDFR proteins revealed that these GbDFRs have a closer relationship to Arabidopsis thaliana DFRs than previously identified GbDFRs. Expression analyses revealed that GbDFR4 and GbDFR6 were preferentially expressed in leaves and fruit, respectively. Young and mature leaves shared similar gene expression patterns, while fruit harvested in May had significantly higher expression levels of GbDFRs than fruit harvested in June. Transgenic tobacco plants ectopically overexpressing GbDFR genes showed higher DFR enzymatic activity levels, higher anthocyanin contents and darker flowers than the control. Ginkgo seedlings treated with NaCl showed increased total flavonoid and anthocyanin contents, and the expression levels of GbDFR genes were also induced. In contrast, UV-B irradiation increased the total flavonoid content but decreased both the anthocyanin content and GbDFR expression levels. A salicylic acid treatment induced and inhibited the total flavonoid content with and without light, respectively. In contrast, both the GbDFR expression levels and the anthocyanin content were inhibited by the salicylic acid treatment regardless of the light conditions. This research validated the functions of GbDFRs and expands our understanding of the environmental stress-response mechanisms of ginkgo trees.