Ginkgo biloba DFR2 Gene Remodels the Flavonoid Metabolic Network in Transgenic Nicotiana benthamiana

Dihydroflavonol 4-reductase (DFR) plays a pivotal role in regulating flavonoid and anthocyanin biosynthesis, governing the accumulation of plant secondary metabolites. This study aimed to characterize the DFR gene family in Ginkgo biloba and elucidate the function of the predominant gene GbDFR2 in the flavonoid metabolic network. Through transcriptome analysis, three differentially expressed GbDFR genes were identified. Bioinformatic analysis revealed that all three GbDFR proteins are hydrophilic and acidic and belong to the NADB_Rossmann superfamily. RT-qPCR analysis of different tissues of ginkgo revealed that all three GbDFR genes exhibited the highest expression levels in the leaves. An overexpression vector of GbDFR2 was constructed and stably transformed into Nicotiana benthamiana. Metabolomic and qPCR analyses showed that heterologous GbDFR2 expression significantly remodeled the flavonoid profile, upregulating sakuranetin and 3,7-Di-O-methylquercetin while downregulating narcissin and naringenin chalcone. Additionally, it upregulated endogenous NbCHI and NbDFR, and suppressed the transcription factors NbMYL2b and NbERF4a. These findings suggest that GbDFR2 can act as a regulator of flavonol biosynthesis and provide a candidate gene for the metabolic engineering of flavonoids in woody plants.