GbbHLH65 broadly regulates flavonoid biosynthesis structural genes in Ginkgo biloba

Flavonoids synthesized in Ginkgo biloba leaves are well-documented to confer significant health benefits. However, the molecular mechanisms regulating the expression of flavonoid structural genes in this species remain poorly understood. In this study, we isolated and characterized seven GbCHS genes from the ginkgo genome. Synchronous expression changes of these genes were observed across different seasons and between genders. To identify a common regulator, a systematic yeast two-hybrid screen revealed that GbbHLH65 interacts with both R2R3-MYB and WDR proteins. Notably, GbbHLH65 exhibited the highest number of interactions with R2R3-MYB proteins, including some that lack conserved interaction motifs. The positive regulatory role of GbbHLH65 in GbCHS expression was initially suggested by co-expression patterns and further confirmed through virus-induced gene silencing (VIGS), which suppressed the expression of all examined GbCHS genes. Promoter analysis revealed no common E-box motifs among GbCHS genes, suggesting that GbbHLH65 may regulate their expression indirectly. Consistent with this, suppression of GbbHLH65 also reduced transcript levels of additional flavonoid structural genes. Collectively, these findings establish GbbHLH65 as a broad-spectrum regulator of flavonoid biosynthesis in ginkgo leaves. • Isolate and characterize seven GbCHS genes in ginkgo genome. • Synchronous expression changes of GbCHS genes indicates a common regulator. • GbbHLH65 interacts with both R2R3-MYB and WDR proteins. • Some GbbHLH65 interacting MYB proteins lack conserved interaction motifs. • Silencing GbbHLH65 reduced both the expression of flavonoid structural genes and the total flavonoid content.