The GbGLABRA3-2-GbMYBR1 module regulates proanthocyanidin biosynthesis in Ginkgo biloba

Proanthocyanidins (PAs), accounting for approximately 7% of the standardized Ginkgo biloba leaf extract EGb761, possess distinct pharmacological properties. Nevertheless, the regulatory mechanisms governing PA biosynthesis in G. biloba remain poorly characterized compared to those of co-occurring flavonol glycosides. Histochemical analysis revealed that PAs are broadly distributed across various tissues, with the highest accumulation observed in macrosporophylls, sarcotesta, young leaves, and calli. Integrating these findings and our previous research, two bHLH transcription factors, GbGLABRA3–1 and GbGLABRA3–2 were identified through phylogenetic analysis and co-expression profiling with the previously characterized MYB repressor GbMYBR1 . Protein-protein interaction assays, including yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), confirmed that GbGL3–2 physically interacts with GbMYBR1. Functional analyses in transgenic systems demonstrated that overexpression of GbGL3–2 in Arabidopsis thaliana (Col-0) altered flavonoid partitioning by reducing the flavonols content while increasing the levels of anthocyanins and PAs. In transgenic G. biloba calli, an antagonistic regulatory relationship was uncovered: GbMYBR1 suppresses PA biosynthesis, whereas GbGL3–2 promotes it. Mechanistically, yeast one-hybird (Y1H) and dual luciferase reporter assays showed that both transcription factors bind to the promoter of GbLAR . Collectively, these findings advance our understanding of PA biosynthesis in G. biloba and provide the first evidence of a repressor-activator module (GbMYBR1–GbGL3–2) in gymnosperms. This work establishes a transcriptional engineering framework for the targeted manipulation of PA accumulation in G. biloba , with promising implications for the production of functional G. biloba PAs.