Development and application of a Ginkgo biloba L. callus-derived protoplast transient expression system for exploring the roles of GbMYB11 and GbbHLH3 in flavonoid metabolism

Flavonoids are characteristic metabolites of plants and function as a central biosynthetic component essential for life in terrestrial environments. However, the molecular mechanisms of flavonoids in ginkgo remained unclear due to the lack of an adequate genetic transformation system. Here we successfully developed a reliable and efficient method for isolating protoplasts, along with a PEG-mediated transient protoplast transformation system utilizing ginkgo callus. Additionally, in conjunction with the metabolomic approach, the levels of various flavonoid metabolites increased to varying extents in protoplasts following transient transformation with GbMYB11, GbbHLH3, or both. Protoplast subcellular localization results demonstrated that GbMYB11 and GbbHLH3 localized to the nucleus. Protein-protein interaction and qRT-PCR results indicated that GbMYB11 may interact with GbbHLH3 to bind to the promoters of multiple structural genes in anthocyanin metabolism, particularly GbLAR , GbANS , and GbDFR , thereby increasing anthocyanin accumulation. Our research establishes a foundation for elucidating the mechanisms of flavonoid metabolism, thereby facilitating genetic improvement in ginkgo. • An efficient method for protoplast isolation and a transient transformation system was developed in the ginkgo callus. • A metabolomic approach was used to explore gene functions and regulatory mechanisms in ginkgo flavonoid biosynthesis. • Combining protoplast transient expression and metabolic analysis to explore flavonoid metabolism and gene expression links. • This study established a ginkgo protoplast system to clarify biochemical mechanisms, aiding genetic enhancement and breeding.