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Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus  Epimedium  plants, several species of which are used as materials for  Herba Epimedii , an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from  Epimedium sagittatum , but a  R2R3-MYB  transcription factor regulating the flavonol synthesis has not been functionally characterized so far in  Epimedium  plants. In this study, we isolated and characterized the  R2R3-MYB  transcription factor  EsMYBF1  involved in regulation of the flavonol biosynthetic pathway from  E. sagittatum . Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that  EsMYBF1  strongly activated the promoters of  EsF3H  (flavanone 3-hydroxylase) and  EsFLS  (flavonol synthase), but not the promoters of  EsDFRs  (dihydroflavonol 4-reductase) and  EsANS  (anthocyanidin synthase) in transiently transformed  Nicotiana benthamiana  leaves. Both yeast two-hybrid assay and transient reporter assay validated  EsMYBF1  to be independent of  EsTT8 , or  AtTT8 bHLH  regulators of the flavonoid pathway as cofactors. Ectopic expression of  EsMYBF1  in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the  EsMYBF1  overexpression lines, including  NtCHS  (chalcone synthase),  NtCHI  (chalcone isomerase),  NtF3H  and  NtFLS , whereas the late biosynthetic genes of the anthocyanin pathway ( NtDFR  and  NtANS ) were remarkably downregulated, compared to the controls. These results suggest that  EsMYBF1  is a flavonol-specific  R2R3-MYB  regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in  E. sagittatum . Thus, identification and functional characterization of  EsMYBF1  provide insight into understanding the biosynthesis and regulation of the flavonol-derived BCs in  Epimedium  plants, and also provide an effective tool gene for genetic manipulation to improve the flavonol synthesis.

A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum