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Herba epimedii ( Epimedium ), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway.  Epimedium  species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in  Epimedium . Here, we reported the isolation and functional characterization of the first  R2R3-MYB  TF ( EsMYBA1 ) from  Epimedium sagittatum  (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes  Arabidopsis  AtPAP1, apple MdMYB10 and legume MtLAP1.  EsMYBA1  was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of  EsMYBA1  resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase ( DFR ) and anthocyanidin synthase ( ANS ). In both transgenic tobacco and  Arabidopsis , overexpression of  EsMYBA1  induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of  EsMYBA1  in  E. sagittatum  leaves by  Agrobacterium  infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in  Epimedium  species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

A R2R3-MYB Transcription Factor from Epimedium sagittatum Regulates the Flavonoid Biosynthetic Pathway