Abstract
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.
Highlights
Flavonoids are a large group of diverse plant secondary metabolites that are derived from phenylalanine and malonylcoenzyme A, including anthocyanins, flavonols and proanthocyanins (PAs, known as condensed tannins) that accumulate in a wide variety of plant tissues [1]
Isolation and Sequence Analysis of EsMYBA1 Gene Degenerate PCR primers, based on conserved residues of R2 and R3 MYB domains, amplified a 233-bp band from mRNA isolated from E. sagittatum leaves
The EsMYBA1 full-length cDNA (FLC) used for functional analysis contains an ORF of 714 bp that encodes a R2R3-MYB transcription factors (TFs) comprised of 237 amino acids
Summary
Flavonoids are a large group of diverse plant secondary metabolites that are derived from phenylalanine and malonylcoenzyme A, including anthocyanins (red to purple pigments), flavonols (colorless to pale pigments) and proanthocyanins (PAs, known as condensed tannins) that accumulate in a wide variety of plant tissues [1]. The flavonoid biosynthetic pathway is one of the most extensively studied pathways of plant secondary metabolites [4,5]. The structural genes of the flavonoid biosynthetic pathway are largely regulated at the level of transcription. The maize MYB gene (ZmC1) regulates the anthocyanin pathway by interacting with a bHLH partner (ZmR or ZmB) to activate the DFR (ZmA1) promoter [10]
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