Cloning, molecular characterization and functional analysis of a putative R2R3-MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba

Abstract

The water-soluble phenolic acids of S. miltiorrhiza Bge. f. alba can protect against herbivores or pathogenic bacteria and have numerous pharmacological activities that are beneficial to human health. The main phenolic acids in S. miltiorrhiza, including rosmarinic acid and salvianolic acid B, are derived from the rosmarinic acid biosynthetic pathway, which has been partially characterized. However, little is known about the endogenous bHLH and MYB transcription factors that function in this pathway. In this paper, we describe the cloning and functional characterization of SmPAP1, a cDNA of the R2R3-MYB transcription factor isolated from S. miltiorrhiza Bge. f. alba. SmPAP1 contains an open reading frame of 645 bp in length and encodes a putative 214-amino acid protein. The deduced amino acid sequence of the N-terminal R2R3 repeat sequence of SmPAP1 shares high identity with other DNA-binding domains of plant MYB-type proteins. Expression studies have indicated that SmPAP1 is mainly expressed in leaves. The expression of SmPAP1 was induced by 0.1 mM methyl jasmonate, salicylic acid and abscisic acid in the hairy roots of S. miltiorrhiza Bge. f. alba. Yeast two-hybrid assays have demonstrated that SmPAP1 can interact with the bHLH factor SmMYC2 and heterologous AtMYC2, which are TFs known to induce anthocyanin synthesis in Arabidopsis. A transient co-expression experiment in tobacco leaves showed that SmPAP1 activated the promoters of two key rosmarinic acid biosynthetic pathway genes of S. miltiorrhiza, SmPAL1 (phenylalanine ammonia lyase) and SmC4H (cinnamic acid 4-hydroxylase). The overexpression of SmPAP1 induced the substantial accumulation of rosmarinic acid, salvianolic acid B, total phenolics and total flavonoids in the roots of transgenic S. miltiorrhiza Bge. f. alba. These data suggest that SmPAP1 is involved in the regulation of the phenolic acid biosynthetic pathway by interacting with bHLH TFs and activating genes encoding key enzymes in S. miltiorrhiza Bge. f. alba.