Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots.

A Salvia miltiorrhiza R2R3-MYB gene, SmMYB9b , has been cloned and characterized. Overexpression of SmMYB9b resulted in a significant improvement of tanshinones, the lipophilic active ingredients in danshen hairy roots. Plant R2R3-MYB transcription factors play important roles in various physiological and biochemical processes. Danshen (Salvia miltiorrhiza bunge) is a valuable medicinal herb with tanshinones and salvianolic acids as the principal bioactive ingredients. A number of putative R2R3-MYB transcription factors have been identified in the plant, but their function remains to be studied. Here, we report the cloning of SmMYB9b, an S20 R2R3-MYB member and its regulatory properties. SmMYB9b contains an open reading frame of 792 bp in length and encodes a 264-amino acid protein. Its transcripts were most abundant in blooming flowers (except for calyces) and increased with flower development. Exogenous abscisic acid strongly activated its transcription. Gibberellins and methyl jasmonate also showed a time-dependent activation effect on its transcription, but to a weaker degree. Overexpression of SmMYB9b in danshen hairy roots enhanced tanshinone concentration to 2.16 ± 0.39 mg/g DW, a 2.2-fold improvement over the control. In addition to increased tanshinone concentration, the hairy root growth and lateral hairy root formation were also suppressed. KEGG pathway enrichment analysis with de novo RNAseq data indicated that stress-response-related metabolic pathways, such as the terpenoid and plant hormone signal transduction pathways, were significantly enriched, implying possible implication of SmMYB9b in such processes. Quantitative RT-PCR analysis showed that the transcription of terpenoid biosynthetic genes SmDXS2, SmDXR, SmGGPPS, and SmKSL1 was significantly up-regulated in danshen hairy roots over expressing SmMYB9b. These data suggest that overexpression of SmMYB9b results in enhanced tanshinone concentration through stimulation of the MEP pathway. The present findings shed new light on elucidating the roles of R2R3-MYB in the biosynthesis of diterpenoids in S. miltiorrhiza.