The R2R3-MYB Transcriptional Activator MYB75 And MVB90 Are Involved In The Regulation Of The JA-Induced Phenolic Acid Synthesis In Salvia Miltiorrhiza

Danshen,the dried root or rhizome of Salvia miltiorrhiza Bunge,is a traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases.R2R3-MYB transcription factor is one of the largest transcription factors of the plant kingdom.R2R3-MYB transcription factors(TFs)are key regulators of the phenylpropanoid pathway which was the upstream pathway of anthocyanins,proanthocyanidins,flavonols,condensed tannins,lignins and phenolic acids in plants.Previous reports showed that MYB,b HLH and WD40 formed a MBW complex(MYBb HLH-WD40 complex)involved in the JA signal pathway and regulated the accumulation of secondary metabolites by regulating the downstream genes.Me JA dramatically improved the accumulation of salvianolic acid in S.miltiorrhiza,but the molecular mechanism is still unclear.This paper identified two novel R2R3-MYB TFs SmMYB75 and SmMYB90 from S.miltiorrhiza cDNA library that were remarkably up-regulated by Me JA.Quantitative real-time PCR(q RT-PCR)was used to determine the expression patterns of SmMYB75,SmMYB90 and tissue expression patterns indicated that SmMYB75 and SmMYB90 were expressed ubiquitously in all tissues and the highest expressions were in stem,leaf and petiole.Induction patterns indicated that SmMYB75 and SmMYB90 were increased in response to YE,ABA,SA,Me JA,GA3 and Eth treatment at different levels and more sensitive to Me JA than others.These expression patterns were consistent with the genes patterns involved in phenolic acids synthesis.Promoter analysis showed the promoter region of SmMYB75 and SmMYB90 contains cis-acting regulatory element involved in the Me JA-responsiveness,indicating that SmMYB75 and SmMYB90 might be involved in the JA metabolism pathway.Subcellular localization results showed that SmMYB75 and SmMYB90 are located in the nucleus.Genetic transformation experiments showed that overexpression of SmMYB75 and SmMYB90 increased phenolic acids accumulation by regulating the expression of phenolic acids synthesis-related genes in S.miltiorrhiza,indicating that SmMYB75 and SmMYB90 were the transcription activators of phenolic acids biosynthesis and the transcription repressor of tanshinones synthesis.Meanwhile,SmMYB75 and SmMYB90 improved the accumulation of anthocyanins.Yeast-one hybrid(Y1H)showed SmC4 H and SmHPPR were selfactivated and this experiment should to be further optimized.Yeast two hybrid(Y2H)suggested that SmMYB75 and SmMYB90 proteins could only interact with Sm JAZ9,a JAZ proteins that could improve the accumulation of tanshinones.And SmMYB75 and SmMYB90 proteins also interact with SmGL3 and Sm WD40 proteins indicated that SmMYB75 and SmMYB90 might formed a MBW complex involved in the JA signal pathway.In summary,SmMYB75 and SmMYB90 might interact with SmGL3 and Sm WD40 form a MBW complex to improve the phenolic acids synthesis by regulating the key genes of phenolic acids synthesis pathway.