SmbHLH60 and SmMYC2 antagonistically regulate phenolic acids and anthocyanins biosynthesis in Salvia miltiorrhiza

Salvia miltiorrhiza is a renowned traditional Chinese medicinal plant with extremely high medicinal value, especially for cardiovascular and cerebrovascular diseases. The jasmonic acid (JA) signaling pathway plays an important role in the improved biosynthesis of secondary metabolites, which is mediated by a major transcriptional regulator, MYC2. However, the JA regulatory mechanism of secondary metabolites biosynthesis in S. miltiorrhiza is still largely unknown. Our work focuses on the dissection of the molecular mechanism of transcriptional regulation in MeJA-mediated biosynthesis of medicinal components of S. miltiorrhiza. We examined the role of MeJA-responsive bHLH transcription factors (TFs) in improving bioactive secondary metabolites accumulation in S. miltiorrhiza. Hairy root transformation based on CRISPR/Cas9 technique was used to decipher gene function(s). Changes in the content of phenolic acids were evaluated by HPLC. Y1H, EMSA and dual-LUC assays were employed to analyze the molecular mechanism of SmbHLH60 in the regulation on the biosynthesis of phenolic acids and anthocyanins. Y2H, BiFC and pull-down affinity assays were used to corroborate the interaction between SmbHLH60 and SmMYC2. Being one of the most significantly negatively regulated bHLH genes by MeJA, a new transcription factor SmbHLH60 was discovered and characterized. Over-expression of SmbHLH60 resulted in significant inhibition of phenolic acid and anthocyanin biosynthesis in S. miltiorrhiza by transcriptionally repressing of target genes such as SmTAT1 and SmDFR, whereas CRISPR/Cas9-generated knockout of SmbHLH60 resulted in the opposite effect. In addition, SmbHLH60 and SmMYC2 formed a heterodimer to antagonistically regulate phenolic acid and anthocyanin biosynthesis. Our results clarified that SmbHLH60 is a negative regulator on the biosynthesis of phenolic acids and anthocyanins. SmbHLH60 competed with SmMYC2 in an antagonistic manner, providing new insights for the molecular mechanism of MeJA-mediated regulation on the biosynthesis of secondary metabolites in S. miltiorrhiza.