The SmWRKY32-SmbHLH65/SmbHLH85 regulatory module mediates tanshinone biosynthesis in Salvia miltiorrhiza

Tanshinones are valuable compounds found in Salvia miltiorrhiza, and gaining a deeper understanding of their transcriptional regulation mechanisms is a key strategy for increasing their content. Previous research revealed that SmWRKY32 acts as a repressor of tanshinone synthesis. This study identified the SmbHLH65 transcription factor, whose expression was significantly reduced in the SmWRKY32 overexpression transcriptome. Overexpression of SmbHLH65 stimulated tanshinone accumulation, while its silencing resulted in a decrease in tanshinone content. However, SmbHLH65 does not directly target the key enzyme genes involved in tanshinone synthesis. Subsequently, we discovered the SmbHLH65-interacting protein SmbHLH85. SmbHLH85 facilitates tanshinone biosynthesis by directly upregulating SmDXS2 and SmCPS1. Further investigation demonstrated that SmbHLH65 not only promotes the expression of SmbHLH85 but also enhances its binding to the promoters of SmDXS2 and SmCPS1, thereby amplifying the activation of these biosynthetic genes. Additionally, SmWRKY32 directly binds to the SmbHLH65 promoter to suppress its activity. In summary, these findings reveal that the regulatory module SmWRKY32-SmbHLH65/SmbHLH85 controls tanshinone synthesis in S. miltiorrhiza. This study uncovers a novel transcriptional regulatory mechanism, offering fresh insights into the complex network controlling tanshinone biosynthesis.