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

Tanshinones are valuable compounds found in <i>Salvia miltiorrhiza</i>, 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 <i>SmWRKY32</i> overexpression transcriptome. Overexpression of <i>SmbHLH65</i> 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 <i>SmDXS2</i> and <i>SmCPS1</i>. Further investigation demonstrated that SmbHLH65 not only promotes the expression of <i>SmbHLH85</i> but also enhances its binding to the promoters of <i>SmDXS2</i> and <i>SmCPS1</i>, thereby amplifying the activation of these biosynthetic genes. Additionally, SmWRKY32 directly binds to the <i>SmbHLH65</i> promoter to suppress its activity. In summary, these findings reveal that the regulatory module SmWRKY32-SmbHLH65/SmbHLH85 controls tanshinone synthesis in <i>S. miltiorrhiza</i>. This study uncovers a novel transcriptional regulatory mechanism, offering fresh insights into the complex network controlling tanshinone biosynthesis.