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

Abstract

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.

Acknowledgements

We sincerely thank Ningjuan Fan, Xiyan Chen, Hui Duan, and Lanlan Wei from the Teaching and Research Core Facility at the College of Life Sciences, North West A&F University, for their technical support. This study was supported by the National Natural Science Foundation of China (31670301).

Author contributions

Juane Dong and Bin Zhang designed the research and made the final modifications. Xiumin Nie performed the experiments, analyzed the data, and wrote the manuscript. Xueying Li, Bingbing Lv, and Shuai Shao revised the paper.

Data availability

The data that support the results of this study can be found in this paper and its supplementary materials.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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