New gene interaction network opens path to higher tanshinone yield in medicinal Danshen
image: A working model of the SmWRKY32-SmbHLH65/SmbHLH85 regulation of tanshinone biosynthesis. SmWRKY32 binds to the SmbHLH65 promoter, suppressing its expression. SmbHLH65 positively regulates tanshinone biosynthesis by forming a complex with SmbHLH85, which promotes synthesis by increasing SmDXS2 and SmCPS1 transcription. Additionally, SmbHLH65 stimulates the expression of SmbHLH85. SmWRKY32 indirectly inhibits tanshinone biosynthesis by suppressing the activation complex formed by SmbHLH65 and SmbHLH85. Image link: https://academic.oup.com/hr/article/12/7/uhaf096/8093018
Credit: Horticulture Research
Salvia miltiorrhiza (Danshen) is an important medicinal herb valued for tanshinones, which show notable therapeutic activity in treating cardiovascular disorders. Tanshinones are synthesized through a multi-step pathway involving key genes such as SmDXS2, SmCPS1, SmGGPPS1, and diverse cytochrome P450s. While individual transcription factors regulating this pathway have been identified, the interactive regulatory complexes that coordinate tanshinone synthesis remain unclear. Understanding how transcription factors jointly activate biosynthetic genes is critical for improving yield through bioengineering. Due to these challenges, deeper studies are needed to elucidate regulatory modules controlling tanshinone biosynthesis.
A research team from Northwest A&F University reported (DOI: 10.1093/hr/uhaf096) on March 25, 2025, in Horticulture Research, that they uncovered a regulatory module—SmWRKY32–SmbHLH65/SmbHLH85—that orchestrates tanshinone biosynthesis in Salvia miltiorrhiza. Through multi-omics screening, transgenic assays, yeast-based interaction tests, and dual-luciferase validation, the authors demonstrated that SmbHLH65 and SmbHLH85 promote tanshinone accumulation, while SmWRKY32 inhibits this pathway by repressing SmbHLH65 expression.
The researchers first identified SmbHLH65 from a transcriptome dataset of SmWRKY32-overexpressing lines, where SmbHLH65 expression was markedly reduced. Overexpressing SmbHLH65 increased total tanshinone content up to 2.9-fold, whereas RNA interference reduced production by ~45%. Because SmbHLH65 did not directly bind the biosynthetic promoters, interaction screening led to the identification of SmbHLH85, which strongly enhanced tanshinone biosynthesis. Overexpressing SmbHLH85 elevated dihydrotanshinone (DT), cryptotanshinone (CT), tanshinone I (TI), and tanshinone IIA (TIIA) by up to 250%, while silencing decreased total content by 31–60%. Yeast one-hybrid and EMSA assays confirmed that SmbHLH85 directly binds the promoters of SmDXS2 and SmCPS1 through E-box elements, activating transcription. Dual-luciferase assays revealed that SmbHLH65 enhances SmbHLH85-mediated promoter activity, forming a synergistic activation complex. Further experiments showed that SmWRKY32 directly binds the SmbHLH65 promoter via a W-box motif and represses transcription, suggesting an upstream inhibitory role. The authors propose a model in which SmWRKY32 suppresses SmbHLH65, while SmbHLH65 and SmbHLH85 cooperatively activate SmDXS2 and SmCPS1, collectively shaping tanshinone biosynthesis.
“The discovery of the SmbHLH65–SmbHLH85 activation module provides new insight into transcriptional coordination in tanshinone metabolism,” the research team stated. “By confirming that SmbHLH85 directly activates SmDXS2 and SmCPS1 while SmbHLH65 enhances this activation, we reveal a synergistic mechanism previously unknown in Salvia miltiorrhiza. Additionally, the inhibitory role of SmWRKY32 offers a regulatory layer that can potentially be targeted to boost metabolite production. Our work opens opportunities for metabolic engineering of medicinal plants.”
This regulatory module provides a strategic gene combination for boosting tanshinone production in medicinal plant cultivation. Upregulating SmbHLH65 or SmbHLH85, or suppressing SmWRKY32, could be applied in breeding, CRISPR editing, and synthetic biology systems to increase compound yield. The study highlights that coordinated transcription control is a promising approach for improving natural product output, offering a blueprint for enhancing other high-value phytochemicals. These results lay the foundation for developing high-tanshinone cultivars and scalable biosynthesis, advancing pharmaceutical resource supply.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhaf096
Funding information
This study was supported by the National Natural Science Foundation of China (31670301).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.