Tanshinones are major bioactive components in Salvia miltiorrhiza and are widely used in cardiovascular therapies. However, their naturally low content limits pharmaceutical utilization. This study reveals a transcriptional regulatory module involving SmWRKY32, SmbHLH65, and SmbHLH85 that directly shapes tanshinone biosynthesis. The researchers demonstrate that SmbHLH65 and SmbHLH85 act as positive regulators promoting tanshinone accumulation, while SmWRKY32 functions as a suppressor by downregulating SmbHLH65. Overexpressing SmbHLH65 or SmbHLH85 significantly increases tanshinone levels, whereas silencing these factors decreases production. These findings uncover a coordinated gene–protein interaction network providing new molecular targets for metabolic engineering to enhance tanshinone yield.

Terpenoids are among the most pharmacologically valuable plant metabolites, yet their biosynthetic gene clusters in Euphorbiaceae have remained largely unexplored. This study establishes a comprehensive genome-wide identification framework and analyzes terpene gene clusters using multi-omics data. A total of 1824 candidate clusters were detected in seven Euphorbiaceae species, and 16 were confirmed as high-confidence terpene clusters after strict screening based on TPS/CYP pairing, copathway linkage, and coexpression patterns. Notably, casbene and casbene-derived diterpenoid gene clusters were identified, providing new clues to the biosynthesis of bioactive compounds such as neocembrene, ingenanes, and jatrophanes. This work lays a foundation for metabolic engineering and drug development linked to Euphorbiaceae terpenoids.