Identifying potential transcription factors involved in low-phosphorus stress-induced biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge is valued for its primary bioactive compounds, tanshinones (TAs) and phenolic acids (PHAs). Low phosphorus (LP) stress, as a critical environmental factor, significantly influences the TAs and PHAs biosynthesis. However, the regulatory mechanisms underlying this process remain largely elusive. In this study, S. miltiorrhiza hairy roots were subjected to LP treatment and it was found that LP stress promoted the accumulation of PHAs whereas suppressed TAs biosynthesis. Integrating transcriptome data with co-expression network analysis, eight potential candidate transcription factors (TFs) responsive to LP stress were identified to be associated with the biosynthesis of TAs and PHAs. Among them, both SmHY5b and SmERF1 were validated to directly bind to the promoter of SmTAT1, a critical gene in the PHAs biosynthetic pathway, using yeast one-hybrid (Y1H) assay. Dual‑luciferase (Dual‑LUC) assay revealed that SmERF1 activates, whereas SmHY5b represses, the transcriptional activity of the SmTAT1 gene. Additionally, Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay revealed that SmERF1 interacted with phosphorus starvation response protein SmSIZ2. Collectively, this study mines several candidate transcriptional regulators directly linked to LP stress-induced modulation of TAs and PHAs biosynthesis, and lays the foundation for breeding high-quality S.miltiorrhiza varieties through metabolic engineering.