Discovery of the roles of <scp>SmMAPK4</scp> in regulating tanshinone biosynthesis in <i>Salvia miltiorrhiza</i>

Tanshinones, the primary bioactive components found in Salvia miltiorrhiza, are widely utilized in the treatment of cardiovascular diseases; however, the molecular mechanisms that govern their biosynthesis remain unclear at the protein phosphorylation level. The proteomic profiles of roots, stems, leaves, and flowers of S. miltiorrhiza identified a total of 8301 proteins. A novel MITOGEN ACTIVATED PROTEIN KINASE 4 (SmMAPK4) was identified to be positively correlated with the accumulation of tanshinones in S. miltiorrhiza through tissue-specific metabolomics and proteomics analyses. Meanwhile, through phosphoproteomic analysis of SmMAPK4 overexpression lines, the BASIC HELIX-LOOP-HELIX family transcription factor, BASIC HELIX-LOOP-HELIX TANSHINONE SYNTHESIS 1 (SmBTS1), was screened. The positive regulatory role of SmBTS1 in tanshinone biosynthesis was confirmed through transgenic assays. Multiple lines of evidence demonstrated that SmMAPK4 could interact with and phosphorylate SmBTS1 in a serine 156- and serine 159-dependent manner. SmMAPK4-phosphorylated SmBTS1 exhibited enhanced nuclear accumulation and increased transcriptional activation, promoting the expression of the tanshinone-biosynthetic gene COPALYL DIPHOSPHATE SYNTHASE 1 (SmCPS1) through its specific binding to the E2-box of the SmCPS1 promoter region. Taken together, these findings open new possibilities for biotechnological strategies to boost tanshinone production, providing new insights into its metabolic regulation at the protein phosphorylation level.