Background: The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites. Results: In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes’ behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones. Conclusion: The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Phenolic acids are the main active ingredients in Salvia miltiorrhiza, which can be used for the treatment of many diseases, particularly cardiovascular diseases. It is known that salicylic acid (SA) can enhance phenolic acid content, but the molecular mechanism of its regulation is still unclear. The nonexpresser of PR genes 1 (NPR1) plays a positive role in the SA signaling pathway. In this study, we identified a SmNPR1 gene that responds to SA induction and systematically investigated its function. We found that SmNPR1 positively affected phenolic acid biosynthesis. Then, we identified a novel TGA transcription factor, SmTGA2, which interacts with SmNPR1. SmTGA2 positively regulates phenolic acid biosynthesis by directly up-regulating SmCYP98A14 expression. After double-gene transgenic analysis and other biochemical assays, it was found that SmNPR1 and SmTGA2 work synergistically to regulate phenolic acid biosynthesis. In addition, SmNPR4 forms a heterodimer with SmNPR1 to inhibit the function of SmNPR1, and SA can alleviate this effect. Collectively, these findings elucidate the molecular mechanism underlying the regulation of phenolic acid biosynthesis by SmNPR1-SmTGA2/SmNPR4 modules and provide novel insights into the study of the SA signaling pathway regulating plant secondary metabolism.

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca²⁺ signaling is essential for the LPS-triggered immune response, Ca²⁺ channel blocker LaCl₃ and CaM antagonist W-7 were used to investigate the role of Ca²⁺ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl₃ and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca²⁺ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca²⁺ signaling. Ca²⁺ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza.

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.

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H⁺-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H⁺-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H⁺-ATPase isoforms were identified and named SmPHA1–SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H⁺-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H⁺-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.