Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza

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.