DNA methylation controls the expression of tanshinone synthesis genes and the tanshinone accumulation in Salvia miltiorrhiza and Salvia bowleyana

DNA methylation plays pivotal roles in regulating gene expression and the secondary metabolism in plants. Salvia miltiorrhiza and Salvia bowleyana are traditional Chinese medicinal plants with roots enriched with tanshinone components. However, the regulatory mechanism of DNA methylation on tanshinone production remains elusive. Here, we analyzed 30-day-old hairy roots of S. miltiorrhiza and S. bowleyana using targeted high-performance liquid chromatography analysis and found significantly higher tanshinone content in S. miltiorrhiza. Whole-genome bisulfite sequencing revealed elevated DNA methylation levels in S. miltiorrhiza, potentially due to the upregulation of methylation-related genes, including DOMAINS REARRANGED METHYLTRANSFERASE 1 (DRM1), DECREASE IN DNA METHYLATION 1 (DDM1), CHROMOMETHYLASE 2 (CMT1), and CHROMOMETHYLASE 3 (CMT3), alongside the low expression of the demethylase gene REPRESSOR OF SILENCING 1 (ROS1) in S. miltiorrhiza. Additionally, four genes that are involved in tanshinone biosynthesis, including 1-DEOXY-D-XYLULOSE-5-PHOSPHATE REDUCTASE (DXS1), GERANYLGERANYL DIPHOSPHATE SYNTHASE (GGPPS2), 4-HYDROXY-3-METHYLBUT-2-ENYL PYROPHOSPHATE REDUCTASE (HDR2), and COPALYL PYROPHOSPHATE SYNTHASE (CPS3), showed lower methylation levels in the promoters of DXS1, GGPPS2, and CPS3 and a higher DNA methylation level in the gene body of HDR2 in S. miltiorrhiza, which may lead to their high expression and the accumulation of tanshinones. Consistently, overexpression of the SmCMT3 in S. miltiorrhiza significantly reduced the contents of cryptotanshinone, tanshinone I, and tanshinone IIA. Transcriptomic and methylome analyses confirmed that the expression levels of the tanshinone biosynthesis-related genes, including SmMK, SmCPS1, SmDXS2, and SmAACT1, were correlated with their promoter or gene body DNA methylation levels. Our findings reveal that DNA methylation critically regulates tanshinone biosynthesis in S. miltiorrhiza and S. bowleyana, offering valuable insights for breeding.