Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analyzed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes had always higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza. DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2, CMK, IDI1, HMGR2, DXR, MDS, CYP76AH1, 2OGD25 and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.

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丹参甲基化组特征及丹参酮生物合成中DNA甲基化的调控机制

丹参是一种模式药用植物，具有重要的经济和药用价值。它的根产生一组二萜类亲脂性生物活性成分，称为丹参酮。丹参酮的生物合成和调控引起了广泛的兴趣。然而，丹参的甲基化尚未被分析，并且丹参酮生产中DNA甲基化的调控机制在很大程度上是未知的。在这里，我们报告了根和叶的单碱基分辨率 DNA 甲基化组。比较分析揭示了 CG、CHG 和 CHH 背景的差异甲基化模式以及 DNA 甲基化与基因和小 RNA 表达之间的关联。低甲基化基因总是具有较高的表达水平，24 核苷酸 sRNA 可能是丹参 RdDM 途径的关键参与者。DNA甲基化变异分析表明CHH甲基化是造成差异的主要原因。Go富集分析表明，与March_root相比，July_root中二萜生物合成过程与hypoCHHDMR下游重叠的基因显着富集。与3月份相比，7月份收集的根部基因启动子或下游区域的丹参酮生物合成相关酶基因，如DXS2、CMK、IDI1、HMGR2、DXR、MDS、CYP76AH1、2OGD25和CYP71D373的CHH甲基化程度较低。一致地，与 3 月份相比，7 月份收集的丹参根中的基因表达上调，并且 DNA 甲基化抑制剂 5-氮杂胞苷的处理显着促进了丹参酮的产生。这表明DNA甲基化通过改变关键酶基因启动子或下游的CHH甲基化水平在丹参丹参酮生物合成中发挥重要的调控作用。