Background DNA methylation plays a key role in regulating plant growth and development, secondary metabolism, and environmental adaptability and is affected by DNA methyltransferase (C5-MTase) and demethylase (dMTase). However, the response of C5-MTase and dMTase genes to abiotic stress in Isodon rubescens remains largely unknown. Results In this study, eight IrC5-MTase genes and five IrdMTase genes were identified within the genome of I. rubescens . The IrC5-MTases were phylogenetically divided into four subfamilies (CMT, DRM, MET and DNMT2), and the IrdMTase were divided into two subgroups (ROS and DML) on the basis sequence similarity, conserved motif and domain architecture. The IrC5-MTase and IrdMTase genes were unevenly distributed on 5 and 4 chromosomes, respectively. Cis‑acting element analysis revealed that the IrC5-MTase and IrdMTase genes were enriched with multiple light-, phytohormone- and stress-responsive elements. Importantly, the level of DNA methylation in I. rubescens was influenced by different 5-azacytidine (5-azaC) concentrations or light intensities but unaffected by different NaCl concentrations. Accordingly, the expression of IrC5-MTase and IrdMTase genes were significantly upregulated or downregulated in response to different concentrations of 5-azaC or amounts of light stress, however, no conspicuous differences were observed under different degrees of NaCl stress. Expression correlation analysis revealed that the expression of IrCMT2a , IrMET1 , and IrDML4 was most closely related to the DNA methylation level in I. rubescens . Conclusions Our results reveal the characteristics and differential expression of the IrC5-MTase and IrdMTase genes in response to 5-azaC, light or NaCl stresses and will facilitate future epigenetic studies in I. rubescens .
Ethnopharmacological relevance Isodon rubescens (Hemsl.) Hara, a traditional Chinese medicinal herb, demonstrates tumor inhibiting properties and therapeutic effecacy in treating esophageal squamous cell carcinoma (ESCC). However, its mechanism of action and the differential effects on varying ESCC differentiation grades remain unclear, despite the direct correlation between pathological grade and patient prognosis. Aim of the study To elucidate the effect heterogeneity of I. rubescens aqueous extracts on well-differentiated and poorly differentiated ESCC cell lines at metabolic level. Materials and methods A fusion strategy of network pharmacology combining targeted metabolomics was developed, focusing on key metabolic pathways through a “chemical components – targets – ESCC” network. Then, a targeted analysis method utilizing ultra-high performance liquid chromatography-multiple reaction monitoring tandem mass spectrometry (UPLC-MRM MS/MS) technology was established for core metabolites. The metabolic regulatory patterns of I. rubescens on two ESCC cell lines were determined through cytological experiments and targeted metabolomics analysis, with regulatory target enzymes verified through ELISA experiments. Molecular docking technology identified effective components acting on the regulatory enzymes. Results The primary regulatory pathways of I. rubescens in ESCC treatment encompassed central carbon metabolism, pyruvate metabolism, choline metabolism and retinol metabolism. A targeted metabolomics method analyzing 74 core metabolites in these pathways was established that had high accuracy and precision. The study confirmed heterogeneous effects of I. rubescens on well-differentiated KYSE-30 and poorly differentiated KYSE-150 cell lines. Citrate synthase (CS) expression varied significantly different between the two cell lines, showing upregulation after I. rubescens administration, albeit with varying degree. Molecular docking analysis revealed strong binding affinity between CS and four compounds: oridonin, quercetin, isorhamnetin, and ponicidin. Conclusions I. rubescens exhibits heterogeneous metabolic regulation in ESCC at different differentiation levels. CS was identified and confirmed as a crucial regulatory target, with oridonin, quercetin, isorhamnetin, and ponicidin serving as the primary active components.
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