Integrated Methylome and Transcriptome Analyses Reveal Methylation-Associated Cadmium Stress Responses in Sophora...

Cadmium (Cd) is a highly toxic heavy metal that impairs plant growth, metabolism, and the accumulation of bioactive compounds. To investigate methylation-associated Cd responses in the medicinal plant Sophora tonkinensis, we integrated whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing under three Cd treatments (T0, T2, and T4). Cd stress induced extensive transcriptional reprogramming and widespread DNA methylation changes, with CHH methylcytosines accounting for the largest proportion of methylated sites, whereas CG sites showed the highest average methylation level. Differentially methylated regions (DMRs) were predominantly detected in the CHH context and were frequently located in promoter and flanking regions. Integrated analysis identified 6547 and 6204 differentially methylated genes in T2 vs. T0 and T4 vs. T0, respectively, and 420 and 612 genes, respectively, showing concurrent changes in DNA methylation and transcript abundance. Genes with hypermethylation and reduced expression were more frequent than hypomethylated/upregulated genes and were mainly associated with photosynthesis, carbon fixation, fatty acid metabolism, sulfur-related metabolism, and secondary metabolic pathways potentially related to medicinal quality. Among the hypomethylated/upregulated genes, the hormone-related candidate gene StGH3.1 was selected for functional validation, and heterologous overexpression of StGH3.1 enhanced Cd tolerance in transgenic Nicotiana benthamiana. These results indicate that Cd stress is accompanied by coordinated methylome and transcriptome remodeling in S. tonkinensis and provide methylation-associated candidate genes for further investigation of Cd-responsive adaptation.