DNA methylation changes in Medicago sativa under salt-alkaline stress and the function of 5-azacytidine in enhancing stress tolerance

Salt-alkali soils severely affect the yield and quality of alfalfa (Medicago sativa), yet the epigenetic mechanisms underlying salt-alkali tolerance remain poorly understood in forage plants. Here, we integrate transcriptome profiling with a chemical demethylation approach to reveal how 5-azacytidine (5-AzaC) remodels the methylome and enhances stress performance. Our research revealed that salt-alkali stress induced a decrease in methylation levels at CG and CHG sites, while methylation at CHH sites increased, with notable differences observed particularly in promoter and transposable element regions. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) enhanced alfalfa's tolerance to salt-alkali stress. Transcriptome analysis showed that this was associated with upregulation of genes related to phenylpropanoid metabolism, flavonoid biosynthesis, and glutathione metabolism. McrBC-PCR confirmed the presence of DNA methylation modifications in the promoter regions of key salt-alkali response genes 4CL, GCL, and HCT, and these genes were found to potentially interact with transcription factors such as NAC, bHLH, and ERF. These findings highlight the role of epigenetic regulation in alfalfa's salt-alkali tolerance, providing theoretical guidance for breeding resilient forage cultivars.