Abstract 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.
Gao et al. (2025) studied this question.