CHH Hypomethylation in Promoters of Oxidoreductase Genes May Contribute to Salt‐Alkali Tolerance in Alfalfa ( Medicago sativa L.)

Salt-alkali stress severely impairs the quality and productivity of alfalfa (Medicago sativa), yet the role of epigenetic regulation remains unclear. To study the role of DNA methylation in salt-alkali tolerance, we conducted integrated whole-genome methylome and transcriptome analyses using two alfalfa cultivars with contrasting tolerance. Results showed that global methylation levels were only mildly affected by salt-alkali treatment, while a substantial number of differentially methylated regions emerged in the CHH context, particularly in promoter regions. Notably, the salt-alkali tolerant cultivar exhibited consistently lower CHH methylation in promoter regions than the sensitive cultivar, under both control and salt-alkali stress. Enrichment analysis showed that genes overlapping CHH-DMRs were associated with oxidoreductase activity. Five representative candidate genes-NCED, LOX2, LOX4, CuAO1 and CuAO2-were selected for validation. qRT-PCR and McrBC-PCR assays demonstrated that reduced promoter methylation was closely linked to stress-induced transcriptional activation. To test whether reduced DNA methylation contributes to tolerance, alfalfa seedlings were treated with 5-azacytidine, which enhanced antioxidant capacity under salt-alkali stress and supported a role for DNA demethylation in adaptation. Overall, this study highlights the importance of epigenetic regulation in forage adaptation to stress and provides a theoretical basis for future functional studies and molecular breeding.