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

ABSTRACT 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.