Soil salinization is one of the major ecological challenges threatening global agricultural production, severely restricting crop growth, yield formation, and quality improvement. Alfalfa (Medicago sativa), as an important perennial legume forage, is particularly constrained by salt-alkali stress, while the epigenetic regulatory mechanisms underlying its response remain largely unknown. DNA methylation, as a key epigenetic modification, plays an essential role in plant adaptation to abiotic stresses.

This study aimed to systematically identify DNA methylation-related gene families in alfalfa, characterize their expression patterns under salt-alkali stress, and further explore the role of DNA methylation in salt-alkali tolerance by applying the DNA methylation inhibitor 5-azacytidine (5-AzaC), thereby providing the theoretical insights for the genetic improvement of salt-alkali-tolerant alfalfa.

Based on the reference genome of alfalfa, DNA methyltransferase and demethylase genes were identified genome-wide, and their functions were inferred through phylogenetic analysis and conserved domain annotation. RT-qPCR was employed to analyze the expression patterns of these genes under salt-alkali stress. Using the cultivar Gannong No. 3 as plant material, a hydroponic salt-alkali stress system was established. Different concentrations of 5-AzaC were applied as pretreatments, and the optimal concentration was selected for subsequent assays. Plant growth, physiological, and photosynthetic parameters were then measured to evaluate the regulatory role of 5-AzaC in alfalfa salt-alkali tolerance.

A total of 13 DNA methyltransferase genes and 4 DNA demethylase genes were identified in alfalfa, all of which were localized to the nucleus with complete conserved domains. Expression analysis revealed that MsCMT4, MsCMT6, MsCMT8, and MsDML2 were significantly upregulated under salt-alkali stress, indicating the involvement of both methylation and demethylation processes in stress responses. Physiological analyses showed that 100 μmol·L^-1 5-AzaC significantly alleviated growth inhibition caused by salt-alkali stress, so plant height, fresh weight, and dry weight increased by 12.62%, 23.50%, and 18.67%, respectively, compared with the control. Regarding chlorophyll metabolism, 5-AzaC suppressed the expression of chlorophyll degradation-related genes (PAO, CAO, NYC), thereby delaying pigment degradation. Photosynthetic analysis indicated that 5-AzaC treatment markedly increased net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), as well as the quantum efficiency of photosystem II (YII) and photochemical quenching (qP), suggesting enhanced stability and efficiency of the photosynthetic system. In terms of osmotic adjustment, 5-AzaC promoted soluble sugar accumulation (+37.21%) but had no significant effect on soluble protein. Reactive oxygen species (ROS) measurements showed that 5-AzaC reduced H₂O₂ and O₂^-·levels by 22.8% and 35.8%, respectively, while superoxide dismutase (SOD) and catalase (CAT) activities increased by 13.58% and 21.82%, respectively, indicating that 5-AzaC enhanced antioxidant capacity and alleviated oxidative damage.

This study systematically characterized DNA methylation-related gene families in alfalfa and their responses to salt-alkali stress, revealing the pivotal role of DNA methylation in shaping salt-alkali tolerance. Exogenous application of 5-AzaC improved alfalfa tolerance by maintaining photosystem stability, enhancing photosynthetic efficiency, promoting osmolyte accumulation, and strengthening ROS scavenging capacity. These findings provided the new experimental evidence for understanding the epigenetic mechanisms of forage adaptation to abiotic stress and offer theoretical guidance for the improvement and utilization of salt-alkali-tolerant alfalfa germplasm.