Cold plasma combined with plasma-activated water enhances chilling tolerance in alfalfa by improving antioxidant defense and altering membrane lipid composition

Low-temperature stress severely restricts the growth and productivity of alfalfa (Medicago sativa L.) by disrupting cellular redox homeostasis and membrane lipid stability. Cold plasma (CP) and plasma-activated water (PAW) have emerged as environmentally-friendly technologies for enhancing plant stress tolerance. However, their combined effects on chilling resistance and the underlying lipid-associated metabolic mechanisms remain poorly understood. In this study, alfalfa seeds were treated with CP combined with PAW under chilling conditions, and physiological responses together with lipidomic profiles were comprehensively investigated. The 100 Hz CP + PAW treatment significantly promoted seedling growth and biomass accumulation under non-stress conditions. Compared with the low-temperature control, it also enhanced the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), reduced reactive oxygen species (ROS) accumulation and malondialdehyde (MDA) content, and maintained chlorophyll levels under chilling stress. Lipidomic analysis identified 4779 lipid metabolites and revealed pronounced changes in membrane-lipid composition following CP + PAW treatment. Representative membrane lipids, including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and diacylglyceryl carboxyhydroxymethylcholine (DGCC), showed significant abundance alterations. Pathway enrichment analysis indicated that these differential lipids were mainly associated with unsaturated fatty acid biosynthesis, omega-9 fatty acid metabolism, and stress-related signaling pathways, including MAPK and G protein-related signaling. CP combined with PAW enhanced chilling tolerance in alfalfa by improving antioxidant defense and altering membrane lipid composition, thereby contributing to the maintenance of cellular redox balance and membrane stability under chilling stress. These findings provide new insights into the physiological responses and lipid-associated metabolic changes accompanying plasma-assisted chilling tolerance and support the potential application of CP + PAW as a sustainable and environmentally friendly strategy for improving stress resistance in alfalfa and other forage crops. Cold plasma (CP) combined with plasma-activated water (PAW) markedly improves chilling tolerance in alfalfa. CP+PAW enhances antioxidant defense and alleviates oxidative damage. CP+PAW contributes to the maintenance of membrane lipid homeostasis under chilling stress. Lipidomics reveals key lipid classes associated with cold adaptation. Coordinated redox regulation and maintenance of membrane lipid homeostasis underlie improved cold tolerance.