Contrasting responses of alfalfa (Medicago sativa) accessions to combined water and nutrient stress: integrated morphological, biochemical, and molecular analyses

Combined water deficit and nutritional deficiencies pose significant challenges to the productivity of alfalfa (Medicago sativa). We evaluated natural variation in 14 Tunisian accessions under combined stress, and identifed two contrasting genotypes, Radhouane (tolerant) and Kettana 1 (sensitive), for detailed analysis. Multivariate analyses, including Principal Component Analysis and clustering, showed clear separation of the two genotypes based on growth traits, with Radhouane showing enhanced root biomass and relative water content, while Kettana 1 exhibited reduced stem length. Biochemical assays demonstrated that Radhouane maintained lower oxidative damage, with no increase in hydrogen peroxide in leaves or roots and only a slight rise in malondialdehyde, while showing substantial enhancement in phenolic content (~250%) and antioxidant capacity (~86%). In contrast, Kettana 1 showed marked increase in hydrogen peroxide content and lipid peroxidation with a weaker antioxidant induction. Molecular analysis highlighted strong upregulation of key stress-related genes in Radhouane, including glutathione synthetase (~220% increase), ferredoxin-dependent glutamate synthase (~187%), NAC transcription factors, and notably aquaporin PIP2-1 in roots (~188%), facilitating enhanced water transport. Conversely, Kettana 1 displayed minimal gene induction. This characterisation reveals that Radhouane's tolerance stems from effective integration of morphological resilience, robust antioxidant defence, and coordinated molecular regulation, providing valuable targets for improving alfalfa stress tolerance under complex environmental conditions.