Integrated Transcriptomic and Metabolomic Analyses Reveal Adaptive Mechanisms of Medicago sativa Under Water Stress

Water stress is a major abiotic constraint limiting the growth and productivity of alfalfa (Medicago sativa L.). To elucidate the adaptive mechanisms and identify key drought-tolerance genes, physiological measurements were integrated with multi-omics analyses of cultivar 'Tamu 1' under three water treatments: waterlogging (100% field water capacity), normal irrigation (80% FWC), and drought (light: 60% FWC, moderate: 40% FWC, severe: 20% FWC). Water stress markedly inhibited plant growth, induced oxidative stress, and reduced the photosynthetic capacity. Compared with waterlogging stress (DAMs: n = 71; DEGs: n = 313), drought stress resulted in a substantially greater number of differentially accumulated metabolites (DAMs, n = 1504) and differentially expressed genes (DEGs, n = 8006). Weighted gene co-expression network analysis (WGCNA) identified six key modules and ten hub genes associated with stress responses. Integrated transcriptomic and metabolomic analyses further revealed four major responsive pathways: starch and sucrose metabolism, phenylpropanoid and flavonoid metabolism, glutathione metabolism, and zeatin biosynthesis. Based on integrative criteria, including differential expression (|log₂FC| ≥ 1, adjusted p < 0.05), WGCNA modules significantly associated with drought-related traits (R² > 0.6), as well as functional annotation and protein-protein interaction (PPI) network topology, 28 candidate genes associated with drought tolerance were identified, of which six were further validated by quantitative real-time PCR (qRT-PCR). These findings highlight key metabolic pathways and regulatory modules underlying alfalfa responses to water stress and provide valuable candidate gene resources for improving drought tolerance.