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 0.6), 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.
Song et al. (2026) studied this question.