Physiological and Transcriptomic Analyses Unravel Alfalfa's Response to Cadmium-Salt Stress and the Alleviating Role of Exogenous Salicylic Acid Application

Soil co-contamination with cadmium (Cd) and salinity imposes combined stress on plants in China. This study screened 40 alfalfa varieties, identifying a Cd-tolerant (’Concept’) and a Cd-sensitive (’SR4030’) cultivar, to elucidate their adaptive responses. Under combined Cd-salt stress (100 μM CdCl₂ + 150 mM NaCl), SR4030 exhibited more severe growth inhibition (e.g., biomass reduction up to 25%) and cellular damage than Concept, indicating synergistic stress effects. The cultivars showed distinct ion profiles: Concept accumulated less Cd²⁺ in shoots and had a lower translocation factor (TF) than SR4030. Salt stress increased shoot Cd²⁺ TF by approximately 7-fold in both cultivars. Although antioxidant enzymes were activated, elevated ROS and MDA indicated oxidative damage, exacerbated under combined stress. Transcriptome analysis revealed 1,401 and 1,378 more differentially expressed genes (DEGs) under combined stress in Concept at 12 h and 24 h, respectively, primarily enriched in plant hormone signaling and MAPK pathways. Concept specifically upregulated salicylic acid (SA)-related genes, whereas SR4030 downregulated jasmonic acid pathway genes. Heterologous expression confirmed MsCIPK enhanced yeast tolerance to both Cd and salt stress. Exogenous SA alleviated damage by reducing shoot Cd²⁺ content (by nearly 50% in SR4030) and Na⁺ accumulation, enhancing antioxidant activities, and regulating osmotic substances. These findings demonstrate the roles of MsCIPK and SA in mitigating combined stress, providing a theoretical basis for breeding tolerant alfalfa and managing co-contaminated soils.