Time-Course Transcriptomic Analysis Identifies MsTIFY11B as a Promising Candidate Regulator of Salt–Alkali Tolerance in Alfalfa (Medicago sativa L.)

Soil salinization severely limits forage crop productivity, yet the regulatory networks that govern salt stress adaptation in alfalfa, a moderately salt-tolerant leguminous forage, remain largely unexplored. Here, we examined the physiological and transcriptomic dynamics of alfalfa leaves under 200 mM NaCl stress across three time points. Salt stress induced a progressive elevation of the Na+/K+ ratio, biphasic activation of antioxidant enzymes and concurrent accumulation of malondialdehyde. Time-course RNA-seq analysis identified 3631 differentially expressed genes (DEGs) and 132 core salt-responsive transcription factors (TFs). Pathway and functional annotation analyses indicated that these DEGs were prominently involved in cell wall biogenesis, redox homeostasis, and the carotenoid biosynthesis pathway, with carotenoid accumulation strongly activated under salt stress. Using weighted gene co-expression network analysis (WGCNA), nine distinct co-expression clusters were constructed. Notably, the brown module, which showed a positive correlation with Na+ accumulation and the Na+/K+ ratio, was significantly enriched in the plant hormone signal transduction pathway, within which 72.7% of the enriched genes belonged to the TIFY family. Among them, a core hub gene, MsTIFY11B, was isolated for functional characterization. Subcellular localization demonstrated that MsTIFY11B is exclusively localized to the nucleus. Heterologous expression in yeast showed that MsTIFY11B overexpression enhanced tolerance to salinity and alkalinity, whereas it conferred negligible protection against mannitol-induced drought stress. Taken together, our findings provide a comprehensive temporal framework of the alfalfa transcriptomic response to salinity and suggest that MsTIFY11B may contribute to salt–alkali tolerance, making it a promising candidate for further functional characterization and potential application in the development of stress-adapted alfalfa varieties.