Transcriptomic Profiling and Functional Validation Reveal MYC2-PSK3 Mediating Salt-Alkali Tolerance in Alfalfa (Medicago sativa L.)

Soil salinization severely limits alfalfa productivity; however, the molecular mechanisms governing cultivar-specific differences in salt tolerance remain largely unclear. In this study, two alfalfa cultivars (Zhongmu No.3 and WL440-HQ) were exposed to 200 mM NaCl stress, followed by integrated transcriptome sequencing, weighted gene co-expression network analysis (WGCNA), and functional validation. In total, 3,517 salt-responsive differentially expressed genes (DEGs) were identified, including 795 shared DEGs and cultivar-specific DEGs (1,336 in Zhongmu No.3 and 1,386 in WL440-HQ). GO and KEGG enrichment revealed conserved stress-response pathways, including flavonoid biosynthesis and starch and sucrose metabolism, as well as cultivar-specific patterns, with Zhongmu No.3 strongly enriched in stimulus-responsive genes. WGCNA further identified phenotype-related modules and core hub genes, notably MsWRKY22 and MsPSK3. Overexpression of MsPSK3 enhanced salt-alkali tolerance in alfalfa by activating antioxidant systems. Dual-luciferase and yeast one-hybrid (Y1H) assays verified that MsMYC2 directly binds to and activates the MsPSK3 promoter. This study reveals the molecular regulatory network underlying alfalfa responses to salt–alkali stress and provides key candidate genes for breeding salt-tolerant alfalfa varieties.