Multi-Omics Integrative Analysis Provided New Insights into the Salt Tolerance of Two “Zhongtian” Series Alfalfa (Medicago sativa L.)

Salinity is a major limiting factor for alfalfa production. This study analyzed the differential regulatory mechanisms of ZT1 and ZT2 under salt stress (100 and 200 mM) through physiological and biochemical responses, the photosynthetic system, and transcriptome and metabolome. The results show that ZT1 is more tolerant than ZT2. Under salt stress, root vitality (30.95–66.28%), shoot dry weight (13.23–53.01%), and chlorophyll a (20.00–50.00%) decreased significantly. However, Na+/K+ (0.93–3.62 times), MDA (0.19–2.52 times), and superoxide dismutase (28.94–79.56%) increased significantly. From a physiological perspective, ZT1 and ZT2 can endow plants with salt tolerance by regulating the Na+/K+ balance, inducing osmotic agents, enhancing antioxidant activity, and regulating the photosynthetic system. In omics analysis, there were significant differences in their regulation of the biosynthetic pathways of phenylpropanin and flavonoids. ZT1’s salt tolerance is strengthened by the positive regulation of transcription factors (GRAS) and genes (CHS, POD, CAD, F3H, and PAL), together with the accumulation of (-)-epicatechin, eriodictyol, and butein. In contrast, ZT2 responded positively to salt stress via the regulation of TFs (GRAS, TRAF, and bHLH) and genes (POD, C4H, CHS, and F3′5′H), as well as the accumulation of caffeic acid. The research results will provide new insights into alfalfa cultivation and new variety breeding in saline–alkali land.