Integrated Physiological,Transcriptomic, and MetabolomicAnalyses Reveal the Mechanism Underlying the Response of Alfalfa toCombined Salt and Heat Stress

Salt and heat stresses often occur simultaneously in arid regions, restricting the distribution and productivity of alfalfa (Medicago sativa L.). However, the mechanisms underlying alfalfa responses to combined salt and heat stress remain unclear. Here, we integrated phenotypic, physiological, transcriptomic, and metabolomic analyses to investigate the regulatory mechanisms involved in combined stress responses. Combined stress more strongly inhibited plant height, stem diameter, fresh weight, net photosynthetic rate, and transpiration rate than single stresses. Salt stress had a predominant effect on several agronomic and physiological traits, whereas heat stress mainly affected chloroplast ultrastructure. Multiomics analysis identified flavonoid metabolism, linoleic acid metabolism, and amino acid biosynthesis as key pathways associated with combined stress responses. Moreover, CHS, CHR, P5CS, and LOX genes expression was closely correlated with metabolites such as naringenin, naringenin chalcone, and proline. These findings provide insights into alfalfa adaptation to multiple abiotic stresses.