Unraveling alfalfa molecular adaptation to salinity and <i>Ascochyta medicaginicola</i> infection: insights from differential physiological traits and proteomics profiling

In real-life scenarios, plants are often exposed to biotic and abiotic stresses simultaneously; it is crucial to understand how they respond to single and combined stresses. This study explored the physiological and molecular responses of two contrasting alfalfa varieties, Gabes-2353 (tolerant) and Magna-601 (sensitive), to salinity and/or <i>Ascochyta medicaginicola</i> infection. Both varieties exhibited reductions in stem length, leaf relative water content, and chlorophyll content, with a greater decrease in Magna-601. Label-free LC‒MS/MS analysis identified 128 differentially abundant proteins (DAPs) between the two varieties, of which 14, 60, and 70 proteins were differentially regulated under salinity, Pm8 infection, and combined stresses, respectively. Gene Ontology analyses revealed that Gabes-2353 activated photosynthesis, metabolism, redox balance, and immune pathways, while Magna-601 disrupted carbohydrate metabolism. Pm8 infection induced immune pathways in both varieties, with antioxidant and metabolic proteins expressed in Gabes-2353, while protease inhibitors and defense proteins were expressed in Magna-601. Under combined stress, Gabes-2353 upregulated proteins associated with synergistic integration of metabolic and immune pathways. In contrast, Magna-601 activated broad stress-signaling responses but inhibited primary metabolism, aminoacyl-tRNA biosynthesis, and vitamin metabolism. The PPI and KEGG analyses identified 7-O-methyltransferase as a central hub in Gabes-2353, while Magna-601's PPI network focused on starch mobilization, reflecting differential adaptation strategies.