Integrated ion, hormone, and proteomic analyses reveal the mechanisms of drought resistance in alfalfa (Medicago sativa)

Drought stress is a major environmental limiting factor limiting forage yield. Alfalfa (Medicago sativa) is a crucial perennial leguminous forage, yet its adaptive mechanisms to drought remain insufficiently understood. We compared ion homeostasis, endogenous hormone levels, and protein expression in a drought-resistant (‘WL168’) and a drought-sensitive (‘Gannong No. 3’) alfalfa variety under drought stress and rehydration. The results showed that that dynamic changes in ions, particularly Na+, K+, and Ca2+, were closely associated with the maintenance of cellular homeostasis and water balance under drought. Rehydration effectively restored ion balance, especially for K+ and Ca2+. Hormonal analysis suggested that ZT, GA3, IAA, ABA, and SA in roots participated in the response to drought and subsequent recovery. Integrated proteomic and WGCNA revealed key pathways, including phenylpropanoid biosynthesis, starch and sucrose metabolism, and glutathione metabolism, responding to drought stress. Critical proteins such as MMK1, BSK3, carbonic anhydrase, and MYB11 were identified as contributors to alfalfa’s drought resistance. This study provides new insights into the molecular mechanisms of drought resistance in alfalfa. The identified candidate genes and proteins offer valuable resources for future molecular-assisted breeding aimed at improving drought tolerance.