ABA Accumulation Confers Cross-Adaptation to Saline-Alkali and Cold Stresses in Alfalfa

Combined saline-alkali and cold stresses severely constrain plant growth at middle and high latitudes. Plants have evolved cross-adaptation mechanisms wherein exposure to one stress enhances resistance to another. However, the specific mechanisms driving cross-adaptation between saline-alkali and cold stresses in alfalfa (Medicago sativa L.) remain to be elucidated. Here, we performed integrated transcriptomic and metabolomic analyses coupled with functional validation to elucidate these mechanisms. We found that saline-alkali pretreatment significantly enhanced the tolerance of alfalfa to subsequent cold stress. Compared to cold stress alone, cross-stress conditions increased osmolyte content and photosynthetic efficiency, while alleviating cellular oxidative damage. Integrated omics analyses revealed that cross-stress specifically activated flavonoid biosynthesis, carbohydrate metabolism and abscisic acid (ABA) biosynthesis and signaling pathways. This promoted the accumulation of endogenous ABA, flavonoids, and carbohydrates. Weighted gene co-expression network analysis identified MsNCED3 as a critical hub gene. Exogenous ABA improved photoprotection and sugar accumulation, and enhanced cold tolerance. MsNCED3 overexpression in alfalfa validated its pivotal role in cross-adaptation by elevating ABA levels and mitigating oxidative damage. In conclusion, we found that MsNCED3-mediated ABA accumulation, along with enhanced antioxidant and osmotic adjustment capabilities, serve as key mechanisms underlying the cross-adaptation of alfalfa to saline-alkali and cold stresses.