Metabolo-transcriptomics analyses reveal alfalfa adaptation to combined saline-alkali and low-temperature stress in the field

Alfalfa (Medicago sativa L.) is known as the "King of Forage" because of its high protein content and excellent palatability. Alfalfa productivity is adversely affected in cold regions with saline-alkali soils. However, the mechanisms underlying the response of alfalfa to combined saline-alkali and low-temperature stress under field conditions remain unknown.The resistance of two alfalfa cultivars (resistant ZD, sensitive BM) in saline-alkali soil during cold and freezing periods was investigated using transcriptomic and metabolomic analyses.Metabolomic analysis revealed specific accumulation of amino acids, organic acids, carbohydrates, fatty acids, and flavonoids in ZD compared with that in BM. The core differential metabolites associated with ZD stress resistance included L-isoleucine, fumaric acid, raffinose, 2-hydroxydocosanoic acid, and isovitexin. Transcriptomic analysis revealed that ZD enriched more upregulated differentially expressed genes in galactose, starch, and sucrose metabolism and flavonoid biosynthesis pathways than BM. Integrated metabolo-transcriptomic analysis highlighted flavonoid, carbohydrate, glutathione, and salicylic acid biosynthesis as key pathways in alfalfa stress response.WGCNA identified ten hub genes responsive to combined stress, with MsBAM1 potentially regulating carbohydrate synthesis pathway and its silencing impaired alfalfa resistance to combined saline-alkali and low-temperature stress.