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 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 10 hub genes responsive to combined stress, with MsBAM1 potentially regulating the carbohydrate synthesis pathway and its silencing impaired alfalfa resistance to combined saline‐alkali and low‐temperature stress.

The accumulation of aniline in the natural environment poses a potential threat to crops, and thus, investigating the effects of aniline on plants holds practical implications for agricultural engineering and its affiliated industries. This study combined physiological, transcriptomic, and metabolomic methods to investigate the growth status and molecular-level response mechanisms of rice under stress from varying concentrations of aniline. At a concentration of 1 mg/L, aniline exhibited a slight growth-promoting effect on rice. However, higher concentrations of aniline significantly inhibited rice growth and even caused notable damage to the rice seedlings. Physiological data indicated that under aniline stress, the membrane of rice underwent oxidative damage. Furthermore, when the concentration of aniline was excessively high, the cells suffered severe damage, resulting in the inhibition of antioxidant enzyme synthesis and activity. Transcriptomic and metabolomic analyses indicated that the phenylpropanoid biosynthesis pathway became quite active under aniline stress, with alterations in various enzymes and metabolites related to lignin synthesis. In addition to the phenylpropanoid biosynthesis pathway, amino acid metabolism, lipid metabolism, and purine metabolism were also critical pathways related to rice’s response to aniline stress. Significant changes occurred in the expression levels of multiple genes (e.g., PRX, C4H, GST, and ilvH, among others) associated with functions such as antioxidant activity, membrane remodeling, signal transduction, and nitrogen supply. Similarly, notable alterations were observed in the accumulation of various metabolites (for instance, glutamic acid, phosphatidic acid, phosphatidylglycerol, and asparagine, etc.) related to these functions. Our research findings have unveiled the potential of compounds such as phenylpropanoids and amino acids in assisting rice to cope with aniline stress. A more in-depth and detailed exploration of the specific mechanisms by which these substances function in the process of plant resistance to aniline stress (for instance, utilizing carbon-14 isotope tracing to monitor the metabolic pathway of aniline within plants) will facilitate the cultivation of plant varieties that are resistant to aniline. This will undoubtedly benefit activities such as ensuring food production and quality in aniline-contaminated environments, as well as utilizing plants for the remediation of aniline-polluted environments.