Metabolic Response of Trifolium ambiguum to Low-temperature Stress based on Widely Targeted Metabolomics

Background: Low-temperature stress is a major abiotic factor that significantly impacts plant growth and productivity, especially in cold climatic conditions. Trifolium ambiguum M. Bieb. (Caucasian clover), a perennial legume known for its cold tolerance and it is an ideal model for studying cold adaptation mechanisms. However, the metabolic pathways that underpin its cold tolerance are not well understood. This study used widely targeted metabolomics to examine the metabolic reprogramming of T. ambiguum under low-temperature stress. Methods: Trifolium ambiguum seedlings were grown under controlled conditions and 14-day-old plants were exposed to two treatments: low-temperature stress (4°C) and control conditions (25°C). Samples were collected two hours after treatment. Widely targeted metabolomics profiling was performed to analyze metabolic changes induced by low-temperature stress. Key pathways and metabolites involved in the low-temperature stress response were identified through kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment and metabolic network analysis. Result: Low-temperature stress induced significant metabolic reprogramming in Trifolium ambiguum, with notable alterations observed in fatty acid metabolism. This enhancement improved cell membrane fluidity and stability, thereby strengthening the plant’s towards cold tolerance. Furthermore, the significant accumulation of proline and glutamate underscores their critical roles in osmotic regulation and antioxidative defense mechanisms. Additionally, the upregulation of secondary metabolites like (+)-Forbesione and flavonoids plays a key role in scavenging reactive oxygen species (ROS) and regulating stress-related signaling. KEGG pathway enrichment analysis revealed that C5-Branched dibasic acid metabolism and alpha-Linolenic acid metabolism play central roles in cold adaptation. C5-Branched dibasic acid metabolism provided essential carbon skeletons and energy substrates, while alpha-linolenic acid metabolism contributed to maintaining membrane stability and supporting antioxidative responses. The enrichment of the TCA cycle and glycerophospholipid metabolism further ensured energy supply and membrane integrity under stress conditions.