Dynamic transcriptomic and metabolomic adaptation mechanisms of Trifolium ambiguum under different durations of cold stress.

Cold stress is one of the major environmental challenges faced by plants, severely affecting their growth, development, and yield. With the intensification of global climate change, the impact of cold stress on plants is becoming increasingly significant. Plants respond to cold stress through complex metabolic regulatory networks, including the accumulation of protective metabolites, modulation of antioxidant defenses, and activation of secondary metabolic pathways. This study aims to explore the dynamic metabolic and transcriptomic responses of T. ambiguum under long-term cold stress, and to reveal its adaptive mechanisms. By integrating metabolomics and transcriptomics, this study provides an indepth analysis of gene expression and metabolic responses of T. ambiguum under different cold stress treatments (2 h, 6 h, 12 h), focusing on key metabolic pathways and signal transduction mechanisms involved in cold stress adaptation. To further investigate the relationship between genes and metabolites, weighted gene co-expression network analysis (WGCNA) was applied to construct the gene-metabolite coexpression network under cold stress. Several functional modules that play significant roles in cold response were identified. Notably, the pink module was found to be associated with lipid metabolism, sugar metabolism, and signal transduction pathways, while the black module was closely linked to plant hormone signaling and antioxidant responses. Additionally, KEGG enrichment analysis revealed that key pathways such as glycerophospholipid metabolism, proline metabolism, and plant hormone signal transduction work synergistically in cold stress adaptation, regulating cellular homeostasis and maintaining energy supply under prolonged cold stress. The results indicate that T. ambiguum enhances its cold tolerance through dynamic coordination between its transcriptomic and metabolic responses. This study provides new molecular biological evidence for the cold adaptation mechanisms of T. ambiguum and offers theoretical support for improving cold tolerance in related crops.