This study investigated the physiological and molecular mechanisms underlying the cold tolerance of Caucasian clover （Trifolium ambiguum M. Bieb.）， a perennial forage known for its strong hardiness. Seedlings grown at 20℃ served as the control （CK）， and low-temperature stress （4℃） was applied for 2， 6， and 12 h. Physiological indicators were measured at each time point， and transcriptome sequencing was conducted on the control and 12 h-stressed plants. The results indicated that under 4℃， the plants enhanced their cold tolerance by significantly increasing the activities of antioxidant enzymes （peroxidase， catalase， and superoxide dismutase） and the levels of osmoregulatory substances （soluble protein， soluble sugar， and free proline）. This coordinated response effectively scavenged reactive oxygen species and mitigated membrane lipid peroxidation， as evidenced by reduced malondialdehyde accumulation. Transcriptome analysis identified 15 428 differentially expressed genes （DEGs） after 12 h of cold stress， with 8909 up-regulated and 6519 down-regulated. Key pathways were significantly activated， including the flavonoid biosynthesis pathway， where the up-regulation of pivotal upstream genes （CHS and CHI） facilitated the synthesis of antioxidant compounds such as luteolin， apigenin， and isoflavones. Concurrently， the MAPK signaling pathway was induced， highlighted by the increased expression of genes like MEKK1， suggesting that cold signals are transduced via a phosphorylation cascade. These findings provide crucial insights into the molecular basis of cold adaptation in Caucasian clover.

Trifolium ambiguum  /  Low temperature stress  /  Physiological parameters  /  DGEs