In contrast to the normal condition, p5cs expression is hyperactive during cold acclimation, whereas ProDH expression is inhibited, resulting in the accumulation of more and more free proline in plant cells. In Arabidopsis and other plants, proline levels are mainly determined by balance of biosynthetic and catabolic pathways, controlled by P5CS and ProDH genes, respectively. Nanjo et al. found that proline degradation was inhibited in Arabidopsis transformed with AtProDH, suggesting that free proline levels increased in leaves. Secondary metabolism and its products are also involved in the response to various stresses in plants, representing a process that formed over a long evolutionary period. There is some evidence that secondary metabolic products and environmental factors are closely linked, as in the case of alkaloids, which play an important role in resisting insects and herbivores via chemical defense mechanism. In addition to ‘arginine and proline metabolism’, the DETs were significantly enriched in ‘quinoline alkaloid biosynthesis’ pathway during cold acclimation, based on KEGG pathway analysis. Early in the cold stress period, 40% of transcripts related to quinoline alkaloid metabolism were up-regulated more than 2-fold compared to the 0 h time point, including contig_65006 and contig_65485. This suggests that the up-regulation of transcripts in response to low temperatures may play a crucial role in plant stress tolerance. However, when the duration of cold stress exceeded 6 h, the expression levels of these up-regulated transcripts decreased gradually, dropping to their initial levels by 24 h. This suggests that there may be a relationship between quinoline alkaloid biosynthesis and abiotic factors, although this relationship may not be as simple and direct as the relationship between the biological environment and chemical defense. Further research is needed to TWS119 explore this relationship in depth. Many researchers believe that plants produce secondary metabolites such as alkaloids at the cost of slower growth. However, when biotic and abiotic stresses become severe enough to affect their survival, the plants have no choice but to produce some secondary metabolites for protection against such rigorous stress conditions. Although the expression of peroxidases such as CAT and SOD increased significantly as the duration of cold exposure increased, these enzymes were still unable to completely clear the increased levels of H2O2, resulting in a significant increase in the amount of H2O2 during cold acclimation. In this study, we found that some genes that are known to be involved in the response to other stresses in other plants are also involved in cold acclimation, which could support the hypothesis that the same gene have different functions in different plants. The GO term ‘translational initiation’ was enriched in response to cold acclimation. A total of 254 transcripts were annotated under in this term, and 89 exhibited a greater than two-fold change in expression during the low temperature treatment. Translation initiation in eukaryotes depends on many eukaryotic initiation factors that stimulate both the recruitment of the initiator tRNA.