Abstract
Cold stress severely impacts crop production, making it crucial to dissect the metabolic and transcriptional regulatory mechanisms of cold-resistant plants for breeding cold-tolerant varieties. This study systematically explored the response mechanism of Zizania latifolia to cold stress by integrating widely targeted metabolomics and genome-wide analysis for the first time. Metabolomics analysis revealed that 690 out of 810 metabolites showed significant differences after cold treatment at 4°C, with significant enrichment of flavonoids, amino acid derivatives, and alkaloids, involving key pathways such as antioxidant defense, osmotic adjustment, and signal transduction. This indicates that Z. latifolia copes with cold stress through the coordination of secondary and primary metabolism. A total of 115 bZIP transcription factors (ZlbZIPs) were identified from the Z. latifolia genome, with 18 genes located in known cold-resistant quantitative trait locus (QTL) intervals. Four cold-tolerant candidate genes were screened through collinearity analysis with the rice genome. Expression analysis showed that ZlbZIP005, ZlbZIP075, and ZlbZIP084 were significantly upregulated (29.17-4.10 fold) at 24 hours of cold treatment, and their promoter regions with high-density G-box elements implied strong cold response potential. Phylogenetic and evolutionary analyses showed that the bZIP family of Z. latifolia is highly homologous to that of rice but exhibits subfamily-specific expansion (such as subfamily Ⅶ) and conserved motif variations related to functional differentiation. This study first elucidated the metabolic reprogramming and bZIP transcription factor regulatory network of Z. latifolia under cold stress. The screened key cold-tolerant genes provide important genetic resources for cold-resistant breeding of gramineous crops and lay a foundation for analyzing the molecular mechanism of plant cold resistance and genetic improvement.