Metabolomic response of <i>Zizania latifolia</i> to low-temperature stress and identification of the bZIP transcription factor family

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 <i>Zizania latifolia</i> 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 <i>Z. latifolia</i> copes with cold stress through the coordination of secondary and primary metabolism. A total of 115 bZIP transcription factors (ZlbZIPs) were identified from the <i>Z. latifolia</i> 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 <i>ZlbZIP005, ZlbZIP075</i>, and <i>ZlbZIP084</i> 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 <i>Z. latifolia</i> 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 <i>Z. latifolia</i> 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.