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 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.低温胁迫严重影响作物生产，因此解析耐寒植物的代谢与转录调控机制，对于培育耐低温作物品种至关重要。本研究首次结合广泛靶向代谢组学与全基因组分析，系统解析了菰（Zizania latifolia）对低温胁迫的响应机制。代谢组学分析显示，在810种代谢物中，有690种在4℃低温处理后出现显著差异；其中黄酮类、氨基酸衍生物及生物碱类物质显著富集，涉及抗氧化防御、渗透调节、信号转导等关键通路。这表明菰可通过初生代谢与次生代谢的协同作用应对低温胁迫。本研究从菰基因组中共鉴定出115个碱性亮氨酸拉链（bZIP）转录因子（ZlbZIPs），其中18个基因位于已报道的耐寒数量性状位点（QTL）区间内。通过与水稻基因组的共线性分析，筛选得到4个耐低温候选基因。表达分析显示，ZlbZIP005、ZlbZIP075及ZlbZIP084在低温处理24小时后显著上调（上调幅度为4.10~29.17倍）；其启动子区域含有高密度G-box元件，暗示其具备较强的低温响应潜力。系统发育与进化分析表明，菰的bZIP基因家族与水稻同源性极高，但存在亚家族特异性扩张（如第Ⅶ亚家族）以及与功能分化相关的保守基序变异。本研究首次阐明了菰在低温胁迫下的代谢重编程过程与bZIP转录因子调控网络；所筛选得到的关键耐寒基因可为禾本科作物的耐寒育种提供重要遗传资源，同时为解析植物耐寒分子机制与遗传改良奠定基础。