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Identification and functional analysis of low temperature responsive genes ZmNTL1 and ZmNTL5 in maize

GAO Yuan**, WANG Yu-Qi**, JIANG Jia-Ning, ZHAO Jian-Xiong, WANG Xue-He-Yuan, WANG Hao-Yu, ZHANG Rui-Jia, XU Jing-Yu,HE Lin*   

  1. Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2025-02-19 Revised:2025-06-01 Accepted:2025-06-01 Published:2025-06-10
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31701328) and the Postdoctoral Research Foundation of Heilongjiang Province (LBH-Q21160).

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Abstract:

Low temperature is one of the major abiotic stresses limiting maize yield in Northeast China. Membrane-bound NAC transcription factors play important roles in plant stress responses. Investigating their function provides a theoretical basis for breeding maize with enhanced cold tolerance. In this study, the molecular characteristics of nine maize NTL genes were analyzed using bioinformatics approaches. Analysis of publicly available RNA-seq data revealed that six of these genes were significantly upregulated by low temperature stress in both roots and leaves. Among them, ZmNTL1 and ZmNTL5, which share 80.04% sequence homology, were selected for further study. A double knockout mutant (zmntl1zmntl5) was generated using gene-editing techniques. Under low temperature stress, the ion leakage rate in the leaves of zmntl1zmntl5 was significantly higher than in the wild-type B104. The fresh and dry weights of both aboveground and belowground tissues, as well as chlorophyll content, were significantly lower in zmntl1zmntl5 compared to B104. In contrast, zmntl1zmntl5 exhibited significantly higher levels of O2? and H2O2, while the activities of antioxidant enzymes GST, SOD, POD, and CAT were significantly reduced. Furthermore, the expression levels of ZmGST24, ZmNCED3, ZmDREB1.6, and ZmDREB2A were markedly decreased in zmntl1zmntl5 compared to B104 under cold stress. In summary, the absence of ZmNTL1 and ZmNTL5 compromises cold tolerance in maize, suggesting that both genes function as positive regulators in the cold stress response. This study provides a valuable theoretical foundation and genetic resources for the improvement of cold-tolerant maize varieties and the development of new cultivars.

Key words: maize, low temperature, RNA-seq, NTL, gene editing

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