Zhang Xingzi, Wang Jin, Wang Yaqi, Jiang Caihong, Yang Aiguo, Li Fengxia
Key Laboratory for Tobacco Gene Resources, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 266101, Qingdao, China; Graduate School of Chinese Academy of Agricultural Science, 100081, Beijing, China.
Key Laboratory for Tobacco Gene Resources, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 266101, Qingdao, China.
Plant Physiol Biochem. 2025 Feb;219:109391. doi: 10.1016/j.plaphy.2024.109391. Epub 2024 Dec 6.
Nornicotine is an undesirable alkaloid in tobacco due to its unpleasant taste and potential toxicity. The accumulation of nornicotine in tobacco leaves is related to the development of the leaves, with very low amounts present in green leaves and a dramatic increase after senescence. While it is known that the NtE4 is a key enzyme involved in nicotine to nornicotine conversion in tobacco leaves, the specific genes regulating the expression of NtE4 during leaf senescence remain unclear. In this study, we identified a WRKY transcription factor, NtWRKY33, as being involved in nornicotine accumulation during senescence. NtWRKY33 is a nuclear protein and its expression is induced by senescence. Knocking out NtWRKY33 significantly decreased nornicotine levels in senescent leaves, whereas overexpressing NtWRKY33 significantly increased nornicotine accumulation. RT-qPCR analysis demonstrated that NtWRKY33 positively regulates the expression of NtE4 without significantly affecting other key enzyme genes involved in nornicotine biosynthesis. Yeast one-hybrid (Y1H) and dual-luciferase analysis (DLA) revealed that NtWRKY33 directly promotes NtE4 expression by binding to its promoter. Therefore, NtWRKY33 is a transcription factor involved in senescence-induced nornicotine accumulation. This study provides novel insights into the molecular mechanisms by which senescence induces nornicotine formation and identifies a new target for regulating nornicotine levels.
去甲烟碱是烟草中一种不受欢迎的生物碱，因其味道不佳且具有潜在毒性。烟叶中去甲烟碱的积累与叶片发育有关，绿叶中含量极低，衰老后会急剧增加。虽然已知NtE4是烟叶中参与尼古丁向去甲烟碱转化的关键酶，但在叶片衰老过程中调节NtE4表达的具体基因仍不清楚。在本研究中，我们鉴定出一个WRKY转录因子NtWRKY33，它参与衰老过程中的去甲烟碱积累。NtWRKY33是一种核蛋白，其表达受衰老诱导。敲除NtWRKY33显著降低了衰老叶片中的去甲烟碱水平，而过表达NtWRKY33则显著增加了去甲烟碱的积累。RT-qPCR分析表明，NtWRKY33正向调节NtE4的表达，而对去甲烟碱生物合成中涉及的其他关键酶基因没有显著影响。酵母单杂交（Y1H）和双荧光素酶分析（DLA）表明，NtWRKY33通过与NtE4启动子结合直接促进其表达。因此，NtWRKY33是一种参与衰老诱导去甲烟碱积累的转录因子。本研究为衰老诱导去甲烟碱形成的分子机制提供了新的见解，并确定了一个调节去甲烟碱水平的新靶点。
