College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
Int J Mol Sci. 2024 Mar 26;25(7):3686. doi: 10.3390/ijms25073686.
Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated , acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that transcript levels were induced by aging and hydrogen peroxide (HO) and downregulated upon hydrogen sulfide (HS) treatment. The physiological and biochemical assays revealed that overexpression of (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased HS content, while disruption of by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of HS. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes ( and ) were upregulated in OE lines, while the expression of HS biosynthesis-related genes ( and ) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene () and a chlorophyll degradation-related gene () by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of and and repressed the expression of and , thereby promoting ROS accumulation and impairing the endogenous HS production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.
叶片衰老是叶片发育的终末阶段，其起始和进程受到众多内源性信号和环境刺激的整合调控。已有研究表明，WRKY 转录因子（TFs）在调控叶片衰老过程中发挥着重要作用，但 WRKY 介导的叶片衰老的分子机制在作物中仍缺乏详细的阐述。本研究克隆并鉴定了一个烟草 WRKY TF 基因，命名为 ，其作为一种自然叶片衰老的正调控因子。表达谱分析表明， 转录水平受衰老、过氧化氢（HO）诱导，而受硫化氢（HS）处理下调。生理生化分析表明，过表达 （OE）明显促进了叶片衰老，导致活性氧（ROS）水平升高，HS 含量降低，而利用嵌合阻遏物沉默技术（SRDX）破坏 则显著延缓叶片衰老的发生，导致 ROS 积累减少，HS 浓度升高。定量实时 PCR 分析表明，OE 系中各种衰老相关基因和 ROS 生物合成相关基因（和）的表达水平上调，而 HS 生物合成相关基因（和）的表达受到抑制。此外，酵母单杂交分析（Y1H）和双荧光素酶报告基因检测表明，NtWRKY70b 可通过结合其启动子序列直接上调 ROS 生物合成相关基因（）和叶绿素降解相关基因（）的表达。因此，这些结果表明 NtWYKY70b 通过直接激活 和 的转录水平，并抑制 和 的表达，从而促进 ROS 积累并损害内源性 HS 产生，进而加速叶片衰老。这些发现增进了我们对 WRKY TFs 调控叶片衰老的调控机制的认识，并为通过遗传操作来确保作物高农业生产力提供了一种新的方法。
