Msr介导DNA去甲基化酶SlDML2亚砜化修饰参与番茄果实成熟衰老调控的机制研究

Reactive oxygen species-mediated oxidative damage to macromolecules is considered to be a main cause of aging. DNA demethylases (SlDML2) is one of the critical factors for tomato fruit ripening and senescence, whereas the Msr-mediated sulfoxidation regulation of SlDML2 and its underlying biological significance in relation to tomato fruit ripening and senescence are still largely unknown. Our previous studies have found that the expressions of some SlMsrs and SlDML2 increased cooperatively. In addition, SlDML2 interacts with SlMsrE4 or SlMsrB2 in vitro and in vivo, and sulfoxidated SlDML2 could be partly repaired by SlMsrE4 or SlMsrB2. The project is to investigate the sulfoxidation regulation of SlDML2 by SlMsrs during fruit ripening and senescence. The following issues will be studies: 1) Analyzing the properties of the protein oxidation during fruit ripening and senescence; 2) Exploring the mechanism of the sulfoxidation regulation of SlDML2 by SlMsrs; 3) Verification the transcriptional regulation of sulfoxidation modification in SlDML2 on its targeted genes; 4) Inspecting the effects of sulfoxidation regulation of SlDML2 on the interaction of SlDML2 and its collaboration proteins. The results will contribute to reveal the molecular mechanism of fruit ripening and senescence from the viewpoint of redox regulation.

活性氧对蛋白质的氧化损伤是导致采后果实衰老的重要原因之一，甲硫氨酸亚砜还原酶（Msr）可以修复蛋白质中发生亚砜化修饰的甲硫氨酸残基。DNA去甲基化酶SlDML2是番茄果实成熟衰老的关键调控因子之一。我们最近的研究发现，SlDML2可能为SlMsrE4和SlMsrB2的目标蛋白，且SlMsrs和SlDML2表达在果实成熟衰老过程中协同增加，暗示SlMsrs可对SlDML2亚砜化进行调节，从而参与番茄果实成熟衰老的表观遗传调控。本项目以番茄果实为研究材料，从SlDML2亚砜化修饰角度解析果实成熟衰老的翻译后调控机制。主要内容包括：1）果实成熟衰老过程中蛋白氧化特性；2）Msr对SlDML2亚砜化修饰的调节机制；3）亚砜化修饰对SlDML2调控下游靶基因的影响；4）亚砜化修饰对SlDML2与其它成熟衰老关键调控蛋白协同作用的影响。研究结果可望从蛋白质氧化还原调控角度揭示果实成熟衰老的分子机制。

DNA去甲基化酶是基因表达的重要调控者，在植物的生长发育和胁迫应答中发挥重要调控作用，其自身也受到多种翻译后修饰调节。DNA去甲基化酶SlDML2是番茄果实成熟的关键调控因子之一，但其自身如何被调控尚不清楚。我们发现，在番茄果实成熟过程中ROS累积导致 SlDML2 发生亚砜化修饰（即甲硫氨酸氧化）。SlDML2亚砜化或模拟亚砜化（SlDML2-M1315Q）降低其DNA去甲基化酶活性，而SlMsrE4（A型Msr）和SlMsrB2通过去除SlDML2的亚砜化修饰恢复其DNA去甲基化酶活性。过表达SlDML2促进果实成熟，而过表达模拟亚砜化的SlDML2-M1315Q则抑制果实成熟。RT-qPCR和McrBC-qPCR实验表明大量成熟相关基因的5meC修饰水平显著上调，基因表达下调，与果实成熟表型一致。我们的研究揭示了亚砜化修饰调控SlDML2的DNA去甲基化酶活性，进而调控番茄果实成熟的新机制。