过氧化氢调控植物低温胁迫应答的机制研究

Low temperature is a major environmental factor that limits plant growth, productivity, and geographical distribution. Therefore, it is important for us to understand how plant responses to cold stress. Extensive studies indicate that cold stress induces H2O2 accumulation in plants, while the mechanism underlying H2O2-modulated plant cold stress response and tolerance remains elusive. Our previous work showed that NADPH oxidases RBOHD/F is involved in cold stress-induced H2O2 production, and the mutant rboh-DF exhibited decreased freezing tolerance compared with the wild-type plants. We also found that cold-induced H2O2 can sulfenylate LOS2, a key factor regulating plant response and tolerance to cold stress. Further study showed that H2O2 may trigger LOS2 translocation in the nucleus through sulfenylation. In addition, our ChIP-qPCR and q-PCR experiments showed that LOS2 can bind to CBF1 promoter and CBF1 expression in los2-1 is significantly lower than that in wild type. These results revealed that LOS2 may act upstream of CBF1 to regulate its expression. In this project, we plan to carry our further experiments to demonstrate that H2O2-mediated LOS2 sulfenylation plays an important role in plant freezing tolerance. We will also explore whether LOS2 protein regulates CBF1 expression through directly binding to its promoter and identify the binding motif, investigate the role of H2O2 in LOS2-mediated regulation of CBF1 expression, and isolate other factors that are regulated by LOS2 in plant cold stress response using RNA-seq and ChIP-seq assays. Our research will show that H2O2 positively regulates plant freezing tolerance by sulfenylating LOS2 and inducing its translocation in the nucleus to up-regulate CBF1 expression. This project will advance our better understanding of the mechanisms underlying plant responses to cold stress.

低温胁迫严重影响植物生长、产量及分布，研究植物低温应答调控机制具有重要意义。植物在冷胁迫下会诱导过氧化氢产生，但过氧化氢如何调控低温应答仍不清楚。我们前期研究发现，低温诱导过氧化氢产生依赖RBOHD/F,该酶通过改变过氧化氢积累参与植物低温应答。低温可通过积累过氧化氢，进而次磺酸化修饰冷调控因子LOS2蛋白促使其进入细胞核。此外，ChIP-PCR表明LOS2能与冷应答关键基因CBF1启动子结合，且冷胁迫下los2-1的CBF1表达量低于野生型。我们将进一步分析过氧化氢次磺酸化修饰LOS2在低温应答中的作用，检验CBF1是否为LOS2的直接下游并深入研究它们间的调控关系；探究LOS2对CBF1的调控作用是否受过氧化氢调控；通过RNA-seq和ChIP-seq获得其他因子。上述研究将证明植物可通过过氧化氢次磺酸修饰LOS2，促进其进入细胞核并上调CBF1表达，进而调控下游基因表达应答低温胁迫。

低温胁迫严重影响植物生长、产量及分布，研究植物低温应答调控机制具有重要意义。植物在冷胁迫下会诱导过氧化氢产生，但过氧化氢如何调控低温应答不清楚。本项目在前期研究基础上，分析了H2O2及低温处理前后野生型和rboh-D/F体内LOS2次磺酸化水平的变化，证明了低温诱导的H2O2 可以次磺酸化修饰LOS2的全长翻译产物ENO2参与调控植物低温应答。利用ChIP-seq 实验筛选ENO2(LOS2的全长翻译产物）的靶基因，重点研究了LOS2对其靶基因CBF1/CBF3的调控。运用EMSA技术以双荧光素酶报告系统，体外证明了ENO2能与CBF1/CBF3直接结合并促进CBF1和CBF3的表达；在los2-1突变体中过量表达CBF1或CBF3，并通过分析其抗冻表型、体内ENO2次磺酸化水平，明晰了ENO2与CBF1的遗传关系；获得35S::GFP-MBP los2-1、35S::GFP-ENO2 los2-1和35S::GFP-ENO2C408S los2-1转基因植株，并通过分子遗传学、生物化学等实验手段，进一步确定ENO2的次磺酸化修饰在植物低温胁迫应答中的作用；构建荧光标记的转基因株系、分析ENO2次磺酸化对ENO2亚细胞定位和转录活性的影响，揭示H2O2通过次磺酸化修饰ENO2调控CBF1的表达的分子机制，证明了H2O2诱导的ENO2次磺酸化诱发ENO2形成寡聚体，促进ENO2进入细胞核和的转录活性；通过研究，本项目明确证明了RbohD/ F调控的H2O2产生通过在Cys408位点次磺酸化ENO2，促进Cys408/ Ser15介导的寡聚体化，增强其核转运和转录结合活性调节CBF1和CBF3的表达，从而促进植物的耐寒性。此外还揭示了茉莉酸、ABA调控植物耐寒性的分子机理。本项目鉴定了活性氧参与植物低温应答的新途径，为农作物抗低温品质的遗传改良提供了理论依据。