光周期下拟南芥CAT2蛋白降解的分子机制研究

H2O2 plays important roles in plant development and environmental stresses. In leaves, H2O2 is mainly generated during photorespiration and accurately regulated in a circadian rhythm way, which is induced by light and inhibited at night. Arabidopsis CAT2 is a major enzyme that eliminates H2O2 in photosynthetic mesophyll cells of leaves and the expression of CAT2 is controlled by the circadian clock as well as H2O2. But the underlying mechanisms in posttranslational regulation of CAT2 remains unclear. Our preliminary data indicates that the protein level of CAT2 fluctuated in circadian rhythm way and CAT2 was degraded by 26S proteasome in dark. An E3 ligase that interacts with CAT2 has been successfully obtained by yeast two-hybrid screening of an Arabidopsis cDNA library. In this project, first, the in vivo interaction of E3 ligase and CAT2 will be further verified by BiFC and CoIP experiments. Second, the genetic material lines of E3 will be generated to investigate whether E3 ligase play important roles in the process of regulating CAT2 degradation, catalase activity and H2O2 content. Finally, the ubiquitination of CAT2 by E3 ligase will be studied in vivo and in vitro to reveal the molecular mechanisms underlying CAT2 degradation.

H2O2在植物生长发育和逆境胁迫应答中发挥重要作用。叶片中H2O2主要来源于光呼吸，且受光周期调节，白天增加，夜晚减少。拟南芥CAT2 (CATALASE2) 是叶片中主要分解H2O2的过氧化氢酶，其表达模式呈现出与H2O2一致的昼夜节律，但目前对其翻译后水平的调控机制研究得还不清楚。本项目的预实验表明，光周期下CAT2蛋白含量也表现出同样的昼夜节律性，黑暗时CAT2的降解过程依赖于泛素/26S蛋白酶体途径，并且通过酵母双杂交从拟南芥cDNA文库中筛选到一个与CAT2互作的E3泛素连接酶。为了进一步研究CAT2的降解机制，拟首先通过BiFC和CoIP验证二者的体内互作关系；其次，通过构建E3基因相关遗传材料，分析材料中CAT2蛋白水平、catalase活性和H2O2含量等，确定E3对CAT2的影响；最后通过生化手段验证E3能否在体外和体内直接泛素化CAT2，从而揭示CAT2降解的分子机制。

过氧化氢酶 (Catalase) 是植物体内主要的抗氧化酶之一，负责清除光呼吸和脂肪酸β氧化等过程中产生的过氧化氢 (H2O2)，使细胞免受过多的H2O2毒害。拟南芥中的过氧化氢酶CAT2和CAT3在转录水平上呈现昼夜节律变化，但对其蛋白水平调控的研究较少。本研究发现CAT2蛋白也表现出昼夜节律变化，并且SCF复合体类E3连接酶的F-box蛋白EBF1和EBF2参与CAT2和CAT3的降解，说明CAT2和CAT3的泛素化降解过程在其昼夜节律变化中起重要作用。由于CAT2和CAT3蛋白相似性很高，我们进一步探究其在胁迫条件下的功能是否有所不同，结果显示CAT2和CAT3在盐胁迫及恢复条件下表达模式不同，并且在恢复胁迫恢复条件下CAT3的蛋白水平受泛素化途径调控，说明CAT2和CAT3在植物应对胁迫过程中发挥不同的作用。此外，过氧化氢可以作为一种信号分子调控植物各种生理过程和对胁迫的应答，于是我们利用CRISPR-Cas9方法构建获得了cat1/cat2/cat3三突变体，通过对三突变体的研究发现过氧化氢酶在植物生长发育及维持氧化胁迫方面的重要作用，并通过转录组分析初步探索了过氧化物酶体逆行信号，为后续深入研究过氧化物酶体逆行信号途径提供了基础。