NYE1（SGR1）保守结构域/关键位点的分子功能研究

We previously showed that AtNYE1 and AtNYE2 constitute a bottleneck regulatory node of both chlorophyll (Chl) and light-harvesting complex degradations during leaf senescence in Arabidopsis. Protein sequences of NYEs are highly conserved across plant species, and multiple conserved motifs and particularly multiple essential residues were identified in different species. Nevertheless, their molecular functions have not been explored yet. Our truncation and mutation analyses identified a conserved cysteine rich motif (CRM) in the C-terminal of NYE1 which was critical to its function. It was further found that NYE1 changed its conformation and oligmerization in responding to in vitro redox state. Importantly, by searching for novel NYE1 interacting proteins in the chloroplast, we identified a soluble luminal protein PSB27, which was reported to be involved in repairing the damage of photosystems. With the support of this grant, we would like to extend our study in the following directions: 1) To determine whether and how the CRM mediates the change of NYE1 conformation and/or oligomerization in response to redox change during leaf senescence；2) To reveal the possible role of the conserved motifs/residues in facilitating NYE1’s localization/interactions; and 3) To identify novel NYE1 interacting proteins by using the yeast-two-hybrid system and to explore the biological implication of PSB27-NYE1 interaction.

前期研究表明，AtNYE1与 AtNYE2构成了拟南芥衰老进程中叶绿素及捕光蛋白复合体降解的瓶颈调控节点。NYEs在不同物种中高度保守，多个保守位点的突变导致其蛋白失活，但它们的分子与生化功能不清楚。片段截短和点突变分析发现，NYE1羧基端富含半胱氨酸结构域（CRM）是其蛋白发挥功能所必需的，且发现NYE1体外响应氧化还原状态而发生构象与聚合形态改变。我们还发现NYE1与类囊体腔蛋白PSB27互作，暗示可能介导PSB27的光系统修复过程，也提示NYE1可能跨类囊体膜定位。本项目拟在此基础上开展三方面研究：1）CRM是否介导NYE1响应衰老阶段氧化还原状态而发生构象和聚合形态改变，及其作用模式；2）NYE1保守结构域/关键位点是否介导其蛋白互作和亚细胞精细定位，及其作用机制；3）NYE1与PSB27互作的生物学含义，是否存在新的NYE1互作蛋白。

植物绿色器官衰老过程中，叶绿素降解是最为显著的标志性事件。研究表明，NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) 负责催化叶绿素a降解的第一步反应，具有镁脱螯合酶活性；但是，对于NYE1的功能域信息和编码基因的保守调控元件还知之甚少。本研究中，我们在NYE1的C末端鉴定到一个保守的富含半胱氨酸的基序（CRM, P-X3-C-X3-C-X-C2-F-P-X5-P），并且发现CRM对NYE1发挥功能至关重要。我们通过遗传学分析发现，CRM中的4个半胱氨酸都是不可替代的；通过酶活性分析发现，CRM中的任何一个半胱氨酸突变都会影响NYE1的镁脱螯合活性，可能是影响了NYE1蛋白响应衰老细胞中氧化还原状态引发的构象改变。近期的研究已经阐明组蛋白去甲基化在调节植物开花、抗病、节律应答和种子萌发中的作用机理，但是组蛋白去甲基化如何影响叶片衰老仍不清楚。在本研究中，我们通过酵母单杂交技术筛选到一个NYE1的上游调控因子，组蛋白H3第27位赖氨酸的三甲基化（H3K27me3）去甲基化酶，即RELATIVE OF EARLY FLOWERING6（REF6）能够结合在NYE1的启动子上。通过体内和体外实验分析，我们发现REF6可以通过其锌指结构域直接结合NYE1/2的CTCGYTY元件，并正调控NYE1/2的表达。REF6功能的缺失突变延缓叶绿素降解，而过表达REF6则加速叶绿素降解。我们还发现，REF6可以通过上调关键衰老调控/功能基因的表达，进而促进叶片全局性的衰老进程。值得注意的是，REF6功能的缺失会导致其下游衰老相关靶基因H3K27me3水平升高。本研究揭示了H3K27me3甲基化作为一种表观抑制机制，抑制拟南芥叶片衰老关键调控因子及相关功能基因被过早的转录激活。