Role of protein phosphorylation in the maintenance of photosystem two in plants

光系统2（PSII）蛋白复合物被广泛认为是地球上最显着的分子机器之一。PSII存在于植物、藻类和蓝细菌中，并执行复杂的任务，即利用阳光从高度稳定的水分子中提取电子，以使产氧光合生物生长。同时PSII也产生我们呼吸的氧气。不幸的是，PSII并不是一台完美的机器;它有时会发生故障，特别是当阳光非常明亮时，必须进行修理。如果没有这种特殊的修复机制，PSII将在光照下迅速失活，植物的生长和放氧将受到抑制。尽管PSII修复的生理重要性，修复过程的细节仍然未知。在分子水平上对PSII修复的详细了解将为我们提供重要的知识，以帮助全球努力增强作物植物的光合作用，以便我们能够增加生长，以帮助满足对更多食物和更多生物质的日益增长的需求。在陆地植物的情况下，我们知道，活跃的PSII位于叶片中发现的绿色叶绿体的类囊体膜系统内，但被隔离在膜的特征堆叠区域内，称为基粒。为了修复，受损的PSII复合物向外迁移到修复机器所在的类囊体系统的未堆叠区域。在这里，受损的PSII亚基可以降解，新合成的亚基插入和PSII复合物重新激活。受损的PSII如何被特异性地运送到修复装置，以及受损的亚基如何被特异性地识别以进行替换，目前尚不清楚。多年来积累的证据表明，带负电荷的磷酸基团的存在下，四个PSII核心亚基可能在修复过程中发挥作用，并在重塑膜系统在明亮的光，以提高修复。这些先前的研究依赖于缺乏磷酸化PSII的激酶的突变体的分析。然而，现在清楚的是，这些激酶突变体具有PSII以外的效应，因此仍然不确定PSII磷酸化的特定缺乏是否是激酶突变体中观察到的所有效应的原因。此外，激酶突变体阻断所有PSII磷酸化，这阻止了对单个亚基磷酸化的特定作用的分析。在这个应用程序中，我们建议使用一种新的方法来研究蛋白质磷酸化在维持PSII活性在陆地植物中的作用。我们将不研究激酶突变体，而是利用叶绿体转化技术使烟草植物的每个亚基中缺乏通常磷酸化的氨基酸残基。激酶在这些植物中仍然具有活性，因此现在可以单独研究从PSII中去除一个特定磷酸基团的效果。在背景工作中，我们已经证明这是一种可行的策略，因为我们已经使烟草植物不能磷酸化D1蛋白。对PSII损伤的影响，它的拆卸，它在类囊体膜内的迁移，受损蛋白质的蛋白水解降解，PSII的重新组装以及在各种光照条件下对基粒结构和植物生长的影响将由一组具有该领域专业知识的研究人员使用最先进的方法进行。我们将研究PSII的突变体植物缺乏的四个磷酸化位点的维护，也在工程植物中，我们删除越来越多的磷酸化位点，以测试重叠的功能。总的来说，我们的研究将提供重要的新信息，光合作用的放氧复合物是如何维持在陆地植物和类囊体膜系统的结构是如何调节。

The photosystem two (PSII) protein complex is widely considered to be one of the most remarkable molecular machines on Earth. PSII is found in plants, algae and cyanobacteria and performs the complex task of using sunlight to extract electrons from highly stable water molecules to allow oxygenic photosynthetic organisms to grow. At the same time PSII also produces the oxygen that we breathe. Unfortunately PSII is not a perfect machine; it sometimes breaks down, especially when the sunlight is very bright, and has to be repaired. Without this special repair mechanism PSII would be quickly inactivated in the light and plant growth and oxygen evolution would be inhibited. Despite the physiological importance of PSII repair, the details of the repair process are still unknown. A detailed understanding of PSII repair at the molecular level will provide us with important knowledge to help in the global effort to enhance photosynthesis in crop plants so that we can increase growth to help satisfy the ever increasing demand for more food and more biomass. In the case of land plants, we know that active PSII is located within the thylakoid membrane system of green chloroplasts found in leaves, but is segregated within the characteristic stacked regions of the membrane known as grana. For repair, the damaged PSII complex migrates outwards to the unstacked regions of the thylakoid system where the repair machinery is located. Here damaged PSII subunits can be degraded, newly synthesised subunits inserted and the PSII complex reactivated. How damaged PSII is specifically shuttled to the repair apparatus and how damaged subunits are specifically recognised for replacement is currently unknown. Over the years evidence has accumulated to suggest that the presence of negatively charged phosphate groups on four PSII core subunits might play a role in the repair process and in reshaping the membrane system in bright light to enhance repair. These previous studies have relied on the analysis of mutants that lack the kinase enzymes that phosphorylate PSII. However, it is now clear that these kinase mutants have effects outside PSII so it is still uncertain whether the specific lack of PSII phosphorylation is responsible for all the effects seen in the kinase mutants. In addition the kinase mutants block all PSII phosphorylation which has prevented analysis of the specific role of single subunit phosphorylation. In this application we propose to use a new approach to examine the role of protein phosphorylation in maintaining PSII activity in land plants. Rather than study kinase mutants, we will use chloroplast transformation technology to make tobacco plants that lack the amino-acid residue in each subunit that is normally phosphorylated. The kinase enzyme will still be active in these plants and so the effect of removing just one specific phosphate group from PSII can now be studied in isolation. In background work we have shown that this is a feasible strategy as we have already made tobacco plants unable to phosphorylate the D1 protein. Effects on the damage to PSII, its disassembly, its migration within the thylakoid membrane, the proteolytic degradation of damaged proteins, the reassembly of PSII and the impact on the structure of grana and plant growth under various illumination conditions will be performed use state-of-the-art approaches by a team of researchers with proven expertise in this area. We will study the maintenance of PSII in mutant plants lacking each of the four phosphorylation sites and also in engineered plants in which we remove increasing numbers of the phosphorylation sites to test for overlap of function. Overall our research will provide important new information on how the oxygen-evolving complex of photosynthesis is maintained in land plants and how the structure of the thylakoid membrane system is regulated.