Defining the scope and components of ubiquitin-dependent chloroplast-associated protein degradation

人口正在迅速增长，到2050年将超过90亿。这对粮食安全构成重大挑战，并给自然资源带来越来越大的压力。因此，作物产量增加的动力比以往任何时候都更强，对次优生长条件的适应能力也更强。为了满足这些需求，发展改良作物将是至关重要的。通过对模式植物泰尔水芹的研究，我们最近取得了一些重大突破：我们发现了一种新的调控过程，名为CHLORAD，它控制植物生长的关键方面，包括植物对干旱和盐分等环境胁迫的反应。值得注意的是，改变ChLORAD活性使植物对这种逆境更有耐受性。在这个项目中，我们将定义ChLORAD的分子目标和机制，并在这样做的过程中更好地了解如何使用它来提供新的作物改良策略。CHLORAD(代表“叶绿体相关蛋白降解”)调节植物细胞内称为叶绿体的结构的发育和操作，叶绿体是正常的细胞成分(即细胞器)。它们定义植物，含有绿色色素叶绿素，负责光合作用，利用太阳光能量为细胞的活动和植物的生长提供动力。由于光合作用是向生物世界输入能量的唯一重要机制，叶绿体不仅对植物，而且对地球上的所有生命都具有巨大的重要性。叶绿体在植物对胁迫的反应中也起着关键作用，因此也是改造有韧性的作物的理想靶标。叶绿体由数千种不同的蛋白质组成，其中大部分由细胞核中的基因编码，因此在细胞基质中的细胞器外形成，称为细胞质。由于叶绿体被双膜“包膜”包围，需要复杂的机械设备才能将这些蛋白质输入细胞器。这包括两种膜上的分子机器，称为TOC(叶绿体外膜转运子)和TIC。每台机器由几个协同工作的蛋白质组成。目前，众所周知，ChLORAD作用于TOC机器，分解其组成蛋白质，以控制哪些其他蛋白质被输入细胞器(这反过来影响细胞器的发育和运行)。已知的ChLORAD机制的组成部分是称为SP1、SP2和CDC48的蛋白质。第一个是SP1，它是一种“泛素E3连接酶”，可以标记不需要的蛋白质，以此为靶点将它们移除。SP2蛋白在叶绿体外膜上形成通道，为SP1标记的蛋白质的清除提供了出口途径。最后，CDC48是一个分子马达，驱动不需要的物质被提取到胞浆中，然后在胞浆中被分解。我们未发表的结果表明，在CHLORAD中，额外的蛋白质(或“辅助因子”)与CDC48一起工作。我们认为这些辅因子是CDC48与不需要的TOC蛋白对接以及随后从CDC48释放这些蛋白所必需的。在这里，我们将详细研究这些辅助因子，以更好地了解在CHLORAD中不需要的叶绿体蛋白是如何被去除的。此外，我们有更多的新结果表明，ChLORAD作用于比先前设想的更多数量的靶蛋白(即，不仅仅是TOC蛋白)，包括叶绿体内部的蛋白。我们将系统地识别这些新的靶标，并试图了解它们是如何被CHLORAD处理的，即使在细胞器的深处。通过了解靶标的身份，我们还将探索ChLORAD对植物生长和发育的更广泛的生理意义。总的来说，所获得的知识将加深我们对ChLORAD的理解，并将对发展具有改进叶绿体性能的作物具有重要意义。

The human population is growing rapidly and set to exceed 9bn by 2050. This presents significant challenges to food security, and places ever increasing pressure on natural resources. Thus, the drivers for increased crop yields with resilience to sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crops. Through research on the model plant thale cress, we recently made some significant breakthroughs: We discovered a new regulatory process, named "CHLORAD", that controls vital aspects of plant growth, including plant responses to environmental stresses like drought and salinity. Significantly, modifying CHLORAD activity makes plants more tolerant of such stresses. In this project, we will define the molecular targets and mechanisms of CHLORAD, and in so doing develop a better understanding of how it can be used to deliver novel crop improvement strategies.CHLORAD (which stands for "chloroplast-associated protein degradation") regulates the development and operation of structures inside plant cells called chloroplasts, which are normal cellular constituents (i.e., organelles). They define plants, contain the green pigment chlorophyll, and are responsible for photosynthesis, harnessing sunlight energy to power the activities of the cell and the growth of the plant. As photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of huge importance, not just to plants but to all life on Earth. Chloroplasts also have critical roles in plant responses to stress, and so are ideal targets for engineering resilient crops.Chloroplasts are composed of thousands of different proteins, most of which are encoded by genes in the cell nucleus and so are made outside of the organelle in the cellular matrix known as the cytosol. As chloroplasts are surrounded by a double-membrane "envelope", sophisticated machinery is needed to enable the import of these proteins into the organelle. This comprises molecular machines in both membranes, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several proteins that work cooperatively.Currently, CHLORAD is known to act on the TOC machinery, breaking up its constituent proteins in order to control which other proteins are imported into the organelle (this in turn influences organelle development and operation). Known components of the CHLORAD machinery are proteins called SP1, SP2 and CDC48. The first, SP1, is a "ubiquitin E3 ligase" that labels-up unwanted proteins to target them for removal. The SP2 protein forms a channel in the chloroplast outer membrane, providing the exit route for removal of proteins labelled by SP1. Lastly, CDC48 is a molecular motor that drives extraction of the unwanted to the cytosol, where they are then broken down.Our unpublished results have revealed that additional proteins (or "cofactors") work together with CDC48 in CHLORAD. We believe that these cofactors are required for the docking of CDC48 onto the unwanted TOC proteins, and for the subsequent release of those proteins from CDC48. Here, we will study these cofactors in detail, to better understand how unwanted chloroplast proteins are removed in CHLORAD. Furthermore, we have additional new results showing that CHLORAD acts on a much larger number of target proteins than previously envisaged (i.e., not just TOC proteins), including proteins of the chloroplast interior. We will systematically identify these novel targets, and seek to understand how they are processed by CHLORAD even when deep inside the organelle. Informed by information on the identity of the targets, we will also explore the broader physiological significance of CHLORAD, for plant growth and development.Overall, the knowledge gained will enhance our understanding of CHLORAD, and will be important for the development of crops with improved chloroplast performance.