Chloroplast-Associated Degradation (CHLORAD): Molecular definition of a ubiquitin-dependent system for plastid protein removal in plants

人类人口正在迅速增长，到2050年将达到90亿，自然资源的压力也越来越大。因此，提高作物产量和抵御气候变化和次优生长条件的驱动力比以往任何时候都更强大。为了满足这些需求，必须开发改良的作物品种。通过对模式植物塔勒水芹的研究，我们最近取得了重大突破：我们发现了一个名为SP1的基因，它控制着植物生长的重要方面，包括植物对水分胁迫和高盐度等不利环境条件（统称为非生物胁迫）的反应。通过修饰SP1的表达，可以使塔勒水芹植物对这种胁迫更耐受。最近，我们发现了两个新的基因，它们在与SP1相同的调节途径中起作用-我们现在称之为CHLORAD（“叶绿体相关降解”）。在这个项目中，我们将详细研究这些新基因，阐明它们的功能，并了解它们如何与SP1一起工作，并希望通过这样做来确定作物改良的新策略。与SP1一样，这两个新的CHLORAD基因调节植物细胞内称为叶绿体的结构的发育。叶绿体是正常的细胞成分（即，它们是细胞器），在许多方面它们定义了植物。它们含有绿色色素叶绿素，负责光合作用，捕获阳光能量并利用它为细胞的活动提供动力。由于光合作用是能量输入到生命世界的唯一重要机制，叶绿体不仅对植物而且对地球上的所有生命都非常重要。叶绿体在植物对非生物胁迫的反应中也起着关键作用，因此是作物抗逆工程的理想目标。叶绿体由数千种不同的蛋白质组成，其中大多数由细胞核中的基因编码，因此在细胞器外的细胞基质中合成，称为细胞质。由于每个叶绿体都被一个双层膜所包围，因此需要复杂的机器将这些蛋白质导入细胞器。这包括两个分子机器，一个在每个膜上，称为TOC（“叶绿体外膜上的转位子”）和TIC。每个机器都由几种不同的蛋白质组成，它们协同工作。SP1基因编码一种称为“泛素E3连接酶”的调节因子。这些调节器通过标记不需要的蛋白质来清除它们。SP1 E3连接酶介导TOC组分的去除，从而控制TOC功能，使得叶绿体仅输入所需的蛋白质。这种控制使叶绿体在发育和适应胁迫过程中发生重要的功能变化。但是TOC蛋白深深地嵌入叶绿体外膜中，在被SP1标记后对它们的去除提出了物理障碍。我们对新CHLORAD基因的发现为如何克服这一障碍提供了线索：我们未发表的数据强烈表明，这些基因编码驱动提取不需要的TOC蛋白的分子马达的关键组分。我们将研究这个CHLORAD机制，以更清楚地了解不需要的叶绿体蛋白质是如何被去除的。此外，CHLORAD在环境胁迫耐受性中的作用也将被研究。我们将探讨如何操纵的途径的活动，可用于提高植物的胁迫耐受性。该途径似乎在许多不同的植物物种中起作用，包括主要作物，因此我们的研究结果有可能得到广泛的应用。干旱和盐碱化是影响作物产量的最重要因素之一，每年仅干旱造成的全球损失估计就达100亿美元。我们相信，我们在CHLORAD上的工作可能有助于减轻这种损失。

The human population is growing rapidly and set to reach 9bn by 2050, and there is ever increasing pressure on natural resources. Thus, the drivers for increased crop yields and resilience to climate change and sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crop varieties. Through research on the model plant thale cress, we recently made a significant breakthrough: We discovered a gene called SP1 that controls important aspects of plant growth, including plant responses to adverse environmental conditions such as water stress and high salinity (collectively, abiotic stresses). Thale cress plants can be made more tolerant of such stresses by modifying SP1 expression. Recently, we identified two new genes that function in the same regulatory pathway as SP1 - a pathway which we now term CHLORAD (for "Chloroplast-Associated Degradation"). In this project, we will study these new genes in detail, to elucidate their functions and understand how they work together with SP1, and in doing so we hope to identify new strategies for crop improvement.Like SP1, the two new CHLORAD genes regulate the development of structures inside plant cells called chloroplasts. Chloroplasts are normal cellular constituents (i.e., they are organelles), and in many ways they define plants. They contain the green pigment chlorophyll and are responsible for photosynthesis, capturing sunlight energy and using it to power the activities of the cell. 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 abiotic stress, and so are ideal targets for engineering stress tolerance in crops.Chloroplasts are composed of thousands of different proteins, and most of these are encoded by genes in the cell nucleus and so are synthesized outside of the organelle in the cellular matrix known as the cytosol. As chloroplasts are each surrounded by a double-membrane envelope, sophisticated machinery is needed to bring about the import of these proteins into the organelle. This comprises two molecular machines, one in each membrane, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several different proteins that work cooperatively.The SP1 gene encodes a regulatory factor called a "ubiquitin E3 ligase". Such regulators work by labelling-up unwanted proteins to target them for removal. The SP1 E3 ligase mediates the removal of TOC components, and thereby controls TOC functions so that only the desired proteins are imported by chloroplasts. Such control enables major functional changes of chloroplasts during development and in adaptation to stress. But TOC proteins are deeply embedded in the chloroplast outer membrane, presenting a physical obstacle to their removal following labelling by SP1. Our discovery of the new CHLORAD genes provides a clue as to how this obstacle is overcome: our unpublished data strongly suggest these genes encode key components of a molecular motor that drives the extraction of unwanted TOC proteins. We will study this CHLORAD machinery to understand more clearly how unwanted chloroplast proteins are removed.Moreover, the role of CHLORAD in environmental stress tolerance will be studied. We will explore how manipulating the activity of the pathway may be used to improve stress tolerance in plants. The pathway appears to operate in many different plant species, including major crops, and so our results have the potential to see broad application. Drought and salinity are among the most significant factors affecting crop yields, with annual global losses due to drought alone estimated at $10bn. We believe that our work on CHLORAD may help to alleviate such losses.