Elucidating the role of SP2 and the SP1-SP2 machinery in chloroplast protein degradation

人口正在迅速增长，到2050年将达到90亿，自然资源面临越来越大的压力。因此，提高作物产量、适应气候变化和次优生长条件的驱动力比以往任何时候都更加强烈。为了满足这些需求，开发改良作物品种将是至关重要的。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，包括植物对不利环境条件的反应，如水分胁迫和高盐分(统称为非生物胁迫)。通过改变SP1的表达，可以使水芹更耐受这种逆境。最近，我们发现了另一种名为SP2的基因，它的功能与SP1的调控途径相同。在这个项目中，我们将详细研究SP2基因，阐明它的功能，了解它是如何与SP1一起工作的，并通过在水稻上的研究来探索它在作物改良中的潜在用途。SP1/2基因调控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，捕捉阳光能量，并利用它来为细胞的活动提供动力。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。叶绿体也在植物对非生物胁迫的反应中起着关键作用，因此是作物工程逆境耐受的理想靶标。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，因此在细胞基质中的细胞器外合成，称为细胞质。由于每个叶绿体都被一个双膜层包围，因此需要复杂的机械来将这些蛋白质引入细胞器。这包括两个分子机器，每个膜上一个，称为TOC(叶绿体外膜转运子)和TIC。每台机器由几种不同的蛋白质组成，它们协同工作。SP1基因编码一种被称为“泛素E3连接酶”的调节因子。这样的监管机构通过标记不需要的蛋白质来针对它们进行去除。SP1 E3连接酶介导TOC组分的去除，从而控制TOC的功能，使叶绿体只输入所需的蛋白质。这种控制使叶绿体在发育和适应逆境过程中发生了重大的功能变化。但TOC蛋白深埋在叶绿体外膜中，在SP1标记后，对它们的去除构成了物理障碍。我们对SP2的发现为如何克服这一障碍提供了线索。SP2基因编码一条穿过叶绿体外膜的通道，我们的证据表明，它形成了清除不需要的TOC蛋白的出口门。事实上，我们认为SP1和SP2因子在一个复合体中稳定地相关，从而使协调的蛋白质标记和去除成为可能。我们将研究这种SP1-SP2机制，以更清楚地了解不需要的叶绿体蛋白是如何被移除的。此外，我们还将研究SP2在环境胁迫耐受性中的作用。特别是，我们将操纵SP1-SP2途径的活性，以提高水稻的逆境耐受性。SP1-SP2途径似乎在许多不同的植物物种中发挥作用，包括主要农作物，因此我们的结果具有广泛应用的潜力。干旱和盐碱是影响作物产量的最重要因素之一，仅干旱造成的全球年度损失估计就达100亿美元。我们相信，我们与SP1/2的合作可能有助于减少此类损失。

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 another gene called SP2 that functions in the same regulatory pathway as SP1. In this project, we will study the SP2 gene in detail, to elucidate its function, to understand how it works together with SP1, and to investigate its potential use for crop improvement by conducting studies in rice.The SP1/2 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 SP2 provides a clue as to how this obstacle is overcome. The SP2 gene encodes a channel across the chloroplast outer membrane, and our evidence suggests that it forms the exit gate for the removal of unwanted TOC proteins. In fact, we believe that the SP1 and SP2 factors are stably associated in a complex to enable coordinated protein labelling and removal. We will study this SP1-SP2 machinery to understand more clearly how unwanted chloroplast proteins are removed.Moreover, the role of SP2 in environmental stress tolerance will be studied. In particular, we will manipulate activity of the SP1-SP2 pathway with the aim of improving stress tolerance in rice. The SP1-SP2 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 with SP1/2 may help to alleviate such losses.