Calcium-regulation of nuclear proteins involved in stress tolerance

作物和植物的抗逆性依赖于对主要信号的感知，并转导到细胞机制以产生耐受性反应。在植物和作物中，主要的防线是基因表达的变化来介导耐受性。细胞钙浓度的增加是这一过程中的关键中间体，在环境和细胞核之间进行信息交换。我们以前的工作提供了深刻的知识，哪些基因是由钙调节的，并通过推断，哪些转录因子被使用。然而，我们还不了解细胞核中通过钙信号介导应激诱导耐受性所需的其他蛋白质成分。该项目将在拟南芥中采用全系统蛋白质组学方法来识别这些蛋白质，对它们进行遗传和机械表征，并利用这些信息生产（未来）具有增强反应性抗逆性的作物品系。这项工作的关键假设是，为了调节基因表达，钙需要触发特定调节核蛋白水平的变化。因此，钙很可能通过表达（包括通过调节翻译），或通过调节这些蛋白质进入细胞核的运输，或通过泛素化等机制调节这些蛋白质的稳定性来控制这些蛋白质的水平。为了验证这一假设，将对从植物中提取的样品进行核蛋白质组学研究，这些样品经过化学处理后，与对照组相比，细胞钙以及低温和干旱胁迫等自然信号发生了变化。通过在样品之间进行定量比较，这将允许识别丰度以钙依赖方式变化的蛋白质。一旦蛋白质被确定，钙依赖的丰度调节机制将被确定，以及它们在抗逆性中的作用，以及用于改造小麦以更好地耐受抗逆性的信息。目的：(1)回答以下生物学问题：(i)哪些核蛋白在应激反应中受到钙的大量调节？（ii）什么机制控制单个核蛋白的丰度？（iii）这些蛋白在抗逆性中的作用是什么？(2)今后利用拟南芥的研究成果，使小麦在逆境胁迫下表现更好。方法：为了回答第一个目标的问题(i)，将对钙激动剂处理的拟南芥进行定量蛋白质组学研究，以及在钙拮抗剂存在和不存在的情况下诱导钙反应的胁迫。问题（ii）将通过使用荧光蛋白标记和共聚焦显微镜、免疫沉淀和Western blot分析来测试蛋白质对钙的响应运动来回答。问题（iii）将通过进行基因功能获得和功能丧失分析来测试这些蛋白质在胁迫耐受性和应激基因表达中的作用。为了实现目标2，将利用达勒姆作物改良技术中心（DCCIT）的小麦转化设施创建转基因小麦品系，以过表达和敲除选定的基因，并测试对胁迫基因表达和胁迫耐受性的影响。符合BBSRC的优先事项：这项工作符合BBSRC的战略研究优先事项1：“农业和粮食安全”，具体来说，开发基础知识和应用解决方案以生产更耐旱的作物符合“生产适应未来环境挑战的作物”的目标，这一点很重要，因为全球农业的水资源可用性正在减少。

Stress tolerance in crops and plants relies on perception of the primary signal, and transduction to the cellular machinery to produce a tolerance response. In plants and crops the major line of defence is changes in gene expression to mediate tolerance. Increases in cellular calcium concentration are key intermediates in this process, brokering exchange of information between the environment and the nucleus. Our previous work has provided profound knowledge on which genes are regulated by calcium, and by inference, which transcription factors are used. However, we do not yet have any understanding of other protein components in the nucleus which are required for mediating stress induced tolerance, via calcium signalling. This project will adopt a whole system proteomics approach in Arabidopsis to identify these proteins, characterise them genetically and mechanistically, and exploit this information to produce (in the future) crop lines with enhanced reactive stress tolerance. The key hypothesis for this work is that to mediate gene expression, calcium needs to trigger changes in the levels of specific regulatory nuclear proteins. Therefore calcium is likely to control the levels of those proteins through expression (including by regulating translation), or by regulating transport of those proteins into the nucleus, or by regulating the stability of those proteins through mechanisms such as ubiquitination. To test this hypothesis nuclear proteomics will be performed on samples extracted from plants treated with chemicals leading to changes in cellular calcium, as well as natural signal such as low temperature and drought stress, compared to controls. By making quantitative comparisons between samples this will allow identification of proteins whose abundance changes in a calcium-dependent manner. Once proteins are identified, the mechanism of calcium-dependent regulation of abundance will be determined, as will their role in stress tolerance, and information used to engineer wheat to better tolerate stress.Aims:(1) To answer the following biological questions:(i) Which nuclear proteins are regulated in abundance by calcium in response to stress?(ii) What mechanism controls the abundance of individual nuclear proteins?(iii) What are the roles of these proteins in stress tolerance?(2) To in the future exploit findings from Arabidopsis towards adapting wheat to perform better in response to stress.Methodology: To answer question (i) from the first aim quantitative proteomics will be performed on Arabidopsis treated with calcium agonists, and stresses which induce calcium responses in the presence and absence of calcium antagonists. Question (ii) will be answered by testing the movement of proteins in response to calcium using fluorescent protein tagging and confocal microscopy, immunoprecipitation and Western blot analysis. Question (iii) will be achieved by performing genetic gain of function and loss of function analysis to test the role of these proteins in stress tolerance and stress gene expression. To achieve aim 2, transgenic wheat lines will be created using the wheat transformation facility in the Durham Centre for Crop Improvement Technology (DCCIT) to overexpress and knock down chosen genes and test effects upon stress gene expression and stress tolerance.Fit with BBSRC priorities: This work fits within BBSRC Strategic research priority 1: "agriculture and food security", specifically, developing fundamental knowledge and applied solutions to producing more drought-tolerant crops fits into the aim "generating crops adapted to the challenges of future environments", important due to decreasing water availability for agriculture worldwide.