Functional characterisation of stress-related fibrillin proteins in the thylakoid membranes of plant chloroplasts

本项目旨在阐明质球相关纤维蛋白(FBN)家族中的两个代表蛋白在植物抗逆性中的生化特性和生理作用。质球是蓝藻和叶绿体中与类囊体体相关的脂泡，含有大量的亲脂分子，如生育酚和色素，在细胞对各种胁迫条件的反应中发挥着至关重要的作用。除了上述分子外，质球中还含有多种蛋白质，其中FBN是主要代表。结果表明，FBN参与了应力保护作用。然而，这个蛋白家族的大多数代表的生化和生理功能还没有被描述。所有的FBN主要由所谓的PAP/FBN结构域组成，该结构域与Lipocalins序列相同，结构上也很可能是相似的。已知Lipocalin来自细菌、植物和动物，它们负责通过亲水性隔间运输疏水小分子。因此，可以假设FBN在内质运输过程中发挥作用。至少可以区分该蛋白家族的11个亚群，它们的表达模式和亚细胞定位不同。这些差异表明该蛋白家族的个体代表具有不同的功能。结果表明，在拟南芥的14个FBN中，只有7个是质球的核心成分，而残留的FBN与类囊体膜相关或定位于基质中。在这个项目的过程中，将研究两个在拟南芥中选择的FBN的代表，即FBN9和FBN11，它们既不是本地化的，也不是特征化的。FBN9在光合作用真核生物中具有很强的序列保守性，表明其具有重要的进化老功能。然而，之所以选择FBN11，是因为蛋白激酶结构域与PAP/FBN结构域结合在一起。在本项目中，我将研究选定的FBN的生化性质，并试图阐明它们在胁迫保护和适应中的生理功能。为了实现这一目标，不同的转基因突变系(功能丧失和过度表达)和野生型植物将被暴露在选定的胁迫条件下，并在分子水平上进行比较，重点是叶绿体蛋白质组、抗氧化剂含量和活性氧的产生。此外，将使用异源表达的蛋白在体外测试FBN11的蛋白激酶活性，并通过对叶绿体磷酸蛋白质组的分析来测试在体内的蛋白激酶活性。这将揭示FBN11在叶绿体中胁迫依赖的磷酸化的潜在底物。揭示FBN的分子和生理功能将有助于对叶绿体防御机制的全面了解，并有助于发展提高植物抗逆性的新概念。

This project aims to elucidate the biochemical properties and physiological roles of two representatives from the plastoglobule-associated fibrillin (FBN) protein family in stress tolerance of plants. Plastoglobules are thylakoid-associated lipid vesicles in cyanobacteria and plastids and contain large amounts of lipophilic molecules, e.g. tocopherols and pigments, and play a vital role in the cellular response to various stress conditions. Apart from the aforementioned molecules, plastoglobules contain a variety of proteins, of which the FBN are the major representatives. It was shown that FBN are involved in stress protection. However, the biochemical and physiological functions of most representatives of this protein family have not been characterised yet. All FBN mainly consist of the so-called PAP/FBN domain, which shares sequence and most probably also structural similarity with lipocalins. Lipocalins are known from bacteria, plants and animals, where they are responsible for the transport of small hydrophobic molecules through hydrophilic compartments. Therefore, it can be assumed that FBN play a role in innerplastidic transport processes. At least 11 subgroups of this protein family can be distinguished, which differ in expression patterns and subcellular localisation. These differences indicate diverse functions of the individual representatives of this protein family. It was shown that only 7 of the 14 FBN in Arabidopsis are core components of plastoglobules while the residual FBN are associated with the thylakoid membrane or localised in the stroma. In the course of this project, two selected and neither localised nor characterised representatives of the FBN in Arabidopsis will be investigated, FBN9 and FBN11. FBN9 was chosen for its strong sequence conservation within the photosynthetic eukaryotes indicating an important and evolutionary old function. FBN11, however, was chosen because of the presence of a protein kinase domain in combination with the PAP/FBN domain. In this project I will investigate the biochemical properties of the selected FBN and try to elucidate their physiological function in stress protection and adaptation. To achieve this, different transgenic mutant lines (loss-of-function and overexpressors) and wild type plants will be exposed to selected stress conditions and compared on the molecular level with a focus on the analysis of plastid proteomes, the antioxidant content and the production of reactive oxygen species. Furthermore, the protein kinase activity of FBN11 will be tested in vitro by using heterologously expressed protein and in vivo by analysis of the plastid phosphoproteome. This will reveal potential substrates for stress-dependent phosphorylation in the chloroplast by FBN11. Revealing the molecular and physiological functions of FBN will contribute to a general understanding of the plastid defence mechanisms and to development of novel concepts for increased stress tolerance in plants.