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

本项目旨在阐明质体小球相关蛋白（FBN）家族中两个代表性蛋白的生化特性及其在植物抗逆中的生理作用。质体囊泡是蓝细菌和质体中的类囊体相关的脂质囊泡，并且含有大量的亲脂性分子，例如生育酚和色素，并且在细胞对各种应激条件的响应中起重要作用。除了上述分子外，质体分子还含有多种蛋白质，其中FBN是主要代表。结果表明，FBN参与了应力保护。然而，该蛋白家族的大多数代表的生物化学和生理功能尚未被表征。所有FBN主要由所谓的PAP/FBN结构域组成，其与脂质运载蛋白共享序列并且最可能也具有结构相似性。已知脂质运载蛋白来自细菌、植物和动物，其中它们负责将小的疏水分子转运通过亲水区室。因此，可以假设FBN在质体内运输过程中起作用。该蛋白质家族至少有11个亚组可以区分，它们在表达模式和亚细胞定位上不同。这些差异表明该蛋白质家族的个体代表的不同功能。结果表明，在拟南芥的14个FBN中，只有7个是质体小分子的核心组分，而剩余的FBN与类囊体膜结合或定位于基质中。在这个项目的过程中，两个选择的，既不本地化，也不表征代表FBN在拟南芥将进行调查，FBN 9和FBN 11。选择FBN 9是因为它在光合真核生物中具有很强的序列保守性，表明它具有重要的和进化上的古老功能。然而，选择FBN 11是因为存在与PAP/FBN结构域组合的蛋白激酶结构域。在本研究中，我将研究所选的FBN的生化特性，并试图阐明其在胁迫保护和适应中的生理功能。为了实现这一目标，不同的转基因突变株系（功能丧失和过表达）和野生型植物将暴露于选定的胁迫条件下，并在分子水平上进行比较，重点分析质体蛋白质组，抗氧化剂含量和活性氧的产生。此外，FBN 11的蛋白激酶活性将在体外通过使用异源表达的蛋白进行测试，并在体内通过分析质体磷酸蛋白质组进行测试。这将揭示FBN 11在叶绿体中胁迫依赖性磷酸化的潜在底物。揭示FBN的分子和生理功能将有助于对质体防御机制的全面理解，并有助于开发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.