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

本项目旨在阐明塑料蛋白相关原纤维蛋白（FBN）家族的两个代表蛋白在植物抗逆性中的生化特性和生理作用。质体红蛋白是蓝藻和质体中与类囊体相关的脂质囊泡，含有大量亲脂分子，如生育酚和色素，在细胞对各种应激条件的反应中起着至关重要的作用。除了上述分子外，质体红蛋白还含有多种蛋白质，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蛋白的生化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，该结构域与脂钙蛋白具有相同的序列和结构相似性。脂质钙素是从细菌、植物和动物中发现的，它们负责通过亲水性隔室运输小疏水分子。因此，可以假设FBN在塑性内输运过程中起作用。该蛋白家族中至少有11个亚群可以被区分出来，它们在表达模式和亚细胞定位上有所不同。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，拟南芥中14个FBN中只有7个是质体红蛋白的核心成分，其余FBN与类囊体膜相关或定位于基质中。在这个项目的过程中，将对拟南芥中FBN的两个选定的、既不是局部的也不是特征性的代表进行研究，FBN9和FBN11。之所以选择FBN9，是因为它在光合作用真核生物中具有很强的序列保守性，表明它具有重要的、古老的进化功能。然而，之所以选择FBN11，是因为它存在一个与PAP/FBN结构域结合的蛋白激酶结构域。在本项目中，我将研究所选择的FBN的生化特性，并试图阐明其在逆境保护和适应中的生理功能。为了实现这一目标，将不同的转基因突变系（功能缺失和过表达）和野生型植物暴露在选定的胁迫条件下，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，FBN11的蛋白激酶活性将在体外通过异种表达蛋白检测，在体内通过质体磷酸化蛋白组分析。这将揭示FBN11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普遍理解，并为提高植物的抗逆性提供新的概念。

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.