Characterization of Feedback Regulation of Plant Cell Wall Accumulation Mediated by Receptor-Like Kinases

受体样激酶介导的植物细胞壁积累反馈调节的表征

• 批准号: 242012848
• 负责人: Dr. Rhea Stoppel
• 金额: --
• 依托单位: Feedstocks Division
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/242012848?language=en
• 关键词: Characterization; Feedback; Regulation; Plant; Cell

Plant cell walls are complex structures composed of polysaccharides that influence plant development and differentiation. In addition to playing a key role in plant growth and development, plant cell walls also comprise the most abundant biomaterial on earth and have potential to provide a source of cheap sugars for industrial biotechnology. Intense basic research over the recent years has provided an understanding of many aspects of cell wall biosynthesis and numerous genes and proteins involved in this process. It becomes obvious that plants have sophisticated feedback mechanisms, which enable them to adapt the compositions of their walls in response to environmental changes or biotechnological intervention. These regulatory mechanisms result in large-scale biomass fluxes that are an important aspect of biomass engineering. However, very little is known about these mechanisms that ensure controlled feedback signals from the cell wall to adapt the cell wall biosynthesis to the plant's needs. The major group of protein classes that have been implicated in feedback from the cell wall are the receptor-like kinases (RLKs). My aim is to investigate cell wall sensing processes mediated by RLKs using molecular and biochemical methods. The project has two main research objectives: (1) Characterization of RLKs involved in signaling from the secondary cell wall. (2) Identification of the RLK ligands by co-transformation of candidate ligands and RLK domain swap constructs. Candidate RLKs that may recognize small molecules such as peptides and saccharides were identified in a previous co-expression approach (Oikawa et al., 2010). In addition, strongly co-expressed candidate ligands were shown to be located in the plasma membrane and could represent signals that trigger signaling pathways. The experimental setup of RLK activation by different ligands will be crucial for identification of the ligands. Therefore, the previously characterized RLK proteins EFR and WAK1, implicated in pathogen response and cell wall signaling, respectively, will serve as positive controls. Chimeric RLKs containing a kinase domain from either EFR or WAK1 fused with the candidate receptor domains from the identified RLKs will be constructed. A screen for the kinase ligands will be set up by transient co-transformation of candidate ligands and RLK domain swap constructs in tobacco leaves. Ligand binding will be measured by increased production of ethylene (for chimeric proteins with EFR kinase domain) or oxidative burst (for chimeric proteins with WAK1 kinase domain). Putative oligosaccharide ligands will be investigated by applying the RLK proteins to glycoarrays. Furthermore, ligands and other proteins associated to the RLKs will be identified by co-immunoprecipitation and T-DNA knockout mutants will be analyzed. Findings of this research project will significantly help to better understand the communication between the cell wall and the cell interior.

植物细胞壁是由多糖组成的复杂结构，影响植物的发育和分化。除了在植物生长和发育中发挥关键作用外，植物细胞壁还包括地球上最丰富的生物材料，并有可能为工业生物技术提供廉价糖的来源。近年来，密集的基础研究提供了对细胞壁生物合成的许多方面的了解，以及参与这一过程的众多基因和蛋白质。很明显，植物有复杂的反馈机制，使它们能够适应环境变化或生物技术干预的墙的成分。这些调节机制导致了大规模的生物质通量，这是生物质工程的一个重要方面。然而，人们对这些机制知之甚少，这些机制确保来自细胞壁的受控反馈信号使细胞壁的生物合成适应植物的需要。与细胞壁反馈有关的主要蛋白质类别是受体样激酶(RLKs)。我的目标是用分子和生物化学方法研究RLKs介导的细胞壁感知过程。该项目有两个主要的研究目标：(1)鉴定参与次生细胞壁信号转导的RLK。(2)通过候选配体和RLK结构域交换结构的共转化来鉴定RLK配体。候选的RLK可能识别小分子，如肽和糖，在以前的共表达方法中被发现(Oikawa等人，2010年)。此外，强共表达的候选配体位于质膜上，可以代表触发信号通路的信号。不同配体激活RLK的实验设置将是鉴定配体的关键。因此，分别参与病原反应和细胞壁信号转导的RLK蛋白EFR和WAK1将作为阳性对照。将构建包含来自EFR或WAK1的激动域的嵌合RLKs，与来自已鉴定的RLKs的候选受体结构域融合。通过瞬时共转化候选配体和烟草叶片中的RLK结构域交换结构，将建立一个KK配体的筛选。配体结合将通过增加乙烯(对于具有EFR激活域的嵌合蛋白)或氧化爆发(对于具有WAK1激活域的嵌合蛋白)的产生来衡量。我们将通过将RLK蛋白应用于糖阵列来研究可能的寡糖配体。此外，将通过免疫共沉淀鉴定与RLKs相关的配体和其他蛋白质，并将分析T-DNA敲除突变体。这一研究项目的发现将大大有助于更好地了解细胞壁和细胞内部之间的联系。