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

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，植物细胞壁还包含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的糖源。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以及参与该过程的许多基因和蛋白质的理解。很明显，植物具有复杂的反馈机制，使它们能够适应环境变化或生物技术干预。这些调节机制导致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，对于这些机制知之甚少，这些机制确保来自细胞壁的受控反馈信号以使细胞壁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是受体样激酶（RLK）。本论文的目的是利用分子生物学方法研究RLKs介导的细胞壁感应过程。该项目有两个主要研究目标：（1）参与次生细胞壁信号传导的RLKs的表征。(2)通过候选配体和RLK结构域交换构建体的共转化鉴定RLK配体。在先前的共表达方法中鉴定了可以识别小分子如肽和RNA的候选RLK（Oikawa等人，2010年）。此外，强共表达的候选配体被证明位于质膜中，并且可以代表触发信号传导途径的信号。不同配体激活RLK的实验装置对于配体的鉴定至关重要。因此，先前表征的分别与病原体应答和细胞壁信号传导有关的RLK蛋白EFR和WAK 1将用作阳性对照。将构建含有来自EFR或WAK 1的激酶结构域与来自鉴定的RLK的候选受体结构域融合的嵌合RLK。通过候选配体和RLK结构域交换构建体在烟草叶中的瞬时共转化来建立激酶配体的筛选。将通过乙烯（对于具有EFR激酶结构域的嵌合蛋白）或氧化爆发（对于具有WAK 1激酶结构域的嵌合蛋白）的产生增加来测量配体结合。将通过将RLK蛋白应用于糖阵列来研究推定的寡糖配体。此外，将通过免疫共沉淀鉴定与RLK相关的配体和其他蛋白质，并分析T-DNA敲除突变体。该研究项目的发现将有助于更好地了解细胞壁和细胞内部之间的通信。

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.