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

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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.