Molecular and biochemical tools for plant cell wall glycan analysis

Plant cell walls are complex structures composed of diverse polymers: polysaccharides, proteins and sometimes phenolic compounds, varying in nature, structure and length. A study of the structure and occurrence of cell wall polysaccharides is required to be able to understand their mechanical and biological functions as well as to understand the overall architecture of cell walls. Studying cell wall polysaccharides is challenging mostly because of the limited methods and tools available for their characterisation. Chromatography systems are often used to separate and purify cell wall polysaccharides. In parallel, monoclonal antibodies (mAbs) have been largely used to study cell wall polysaccharide localisation, compositional variation and their potential mechanical properties and biological functions. However, the number of epitopes currently targeted by mAbs is limited and some polysaccharide features cannot be studied using mAbs. The project reported here was focused on the development of new methods, namely Epitope Detection Chromatography and glycan sandwich-ELISA, as well as the raising and characterisation of several new monoclonal antibodies PDT2, PDT5, PDT8, PDT10, PDT13 and PDT17. These new probes recognise various RG-I (PDT10, PDT13), AGP (PDT8, PDT17) and heteroxylan (PDT2, PDT5) specific epitopes. The Epitope Detection Chromatography can perform high resolution glycan analysis by combining the separation of polysaccharides by chromatography and their detection by specific molecular probes such as monoclonal antibodies and carbohydrate-binding modules. Contrary to numerous available polysaccharide analysis techniques EDC analysis can be performed directly on complex mixtures such as cell wall extracts and target one precise defined oligosaccharide or epitope. The development of sandwich-ELISA allows the study of multi-component complexes and inter-linked polysaccharides. The two methods can be combined to study subsets of polysaccharides. Here, the two techniques have been used together with the new set of PDT monoclonal antibodies to study cell wall polysaccharide composition and multi-polysaccharide complexes in a range of biological systems: tobacco seed endosperm, grape fruit and Arabidopsis thaliana. These studies have led to the identification of RG-I sub-families in tobacco seed and A. thaliana, as well as potential xylan-pectin linkages found in A. thaliana stem cell walls.