Gel-permeation chromatography-enzyme-linked immunosorbent assay method for systematic mass distribution profiling of plant cell wall matrix polysaccharides.

Abstract

Cell walls are dynamic and multi-component materials that play important roles in many areas of plant biology. The composition and interactions of the structural elements give rise to material properties, which are modulated by the activity of wall-related enzymes. Studies of the genes and enzymes that determine wall composition and function have made great progress, but rarely take account of potential compensatory changes in wall polymers that may accompany and accommodate changes in other components, particularly for specific polysaccharides. Here, we present a method that allows the simultaneous examination of the mass distributions and quantities of specific cell wall matrix components, allowing insight into direct and indirect consequences of cell wall manipulations. The method employs gel-permeation chromatography fractionation of cell wall polymers followed by enzyme-linked immunosorbent assay to identify polymer types. We demonstrate the potential of this method using glycan-directed monoclonal antibodies to detect epitopes representing xyloglucans, heteromannans, glucuronoxylans, homogalacturonans (HGs) and methyl-esterified HGs. The method was used to explore compositional diversity in different Arabidopsis organs and to examine the impacts of changing wall composition in a number of previously characterized cell wall mutants. As demonstrated in this article, this methodology allows a much deeper understanding of wall composition, its dynamism and plasticity to be obtained, furthering our knowledge of cell wall biology.