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Background: Xylan is a major hemicellulosic component in the cell walls of higher plants especially in the secondary walls of vascular cells which are playing important roles in physiological processes and overall mechanical strength. Being the second most abundant cell wall polymer after cellulose, xylan is an abundant non-cellulosic carbohydrate constituent of plant biomass. Xylan structures have been demonstrated to contribute to plant biomass recalcitrance during bioenergy applications. A critical understanding of xylan composition, structure, and biosynthesis in developing plant stems will allow an increased understanding of how cell walls are put together in this organ in a basic research, and, in applied research, will improve strategies in xylan engineering to reduce biomass recalcitrance for economically feasible biofuel production. Methods: We describe an approach to enable the monitoring of xylan epitope structures in cell walls during the stem maturation process in Arabidopsis. The technique integrates glycome profiling, an in vitro immunoanalytical platform, and in situ immunolocalisation to provide comprehensive details on the presence, relative abundances, and dynamics with which diverse xylan epitope structures are integrated to the cell walls throughout the stem maturation process. Results: Our experimental results and the supporting in silico analysis demonstrated that xylan deposition in stems occurs early on in stem development; however, xylan epitope types (representing substituted and unsubstituted regions on xylan backbone made of β-(1,4)-linked xylose residues) and the strength of their integration into the final wall structure vary during stem maturation. Conclusions: Our novel approach thus provides a method to comprehensively survey the differences in xylan epitope patterning and deposition occurring in stem development and thereby providing a robust tool for characterising altered xylan integration patterns in cell walls during the stem maturation process in diverse plant cell wall biosynthetic mutants. Our findings also suggest that this approach could rapidly and reliably delineate xylan deposition patterns in the cell walls of plants belonging to diverse phylogenetic classes providing novel insights into the functional roles of xylans in overall growth and development. [ABSTRACT FROM AUTHOR]