An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls

Xylan and cellulose are abundant polysaccharides in vascular plants and essential for secondary cell wall strength. Acetate or glucuronic acid decorations are exclusively found on even-numbered residues in most of the glucuronoxylan polymer. It has been proposed that this even-specific positioning of the decorations might permit docking of xylan onto the hydrophilic face of a cellulose microfibril. Consequently, xylan adopts a flattened ribbon-like twofold helical screw conformation when bound to cellulose in the cell wall. Here we show that ESKIMO1/XOAT1/TBL29, a xylan-specific O-acetyltransferase, is necessary for generation of the even pattern of acetyl esters on xylan in Arabidopsis. The reduced acetylation in the esk1 mutant deregulates the position-specific activity of the xylan glucuronosyltransferase GUX1, and so the even pattern of glucuronic acid on the xylan is lost. Solid-state NMR of intact cell walls shows that, without the even-patterned xylan decorations, xylan does not interact normally with cellulose fibrils. We conclude that the even pattern of xylan substitutions seen across vascular plants therefore enables the interaction of xylan with hydrophilic faces of cellulose fibrils, and is essential for development of normal plant secondary cell walls.This work was part supported by the Leverhulme Trust grant for the Centre for Natural Material Innovation. J.W.-R., J.J.L. and O.M.T. are supported by studentships from Conicyt Chile and the Cambridge Trusts, the BBSRC Doctoral Training Partnership BB/J014540/1, and a BBSRC Novozymes iCASE award (BB/M015432/1) respectively. We thank K. B. Krogh for co-supervision of O.M.T. The UK 850 MHz solid-state NMR Facility used in this research was funded by EPSRC and BBSRC (Contract reference PR140003), as well as the University of Warwick including via part funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF)

Evolution of glucuronoxylan side chain variability in vascular plants and the compensatory adaptations of cell wall-degrading hydrolases.

Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation.This work was supported by the Leverhulme Trust Centre for Natural Material Innovation, the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK as part of the OpenPlant Synthetic Biology Research Centre (Reference BB/L014130/1), The UKRI grant underwriting the ERC advanced grant (EVOCATE Function and evolution of plant cell wall architecture for sustainable technologies. EP/X027120/1), Innovate UK, the Cambridge BBSRC-DTP Programme (Reference BB/J014540/1), a Novo Nordisk Foundation grant Oxymist (Grant Number NNF20OC0059697), and a BBSRC iCASE studentship (Reference 648 BB/M015432/1)

Evolution of glucuronoxylan side chain variability in vascular plants and the compensatory adaptations of cell wall–degrading hydrolases

Publication status: PublishedSummary Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30‐family endo‐glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47‐A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae‐specific xylan galactosyltransferase. Simultaneously, GH30 endo‐glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan‐modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate‐active enzyme activities, providing insight into plant–pathogen interactions and facilitating plant cell wall biotechnological utilisation. </jats:p

NMR Data files "Eudicot primary cell wall glucomannan is related in synthesis, structure, and function to xyloglucan"

Zip files contain unprocessed data of solid-state NMR on Arabidopsis wild-type callus and mutant calli, irx9l xxt1 xxt2, csla2 xxt1 xxt2, which were described in the paper published in The Plant Cell. Each zip file contains a different experiment. Microsoft Word file describes the keys for each experiment. Zip file no. 10, 30, and 1000 contain the data of 2D CP-INADEQUATE of irx9l xxt1 xxt2, wild-type, and csla2 xxt1 xxt2, which detect immobile components in the samples. Zip file no. 20 contains 2D DP-INADEQUATE of irx9l xxt1 xxt2, which detects mobile components in the sample. See the main manuscript for more details on sample collection, data acquisition, and interpretation of the data