Altering the substitution and crosslinking of glucuronoarabinoxylans affects cell wall porosity and assembly in Brachypodium distachyon.

O_LIThe Poaceae family of plants provides cereal crops that are critical for human and animal nutrition and also they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion, thus understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. C_LIO_LIWe generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan biosynthesis. Using carbohydrate gel electrophoresis we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. C_LIO_LIBdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are more porous, suggesting the xylan substitutions directed by both BdXAX1A andGUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. C_LIO_LISince xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials. C_LI