Plants represent the largest living source of biomass on Earth. Most of this biomass comes from the polysaccharide-rich secondary cell wall. Industrially, polysaccharides have a number of potential key uses. For these to be possible, it is important to understand how polysaccharides are synthesised and arranged to confer structural properties to the cell wall. The hemicellulose xylan is the second-most abundant secondary cell wall polysaccharide after cellulose. For angiosperms, the xylan backbone consists of repeating xylosyl residues and is decorated along its length by acetate and glucuronic acid (GlcA). It is likely that the position of these decorations, created by Trichome-Birefringence-Like (TBL) and Glucuronic acid substitution of Xylan (GUX) enzymes respectively, are key for the xylan molecule to perform a hypothesised linking function between cellulose and lignin, contributing to the wall’s recalcitrance to enzymatic degradation. While GUX1 is responsible for evenly spaced GlcA decoration, which covers the majority of the xylan backbone, GUX2 positions GlcA in clusters covering a smaller area. This work investigated the source of this activity difference and the impact of GlcA patterning on secondary cell wall structure and phenotype.

The use of site directed mutagenesis in this work allowed investigation of the source of well-documented patterning differences between GUX2 from angiosperms, specifically AtGUX2, and gymnosperms, focusing on PtGUX2, and identified an SRF amino acid motif in PtGUX2, absent in angiosperms, crucial for creating consecutive GlcA decorations on the xylan backbone. Taken together with results from mutagenesis of the predicted active site of AtGUX1 and AtGUX2, this work suggested that the amino acid composition in the GUX catalytic C-terminus, including a GT8 domain, plays a key role in activity and patterning. However, creation of GUX chimeras showed that addition of the AtGUX1 N-terminal amino acid sequence to the C-terminus of AtGUX2 led to a significant increase in activity compared to the full length AtGUX2 enzyme. The chimera also produced an unusual patterning of unevenly spaced GlcA additions, extended along the xylan backbone. Interestingly, this uneven patterning did not lead to an increase in the proportion of xylan adopting a three-fold screw conformation in the plant cell wall. Combined with results from previous literature, this finding suggests that an even acetylation pattern may have more relevance for xylan conformation than GlcA patterning. Analysis of the activity of chimeras with swapped GUX and TBL29 N and C-terminal domains suggested that GUX and TBL29 activity exist in a fine balance, crucial for xylan patterning. Mass spectrometry results indicated that GlcA decorations by AtGUX2 were generally positioned closer to acetate than decorations by AtGUX1. Intriguingly, GlcA decorations by GUX1-2 were surrounded by less acetylation than GlcA by AtGUX2, and vice versa for the GUX2-1 chimera. Altogether, these results heavily suggest that the GUX N-terminus also plays an important, although perhaps indirect, role in determining activity.