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Department of Biochemistry



Xylans are based on a linear β-1,4-linked xylose polymer that can be substituted by arabinosyl (Ara) and/or glucuronyl (GlcA) side chains with the degree and nature of substitution varying between tissues and species (Figure 1). Xylans can be furthermore modified by additional sugar side chains, by methylation, acetylation or feruloylation.


GAX Figure 1 

Figure 1: Simple structure of glucuronoarabinoxylan


Arabinoxylan biosynthesis

Anders et al., 2012

Our recent work on the biosynthesis of arabinoxylan in grasses identified novel players of the glycosyltransferase family GT61, which are required for the addition of arabinosyl side chains to the xylan backbone (named XATs for xylan arabinosyltransferases). Candidate arabinosyltransferases were identified on the basis of an expression bias in monocots versus dicots, which coincides with the respective abundance of arabinoxylan. Localisation of XAT was confirmed to the Golgi apparatus the site of xylan biosynthesis. RNAi knock-down was then used to reduce XAT activity in wheat endosperm in collaboration with Dr Rowan Mitchell at Rothamsted Research and a reduction of α-1,3 linked arabinose side chains in the transgenic lines was detected. In addition, heterologous expression of GT61 wheat and rice genes in Arabidopsis thaliana stem, in which arabinosyl side chains on xylan are absent, resulted in α-1,3 arabinosyl side chain addition to xylan (Figure 2). This demonstrates the key role of the XAT genes in arabinoxylan biosynthesis and in the evolutionary divergence of grass cell walls. 




Figure 2: Analysis of rice (Ta) and wheat (Os) XAT activity in gux1/gux2 (gux) mutant and wildtype (wt) in Arabidopsis thaliana plants. The asterisks mark novel xylan oligosaccharides, that are arabinofuranosidase GH62 sensitive.


Glucuronoxylan biosynthesis in primary and secondary cell wall

Mortimer et al., 2015

We identified members of the GT8 glycosyltransferase family, GUX1 and GUX2 as being required for the addition of glucuronyl acid side chains to the xylan backbone in the secondary cell wall of Arabidopsis thaliana (Mortimer et al., 2010).

Our recent work revealed that another GT8 family member GUX3 is required for the addition of glucuronyl side chains to xylan in the primary cell wall-rich tissues (e.g. roots and young stem). Interestingly, the glucuronyl acid side chains added by GUX3 are further modified with a pentose, constituting a novel type of xylan side chain PUX5 (Figure 3).



Figure 3: (a) PACE analysis of xylanase GH11-digested AIR from mature stem, young stem and roots and respective no-enzyme controls. Xylo-oligosaccharide standard: X1-X5, GH11 only: enzyme only control. Note: PUX5 structure, marked with *, is not present in mature stem, whereas [m]UX4, marked with ┼, is present in all tissues analysed. (b) Structural analysis of the PUX5 oligosaccharide with MALDI-CID.


A plethora of potential xylosyltransferases are required for the synthesis of the xylan backbone (IRX9, IRX9L, IRX10, IRX10-L, IRX14 and IRX14-L), which show diverse degrees of redundancy in secondary cell wall biosynthesis (Hao Z, Mohnen D., 2014). Our work showed that in primary cell wall synthesis, only a subset of these glycosyltransferases, namely IRX9L, IRX10L and IRX14, are essential for xylan biosynthesis.

Understanding these differences in xylan structure and biosynthesis in primary versus secondary cell wall might help us to identify the biological roles of side chains in context of cross-linking and interaction with other cell wall components.


Patterning of glucuronoxylan decorations

Bromley et al., 2013 and Busse-Wicher et al., 2014

In the secondary cell wall of Arabidopsis thaliana glucuronyl acid side chains are added by the glycosyltransferases, GUX1 and GUX2 (Mortimer et al., 2010). Our recent work revealed that the activity of both enzymes, however, is surprisingly distinct. Using a glycosylhydrolase of the GH30 family XynC, which specifically cuts the xylosyl backbone in defined relation to the glucuronyl acid side chain, we found out that GUX1 and GUX2 decorate different domains of xylan (Figure 4). The GUX1-decorated domain is more abundant and the decoration of glucuronyl acid side chains is only on evenly-spaced xylosyl residues. Intervals of eight or 10 residues dominate, but larger intervals are observed. GUX2, in contrast, produces more tightly clustered decorations with most frequent spacing of five, six or seven xylosyl residues, with no preference for odd or even spacing.



Figure 4: (a) Scheme of the glucuronoxylanase XynC action. X, Xyl; U, [Me]GlcA; UXn, [Me]GlcAXyln; R, reducing end; NR, non-reducing end. (b) AIR from Col-0, gux1, gux2 and gux1 gux2 was hydrolysed by XynC and analysed by polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Markers X1 to X6 are shown (M). (c) DNA-sequencer assisted saccharide analysis in high-throughput (DASH) quantification of the relative abundance of each XynC oligosaccharide product.


Using mass spectrometry and NMR we were able to extend this novel finding of patterning to other modification of the xylose backbone: Acetylation of the xylan backbone occurs on every other xylosyl residue.

What is the biological function of xylan patterning? In collaboration with the group of Munir Skaf, Brazil, molecular dynamics simulation was performed. We found that a twofold helical screw conformation of xylan with every other xylosyl residue turned 180 degrees towards the previous one is stable in interaction with both hydrophilic and hydrophobic cellulose faces. Side chains of the xylan backbone are believed to affect negatively the interaction of xylan with cellulose. However, even-spacing of xylan modifications of a two-fold helical screw xylan backbone would expose all modifications on one side only, leaving an unsubstituted surface for a compatible interaction with cellulose through hydrogen bonding.



Figure 3: Molecular model indicating possible roles of xylan in  dicot secondary cell wall architecture. Xylan can interact with hydrophobic surface of cellulose (top view, A), and the compatible domain is also able to tightly associate with hydrophilic face of cellulose (B). The incompatible domains may allow tethering of adjacent cellulose fibrils (C), form loops which dissociate from the fibril and extend into the matrix and associate back to the same or different fibril (D), or span the fibrils and dock onto a different hydrophilic groove of the same fibril (E).

This Cambridge cell wall model highlights that not only the nature of side chains but also their distribution along the backbone might be of importance for the biological function of xylan in cell wall architecture and opens up new ways of thinking about polysaccharide interaction in vivo.

Contact details

Research Group Leader  Paul Dupree


Location  Hopkins Building


The Dupree Group is accepting enquiries from prospective interns, undergraduate students, postgraduate students and postdoctoral researchers.