PMC

GlcA spacing on primary wall xylan. PACE analysis of xylanase GH30-digested AIR from WT, gux1gux2 and gux3 roots. The GUX3-dependent oligosaccharides are marked with *.

The PUX₅ structure is dependent on GUX3, IRX9L, and IRX14/IRX14L. PACE analysis of xylanase (GH11)-digested AIR from young stem of (a) gux mutants, and (b) irx mutants, hydrolysed with GH11 and analysed by PACE, alongside controls. Xylo-oligosaccharide standard: X₁–X₄ positions are shown.

Nuclear magnetic resonance (NMR) analysis of PUX₅. H-1 strip plots from 2D ¹H-¹H TOCSY (red) ROESY (blue) and DQFCOSY (green) spectra, showing the nuclear Overhauser effect (NOE) connectivity arising from the pentose-α-1,2-GlcpA-α-1,2-Xylp and β-Xylp-β-1,4-Xylp glycosidic linkages.

Xylan structure in primary cell wall rich tissues. 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.

Complementation of irx9L and irx10L xylan phenotype with IRX9 and IRX10 respectively.PACE analysis of xylanase (GH11)-digested AIR from (a) irx9L young stems expressing IRX9 under the control of the IRX9L promoter (IRX9L_pro:IRX9) and (b) irx10L young stems expressing IRX10 under the control of the IRX10L promoter (IRX10L_pro:IRX10). Some lanes have been excised for clarity, as shown by the black line. AIR was prepared from young inflorescent stems, hydrolysed with xylanase GH11 and analysed by PACE. Three independently transformed lines are shown. Background bands are indicated by an *.

Structural analysis of the PUX₅ oligosaccharide. (a) Xylanase (GH11)-digested and 2-AA labelled AIR from young WT stem analysed by MALDI-ToF-MS to generate a series of sodiated adducts [M + Na]⁺. Peaks with dots are doubly sodiated. Peaks with stars are trebly sodiated. The peak corresponding to PUX₅ with mass GlcAPent₆ (m/z 1130.2) was selected for structural analysis using high intensity MALDI-CID. (b) The CID spectrum identifies PUX₅ as 1,2-pentose-α-1,2-GlcA on a backbone of five β-1,4-Xyl.

XylT and GlcAT activity in callus culture microsomes.An acceptor (APTS-labelled X₆) and substrates (UDP-Xyl, UDP-GlcA) were incubated for 5 h in the absence (a) or presence (b–g) of callus microsomes, and analysed by capillary electrophoresis (CE). WT Col0 callus gave a ladder of products (b) which following hydrolysis with GH115 α-glucuronidase resulted in a ladder of xylo-oligosaccharides (c). Peaks marked with an * are GH115 sensitive. gux3 callus (d) gave a very similar trace to WT + GH115 implying that little or no GlcA addition occurred. irx10L (e) irx9L (f) and irx14 (g) showed little or no extension of the acceptor implying that xylan synthesis was essentially absent. The acceptor (X₆) peak has been cropped in all traces except (b–d) for clarity.