Elsevier

Carbohydrate Research

Volume 386, 11 March 2014, Pages 23-32
Carbohydrate Research

The reducing end sequence of wheat endosperm cell wall arabinoxylans

https://doi.org/10.1016/j.carres.2013.12.013Get rights and content

Highlights

  • Reducing end (RE) sequence(s) of wheat (monocot) endosperm AXs.

  • β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp.

  • β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-d-GlcAp-(1→2)]-β-d-Xylp.

Abstract

Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of hetero-(arabino)xylans (AXs) (70%) and (1→3)(1→4)-β-d-glucans (20%) with minor amounts of cellulose and heteromannans (2% each). To understand the differential solubility properties of the AXs, as well as aspects of their biosynthesis, we are sequencing the xylan backbone and examining the reducing end (RE) sequence(s) of wheat (monocot) AXs. A previous study of grass AXs (switchgrass, rice, Brachypodium, Miscanthus and foxtail millet) concluded that grasses lacked the comparable RE glycosyl sequence (4-β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp) found in dicots and gymnosperms but the actual RE sequence was not determined.

Here we report the isolation and structural characterisation of the RE oligosaccharide sequence(s) of wheat endosperm cell wall AXs. Walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with hot water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr). Detailed structural analysis of the RE oligosaccharides was performed using a combination of methylation analysis, MALDI-TOF-MS, ESI-QTOF-MS, ESI-MSn and enzymic analysis. Analysis of RE oligosaccharides, both 2AB labelled (from W-sol Fr) and glycosyl-alditol (from KOH-sol Fr), revealed that the RE glycosyl sequence of wheat endosperm AX comprises a linear (1→4)-β-d-Xylp backbone which may be mono-substituted with either an α-l-Araf residue at the reducing end β-d-Xylp residue and/or penultimate RE β-d-Xyl residue; β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp and/or an α-d-GlcpA residue at the reducing end β-d-Xylp residue; β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-d-GlcAp-(1→2)]-β-d-Xylp. Thus, wheat endosperm AX backbones lacks the RE sequence found in dicot and gymnosperm xylans; a finding consistent with previous reports from other grass species.

Introduction

Heteroxylans are major components of the primary walls of grasses and the secondary walls of all angiosperms1, 2, 3, 4 and are important sources of ‘soluble dietary fibre’ in the grains of cereals.5, 6 They are a family of polysaccharides with xylan backbones differing in their type and pattern of substitution with glycosyl (primarily glucuronic acid (GlcA), Araf) and non-glycosyl (O-Acetyl, ferulic acid) residues depending upon tissue type and species. Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of hetero(arabino)xylans (AXs) (70%) and (1→3)(1→4)-β-d-glucans (20%) with minor amounts of cellulose and heteromannans (2% each).7 The heteroxylans of endosperm walls are unique in that their glycosyl substitution is primarily with Araf, with few if any GlcA or O-Ac residues and the presence of ferulic acid substitution on the side chain Araf residues. Thus, endosperm AXs consist of a linear (1→4)-β-d-Xylp backbone which may be variously un-substituted, mono-substituted (2- or 3-) and di-substituted (2, 3-) with α-l-Araf residues.6, 8 In addition, the xylan backbone may be substituted with α-l-Araf residues which bear a feruloyl group at the C(O)5 position through an ester linkage.9 The arabinose/xylose (Ara/Xyl) ratio is about 0.5–0.6 of the water-soluble wheat endosperm AXs, which comprise 25–30% of the total AXs.10 However, there are a wide range of substitution patterns and Ara/Xyl ratios observed for different cultivars and various fractions.11

The RE glycosyl sequence of grass (monocot) AXs has not been determined although Kulkarni et al.12 reported that grass (switchgrass, rice, Brachypodium, Miscanthus and foxtail millet) AXs lack ‘discernible amounts’ of the specific RE sequence (4-β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp) as found in dicots (for example, Arabidopsis thaliana)13 and gymnosperms (for example, spruce (Picea abies)14). However, Kulkarni et al.12 did not determine the actual RE sequence in these grass AXs.

The differential expression of several genes encoding glycosyltransferases (GTs) involved in GAX biosynthesis have been reported for poplar wood (Populus tremula)15 and cotton (Gossypium hirsutum) fibre16 and has been recently reviewed.3, 4 Pena et al.13 demonstrated the requirement of FRA8 and IRX8 genes for the synthesis of specific glycosyl sequence at the RE of Arabidopsis GAXs whereas IRX9 is proposed to be involved in elongation of the GAXs chain. The involvement of genes for the biosynthesis of grass AXs are not well studied, however, based on AX structure and bioinfomatic studies it has been suggested that GTs from families GT43 (IRX9 and IRX14)17, 18, 19 and GT47 (IRX10)19, 20 are involved in xylan backbone incorporation, GT 61 are responsible for arabinosyl (XATs21) and xylosyl (XAX22) side chain incorporation and GT 8 (GUX)23 are involved in GlcA side chain incorporation.

In this paper we report the RE oligosaccharide sequence of wheat endosperm AXs. Endosperm walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with hot water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr). Two different approaches (see summary in Fig. 1) were adopted with 2 aminobenzamide (2AB) labelling of RE oligosaccharides to assist in detection and purification. In the first, intact W-sol AXs were treated with 2AB to tag the RE sugar residue. The 2AB labelled W-sol AXs were then treated with an endoxylanase to generate a mixture of reduced (2AB-labelled) and non-reduced oligosaccharides. In a second approach involving the KOH-sol Fr the endoxylanase was used to first generate oligosaccharides which were subsequently labelled with 2AB. In this scenario the original RE of the KOH-sol AX would not be labelled with 2AB since it had been reduced to the glycosyl-alditol during the alkali extraction that contained the reductant, sodium borohydride (NaBH4). The released oligosaccharides from both W- and KOH-sol Frs were fractionated by reversed-phase (RP) HPLC on a C18 silica column. The oligosaccharides were characterised using a combination of methylation analysis, MALDI-TOF-MS, ESI-QTOF-MS, ESI-MSn and enzymic analysis.

Section snippets

Linkage composition of wheat endosperm AIR and soluble wall fractions

The monosaccharide and derived polysaccharide compositions from linkage analyses of the AIR, W- and 1 M KOH-sol Frs was essentially as previously reported7 (see Supplemental Table S1 and Table 1) with the major polysaccharides being AXs and MLGs. AX was the principal component accounting for 71, 83 and 81 mol % of total wall polysaccharides of each of the AIR, W- and KOH-sol Frs respectively (Table 1). MLGs were the other significant components of AIR, W- and 1 M KOH-sol Frs (12, 6 and 7 mol %

Conclusion

Analysis of RE oligosaccharides revealed that the RE glycosyl sequence of wheat endosperm AX comprises a linear (1→4)-β-d-Xylp backbone which may be mono-substituted with either an α-l-Araf residue at the reducing end β-d-Xylp residue and/or penultimate RE Xyl residue; β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp and/or an α-d-GlcpA residue at the reducing end β-d-Xylp residue; (β-d-Xylp-(1→4)-[α-l-Araf-(1→3)](+/−)-β-d-Xylp-(1→4)-[α-d-GlcAp-(1→2)]-β-d-Xylp

Wheat flour preparation

Wheat (Triticum aestivum L., va. Baxter) caryopses were conditioned at 14% moisture prior to milling. The bran (pericarp, seed coat and aleurone layers) was separated using a Quadumat buhler roller mill and then dry sieved through a 150 μm screen to obtain wheat flour.

Isolation of cell walls and extraction of AXs

The wheat flour (⩽150 μm particle sizes) was dry sieved (75 μm) to separate the starch granules from walls. Walls were prepared as an alcohol-insoluble residue (AIR) essentially as previously described.27, 28 The AIR was sequentially

Acknowledgments

This project was supported by funds from Commonwealth Scientific and Research Organisation Flagship Collaborative Research Program, provided to the High Fibre Grains Cluster via the Food Futures Flagship.

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