The reducing end sequence of wheat endosperm cell wall arabinoxylans

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 1M 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.