Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes

Abstract

BackgroundPlant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow.ResultsA method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.ConclusionsThe DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

“…Assay products, alongside appropriate controls and standards were separated by capillary electrophoresis (CE) according to Li et al . (). Data was analysed using the DASHboard software (Li et al ., ).…”

“…X 6 (20 lg, Megazyme) was derivatised with 8-aminopyrene-1,3,6trisulfonic acid (APTS, Biotium) as described in Li et al (2013). Excess fluorophore was removed using a GlykoClean S Cartridges (Prozyme, www.prozyme.com) and dried in vacuo.…”

Xylan decoration patterns and the plant secondary cell wall molecular architecture

“…In contrast with acetylation, glucuronic acid (GlcA) decorations exist on only one in eight xylose residues and are strictly limited to a glycosidic linkage at the 2-OH position. The GlcA residues themselves may or may not become methylated [Me] at the 4-OH position through the actions of one of the three DUF579 glucuronoxylan methyltransferases (GXM) [14,15]. Methylation occurs after the GlcA moieties have been transferred on to the xylan backbone.…”

Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis

“…DUF579 family members of higher plants are clustered in four clades (Figure a). The three members of the DUF579 family Clade I, GXM1‐3, are 4‐ O ‐methyltransferases using S‐adenosylmethinonine as donor to catalyze the transfer of 4‐ O ‐methyl to glucuronic acid on xylan (Lee et al., ; Li et al., ; Urbanowicz et al., ). In addition, two DUF579 members of Clade II, IRX15 and IRX15L, are implicated in xylan biosynthesis and/or deposition of xylan in secondary cell walls (Brown et al., ; Jensen et al., ).…”