Development of a new high-throughput method to determine the composition of ten monosaccharides including 4-<i>O</i>-methyl glucuronic acid from plant cell walls using ultra-performance liquid chromatography

Abstract

Plant cell walls are an important dietary source for livestock, and could be an enormous resource for production of next-generation bioethanol and more valuable materials. Because polysaccharides are major components of plant cell walls, analysis of their composition is important. In this report, we established a high-throughput method to determine the composition of ten monosaccharides from plant cell walls simultaneously using ultra-performance liquid chromatography with p-aminobenzoic ethyl ester-labeling technology. Complete separation of a mixture of internal standards, 2-deoxyglucose and 3-O-methyl glucose, and ten monosaccharides, consisting of seven neutral and three acidic sugars including 4-O-methyl-D-glucuronic acid, which are frequently found in plant cell wall polysaccharides, can be obtained within 7 min using this system. Relative standard deviations of retention time and peak area value are lower than 1%. Linearity for broader dynamic ranges (0.02-2000 mg l −1 ), faster analysis and higher sensitivity than other traditional methods, including one that employs widely used high-performance anion exchange chromatography, are achieved. We evaluated this new method by analyzing the composition of cell walls from three model plants (Arabidopsis thaliana, rice and hybrid aspen) and confirmed that the obtained results for most monosaccharides are consistent with those in previous studies. These data suggest that our newly developed system could greatly contribute to the study of plant cell walls, especially research requiring highthroughput analysis.

Golgi-localized membrane protein AtTMN1/EMP12 functions in the deposition of rhamnogalacturonan II and I for cell growth in Arabidopsis

“…To determine the monosaccharide composition of cell walls, extracted rosette leaf cell walls were analysed using an ultra-performance liquid chromatography– p -aminobenzyl ethyl ester system ( Sakamoto et al , 2015 ). Approximately 2.00–3.00 mg of AIR were added to a 2 ml microtube, and starch contained in the AIR was digested with 1 ml of an amylase solution containing 500 U ml –1 of α-amylase from porcine pancreas (Megazyme, Ireland) and 0.33 U ml –1 of amyloglucosidase from Aspergillus niger (Megazyme, Ireland) in 0.1 M sodium malate buffer (pH 6.0) at 37 °C for 18 h. Insoluble residues in the amylase suspension were rinsed with absolute ethanol and ultrapure water, then dried completely at 60 °C overnight.…”

“…Monomerized sugars in the neutralized supernatant were labelled with aminobenzyl ethyl ester solution containing 330 mg ml –1 of p -aminobenzyl ethyl ester (FUJIFILM Wako Pure Chemical, Japan), 66 mg ml –1 of sodium cyanoborohydride, 8% (v/v) acetic acid, and 75% (v/v) methanol at 80 °C for 30 min. Chromatographic separation and detection were conducted using an ACQUITY UPLC H-Class system equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.0 mm, 1.7 μm particle size, Waters Corp., USA) and fluorescence detector (ACQUITY UPLC FLR Detector, Waters Corp., USA) as previously described ( Sakamoto et al , 2015 ). For samples without amylase treatment, AIR powder was hydrolysed with H 2 SO 4 ; monosaccharide composition in hydrolysate was analysed as described above.…”

Wood reinforcement of poplar by rice NAC transcription factor

“…The purified cell wall residues were hydrolyzed using a two-step sulfuric acid method as described previously 11 . The monosaccharides in the hydrolysate were labelled with ABEE reagent and quantified using an UPLC system equipped with a fluorescence detector 24 .…”

Identification of enzymatic genes with the potential to reduce biomass recalcitrance through lignin manipulation in Arabidopsis

“…Monosaccharide composition was analyzed based on the UPLC-ABEE system as previously described [34]. AIR powder was hydrolyzed by two-step sulfuric acid hydrolysis, and the hydrolysate was neutralized with calcium carbonate.…”