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Analysis of Monosaccharides from Arabidopsis Seed Mucilageand Whole Seeds Using HPAEC-PAD.

Bio-protocol (2019-12-20)
Gillian H Dean, Kresimir Sola, Faride Unda, Shawn D Mansfield, George W Haughn
ABSTRACT

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
D-(+)-Mannose, wood, ≥99%
Sigma-Aldrich
L-(+)-Arabinose, ≥99%
Sigma-Aldrich
D-(+)-Galactose, BioXtra, pH 5.0-7.0 (20 °C, 1 M in H2O), ≥99%
Sigma-Aldrich
D-(+)-Xylose, BioXtra, ≥99%
Sigma-Aldrich
D-(+)-Galacturonic acid monohydrate, ≥97.0%