Mucilage Monosaccharide Composition Analysis

The non-adherent mucilage and adherent mucilage monosaccharide compositions were analyzed as previously described20 (link). In brief, mucilage extracted from 5 mg of seed were hydrolyzed using 2 N trifluoroacetic acid for 2 hours. After evaporating the trifluoroacetic acid, the hydrolysates were derivatized with 1-phenyl-3-methyl-5-pyrazolone and NaOH at 70 °C for 2 hours. HCl was then added for neutralization. The mixture was extracted with dichloromethane 3 times and then analyzed on a ZORBAX Eclipse XDB-C₁₈ column (2.1 × 250 mm; Agilent) connected to a Agilent 1200 HPLC System at a constant flow rate of 0.5 mL/min. 5 μL sample was injected, eluted with 70% (v/v) ammonium formate buffer (0.1 M, pH 5.5) and 30% (v/v) acetonitrile and monitored by UV A₂₄₅.