Carbopac 1 column

For the non-cellulosic monosaccharide composition analysis, approximately 200 mg of frozen powder was freeze-dried and used for the cell wall extraction, as described in Duran Garzon et al. [54 (link)]. The freeze-dried cell wall material was then digested with amylase according to Foster et al. [58 (link)]. After digestion, 2 mg of destarched dry cell wall (DCW) was hydrolyzed with 2 N trifluroacetic acid (TFA) for 90 min at 121 °C. TFA was then evaporated under a stream of nitrogen. The sample was dissolved in 1 mL H₂O and 100 µL of the sample was injected onto a CarboPac-1 column (Dionex) for high-performance anion exchange chromatography (HPAEC) of non-cellulosic sugars, which were detected using pulsed amperometry (PAD) as described in Duran Garzon et al. [54 (link)]. Analyses were performed in triplicate. For quantification purposes, standard curves for all measured sugars (l-fucose, l-rhamnose, l-arabinose, d-galactose, d-glucose, d-xylose, d-mannose, d-apiose, d-galacturonic acid and d-glucuronic acid) were used from a stock solution with the following concentrations of each sugar at 0.1, 0.3, 0.5, 0.7, 0.2, 0.8, 0.08, 0.9, 0.5 and 0.05 mM, respectively, and diluted to a half and a tenth of the concentration.

Plant cell wall material was prepared from 150 mg of frozen shoot powder as described by Duran Garzon et al. (2020) (link). Dry cell wall material was digested with amylase according to Fleischer et al. (1999) (link). After digestion, the dry cell wall (2 mg) was hydrolyzed, and the samples were injected onto a CarboPac-1 column (Dionex) HPAEC, separated, and detected by PAD. Data were collected and processed using the Chromeleon 6.50 software (Dionex Corp.) (Duran Garzon et al., 2020 (link)).