The composition of non-cellulosic polysaccharides was determined according to Foster et al. (2010) with modifications. Due to shortage of plant material, analysis was performed only for samples harvested at the heading stage and maturity. De-starched AIR material (5 mg) was hydrolysed in 2 M TFA for 90 min at 121 °C. TFA was removed by drying under vaccum. TFA breaks down the cell wall network to release primarily non-cellulosic polysaccharides, and some fractions of cellulose that contain kinks and chain dislocations (amorphous form). The composition of monosaccharides in the filtrate was determined and quantified by high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-iPAD) using Thermo Scientific Dionex ICS5000 as described previously by Głazowska et al. (2018) (link). The system was calibrated with standards (l -Ara, d -Xyl, d -Gal, d -Glc, d -GlcA, and d -GalA) (Sigma). All calculations were done in Chromeleon CDS software.
D xyl
Manufactured by Merck Group
Sourced in United States
D-Xyl is a laboratory equipment product manufactured by Merck Group. It is a high-performance liquid chromatography (HPLC) column used for the analysis and separation of D-xylose and related carbohydrates. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
D glc, by Merck Group (5 mentions)
D gal, by Merck Group (5 mentions)
L fuc, by Merck Group (4 mentions)
L rha, by Merck Group (4 mentions)
D gala, by Merck Group (3 mentions)
D glca, by Merck Group (3 mentions)
L ara, by Merck Group (3 mentions)
Carbopac pa20 column, by Thermo Fisher Scientific (2 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (1 mentions)
Pulsed amperometric detector, by Thermo Fisher Scientific (1 mentions)
D fru, by Merck Group (1 mentions)
Dionex ics 2500, by Thermo Fisher Scientific (1 mentions)
Acid debranched wheat arabinoxylan, by Megazyme (1 mentions)
Xylotriose x3, by Megazyme (1 mentions)
Oligonucleotide primers, by Eurogentec (1 mentions)
Xylopentaose, by Megazyme (1 mentions)
Xylobiose x2, by Megazyme (1 mentions)
Xylohexaose x6, by Megazyme (1 mentions)
Xylotetraose x4, by Megazyme (1 mentions)
Galactomannan, by Megazyme (1 mentions)
7 protocols using d xyl
1
Quantifying Non-Cellulosic Polysaccharides
2
Monosaccharide Composition Analysis of Plant Cell Walls
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To determine monosaccharide composition, 3 replicates of cell walls from 10 plants were used. 1 mg of dry de-starched cell wall was hydrolyzed with 2 N trifluoroacetic acid at 120 °C for 2 h. The hydrolysates were dried at 50 °C, re-dissolved in water, and analyzed by high-performance anion-exchange chromatography with pulsed-amperometric detection using a CarboPac PA-20 column (3 mm × 150 mm; Dionex, Sunnyvale, CA, USA) as described earlier [99 (link)]. Monosaccharides were separated using a gradient of 100 mM NaOH in water at 0.5 mL min−1 under the following conditions: 0–0.05 min—12 mM NaOH; 0.05–26 min—0.65 mM NaOH; 26–46 min—300 mM NaOH; 46–55 min—12 mM NaOH. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D Man, D-GalA, and D-GlcA (all from Sigma–Aldrich, St. Louis, MO, USA). To determine response factors, standard curves were created using mixtures of all standard monosaccharides at different concentrations.
3
Monosaccharide Composition Analysis of Polysaccharides
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Samples (2 mg) of six polysaccharide components were hydrolyzed with 3 mL 2 M trifluoroacetic acid (TFA) at 110 °C for 4 h. After hydrolysis, hydrolysates were dried with a Termovap sample concentrator, and then 3 mL methanol was added and dried repeatedly three times until the TFA was completely removed. The monosaccharide compositions were determined by a high-performance anion exchange chromatography (HPAEC) system (Dionex ICS-2500, Dionex, Sunnyvale, CA, USA) equipped with a CarboPac™ PA20 column (3 mm × 150 mm, Dionex, USA) and a pulsed amperometric detector (Dionex, USA). The column was eluted with 2 mM NaOH (0.45 mL/min) followed by 0.05 to 0.2 M NaAc at 30 °C. The monosaccharide compositions and content of polysaccharide components were determined using d -Gal, d -Glc, d -Ara, l -Fuc, l -Rha, d -Man, d -Xyl, d -Fru, d -Rib, d -GluA, and d -GalA (Sigma-Aldrich, St. Louis, MO, USA) as the standards.
4
Carbohydrate Characterization and Enzyme Assays
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Beechwood GX, d -Xyl, and most other reagents were purchased from Sigma-Aldrich (Darmstadt, Germany). Low-viscosity wheat arabinoxylan (Ara:Xyl = 38:62; LVWAX), acid-debranched wheat arabinoxylan (Ara:Xyl = 22:78; ADWAX), enzyme-debranched wheat arabinoxylan (Ara:Xyl = 30:70; EDWAX), rye arabinoxylan (Ara:Xyl = 38:62; RAX), galactomannan (carob; low viscosity), xyloglucan (tamarind), arabinan (sugar beet), β-glucan (barley; medium viscosity), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), p-nitrophenyl-β-d -xylopyranoside (pNPX), p-nitrophenyl-β-d -xylobiose (pNPX2), p-nitrophenyl-β-d -xylotriose (pNPX3), and p-nitrophenyl-β-d -xylotetraose (pNPX4) were all purchased from Megazyme (Bray, Ireland). Cellulose nanocrystals (nanocellulose) from cotton linters were prepared as previously described (56 (link)). Oligonucleotide primers were purchased from Eurogentec (Liège, Belgium) (Table 3 ).
The GenBank accession number for the clone containing Pm25 isHF548280.1 , and the protein ID for Pm25 is CCO21036.1 .
The GenBank accession number for the clone containing Pm25 is
5
Allium roseum Leaf Monosaccharide Analysis
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Leaves of Allium roseum were collected from the region of Monastir (Tunisian Sahel; coordinates: lat 35°73′ N; long 10°76′ E) during the flowing period (Marsh), 2017; next, washed with distilled water and ground by a mixer blender and freeze-dried, with the dry powder stored until used. The botany department (Faculty of Pharmacy of Monastir) identified the plant. Trifluoroacetic acid (TFA) and monosaccharide standards (D-Gal, L-Fuc, L-Rha, D-Man, D-Xyl, and D-Glc) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
6
Habituated Stem-Cell-Like Suspension Culture from A. thaliana Roots
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Habituated stem-cell-like suspension culture used in this study was derived from roots of A. thaliana[17] (link). The cells were grown on an orbital shaker in the dark in full-strength MS liquid media. Commercially available MS salts with vitamins (Duchefa, Haarlem, Netherlands) were prepared and autoclaved prior to the addition of the carbon source. Filter sterilized stock solutions of Suc and the pentose D-(+)-Xyl (Sigma-Aldrich) were added to the liquid media to reach a final concentration of 87 mM. The cells were subcultured every 7 d through harvesting cells by centrifugation for 2 min at 200xg, measuring the packed cell volume (PCV), washing the cells with fresh media and subdividing the culture in additional fresh media with a ratio 1∶2 (Xyl-grown) or 1∶8 (Suc-grown). To estimate the effect of the C-source on the growth, A. thaliana cells, conditioned for growth on either Suc- or Xyl-containing MS media, were subcultured into the respective Suc- or Xyl-containing media and incubated for 7 days in the dark. Every day, samples were taken and the PCV of each sample was estimated after centrifugation for 2 min at 200xg. Subsequently, the media was removed through vacuum filtration and the cells were washed twice using full strength MS-media without C-source. The dry cell pellet was immediately frozen in liquid N2 and stored at −80°C for further analysis.
7
Monosaccharide Profiling by HPAEC-PAD
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Cell wall monosaccharide was determined by HPAEC-PAD of hydrolysed material. A PA20 column (Dionex, USA) was used at a flow rate of 0.5 ml/min. The column was washed with 200 mM NaOH for 10 min before the injection of each sample and then equilibrated with 10 mM NaOH for 10 min.
The elution programme consisted of an isocratic elution with 10 mM NaOH from 0 to 37 min, followed by a linear gradient up to 800 mM NaOH from 37 to 43 min, and finally down to 10 mM NaOH from 43 to 45 min. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D-Man, D-GalUA and D-GlcA (Sigma, UK); the standard mixture concentration ranges from 0.001 μg/10 μl to 0.1 μg/10 μl. A standard mixture run was performed before analysis of a batch of samples for verification of the response factors.
The elution programme consisted of an isocratic elution with 10 mM NaOH from 0 to 37 min, followed by a linear gradient up to 800 mM NaOH from 37 to 43 min, and finally down to 10 mM NaOH from 43 to 45 min. Monosaccharide standards included L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D-Man, D-GalUA and D-GlcA (Sigma, UK); the standard mixture concentration ranges from 0.001 μg/10 μl to 0.1 μg/10 μl. A standard mixture run was performed before analysis of a batch of samples for verification of the response factors.
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