Flasks with N. crassa wild type strain (FGSC #2489) were pre-grown for 16 h on 2% sucrose medium, then washed three times with 1× Vogel’s salt solution (no carbon source added) for a total duration of 30 min before transferring to their respective carbon source: 1 mM galacturonic acid (D -GalA; Sigma Aldrich) plus 1 mM rhamnose (L- Rha; Sigma Aldrich), referred to as GalAR (D -GalA + L -Rha), 2 mM glucose (D -Glc; Sigma Aldrich), 2 mM xylose (D -Xyl; Sigma Aldrich), 0.5% cellobiose (Cel; Sigma Aldrich) and 2 mM 1,4-ß-D -glucosyl-D -mannose plus 1,4-ß-D -mannobiose (referred to as Glucomannodextrins or Gm; Megazyme). Samples for global proteome and phosphoproteome were incubated for additional 2 min. No carbon condition (NC) was incubated for 1 h after the medium switch (Figure 1A ). More details about the experimental methodology for Mass Spectrometry can be found in the Supplementary Material (Supplementary Table S1) .
L rha
Manufactured by Merck Group
Sourced in United States
L-Rha is a laboratory equipment product. It serves as a core function in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
D glc, by Merck Group (5 mentions)
L fuc, by Merck Group (5 mentions)
D gal, by Merck Group (5 mentions)
D xyl, by Merck Group (4 mentions)
D gala, by Merck Group (3 mentions)
L ara, by Merck Group (3 mentions)
Carbopac pa20 column, by Thermo Fisher Scientific (2 mentions)
D glca, by Merck Group (2 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)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Carbopac pa1 column, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Pa20 column, by Thermo Fisher Scientific (1 mentions)
6 protocols using l rha
1
Proteomic Analysis of N. crassa Responses to Carbon Sources
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
Sequential Extraction of CW Polysaccharides
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The sequential extraction of CW polysaccharides was performed in four steps and detailed in [16 (link)]. In summary, 100 mg of a deproteinized CW fraction were used. Four successive extractions were carried out to obtain extracts enriched in pectins (E1 and E2, respectively using CDTA 50 mM and 50 mM Na2CO3) and hemicelluloses (E3 and E4, respectively using 20 mM NaBH4 and 4 M NaOH). Each extract was hydrolyzed in 2 N TFA for 1 h at 120 °C. After 10× dilution in UHQ water, monosaccharides were analysed by High-Performance Anion-Exchange Chromatography coupled to Pulsed Amperometric Detection (HPAEC-PAD; Dionex, Sunnyvale, California, USA) using a CarboPac PA1 column (Dionex). L-Fuc, L-Rha, L-Ara, D-Gal and GalA (Sigma-Aldrich); D-Glc (Merck, Darmstadt, Germany); D-Xyl (Roche, Mannheim, Germany) were used as standard monosaccharides for identification and quantification. Data are described in [43 (link)]. CW polysaccharides reconstruction was performed using formula previously described [16 (link),43 (link)] and adapted from [47 (link)].
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
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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