Carbopac pa 100 column

Sugar samples were analyzed on a Dionex 5000 (Thermofisher Scientific, Waltham, Massachusetts, USA) HPAEC-IPAD. Ninety mM NaOH was used as mobile phase with a flow rate of 0.25 mL min⁻¹, analyzing 6 µL sample on a CarboPac^TM PA100 column (Thermofisher Scientific, Waltham, MA, USA). Small soluble sugars were measured using an isocratic method of 90 mM NaOH for 10 min, while fructan samples were analyzed using a concentration gradient with NaOAc as described by Vergauwen et al. [99 (link)]. As an internal standard, rhamnose (Sigma-Aldrich, St. Louis, MO, USA) was added to all samples at a concentration of 20 µM. Glc, Fru and Suc standards of 10 µM (Acros Organics, Morris Plains, NJ, USA) were run alongside samples for peak identification and quantification.

The production of XOS was carried out using as a substrate commercial beechwood xylan (Megazyme) at a 1% concentration in a 50 mM Tris–HCl buffer, pH 9.0. Xylanases, Xyn11_Ec or Xyn11_Nb, were added at 0.75 µg/mL and incubated at 90ºC for different times. The reactions were stopped by placing the tubes on ice. XOS were analyzed by exchange ion chromatography, using a DIONEX instrument equipped with a CarboPac PA100 column and a pulsed amperometric detector (Dionex, Thermo Fisher Scientific). Xylose (Sigma-Aldrich) and xylooligosaccharides, from two to six units (Megazyme), were used as the chromatographic standards.

Soluble sugars were extracted and quantified as previously explained [40 (link)]. Frozen leaf material was grinded in liquid nitrogen using a mortar and pestle, and 100 mg extracted in 1 mL ddH₂O by boiling for 15 min at 95 °C. Samples were vortexed and centrifuged for 10 min at 15,000× g to remove plant debris and supernatant was desalted by applying 200 µL to a Dowex anion and cation exchange column prepared in glass Pasteur pipettes. Columns were washed 6 times with 200 µL ddH₂O and the flow through diluted 1:1 in 20 µM rhamnose H₂O serving as internal standard. Samples were quantified by injecting 6 µL on a High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) Dionex 5000 (Thermo Scientific, Whaltam, MA, USA) with separation on a CarboPac PA100 column (Thermo Scientific, Whaltam, MA, USA) and a mobile phase of 90 mM NaOH, as described previously [47 (link)]. Concentrations were calculated using standards of 10 µM of each sugar running alongside.

Enzyme activity was assayed as previously described [51 (link)], using 200 ng α-1,6-mannobiose (Dextra Laboratories, Reading, UK) as the rhamnose acceptor and 500 µM UDP-L-rhamnose (Chemily Glycoscience; Peachtree Corners, GA, USA). The reaction products were analyzed by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) with a Dionex system (Thermo Fisher Scientific), using a CarboPac PA-100 column (4.6 × 250 mm) and a linear gradient of 10–100 mM sodium acetate in 100 mM NaOH at a flow rate of 0.8 mL min⁻¹ for 30 min. [52 (link)]. For assays including treatment with α-L-rhamnosidase, the reaction product of rhamnosyltransferase activity was mixed with 1 U enzyme (Megazyme, Bre, Ireland) and incubated for 60 min at 50 °C. The reactions were stopped by boiling for 10 min and subjected to monosaccharide separation by HPAEC-PAD using the following conditions: a CarboPac PA-200 analytical column (3 × 250 mm) with a CarboPac PA-200 guard column (3 × 50 mm) and an isocratic gradient of 3.2 mM NaOH with a flux rate of 0.15 mL min⁻¹ for 25 min [53 (link)]. Fluorescence-assisted carbohydrate analysis (FACE) was performed essentially as previously described [54 (link)]. UDP-glucose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-galactose were from Sigma-Aldrich.

TLC was performed by loading samples on silica gel 60F254 plates (Merck, Germany). The loaded samples were developed by a mixture of 95% ethanol/1M acetic acid (5:2, pH 7.5; Kaminski and Eichler, 2014 (link)) and visualized by spraying the plate with 0.5% (w/v) 3,5-dihydroxytoluene in 20% (v/v) sulfuric acid and heating it at 120°C for 5min.
HPLC was performed on an Agilent 1,260 series HPLC system coupled with a UV detector (Agilent Technologies, Inc. United States) using the CarboPac^™ PA-100 column (4×250mm, 4μm particle size, Thermo Fisher Scientific, United States). The sample was eluted with a gradient concentration of ammonium acetate, set as 0–30mM (0–12min), 30–60mM (12–22min), 100mM (22–32min), and 0 (32–37min), as the mobile phase at a flow rate of 1.0ml/min and detected at 260nm. The corresponding fractions were combined and concentrated through lyophilization.