Preparation of a potato cell wall fraction enriched in RG-I oligosaccharides
Potato pulp (Solanum tuberosum L., obtained from Roquette, Lestrem, France) (100 g) was de-starched using an α-amylase (Termamyl 120 L, Novozyme) (10 mL at 15 U/mL) in 80 mM sodium phosphate buffer pH 6 (2 L) for 25 min at 90 °C. The suspension was cooled down to 30 °C, and the pH was brought to 4.5 by 1 M HCl. The suspension was then incubated with amyloglucosidase (Aspergillus Niger, A. 3042 Sigma, 1 mL, 6000 U/mL) for 17 h at 60 °C. Fresh amyloglucosidase (1 mL, 6000 U/mL) was added at 30 min and 60 min. The mixture was then cooled and the pH adjusted to 7.8 with 1 M NaOH. The de-starched potato pulp was mixed with 0.1 M NaOH (3 L) at 90 °C for 2 h. These conditions favour the β-elimination of methyl-esterified HG, resulting in the degradation of part of the HG component and enrichment in pectic RG-I. After filtration and NaOH neutralisation with 1 M HCl, the solution was concentrated to 1.5 L by rotary evaporation at 40 °C, and polysaccharides were recovered by precipitation with 70 % (v/v) ethanol overnight at 4 °C. The suspension was centrifuged, and the pellet was recovered and dissolved in water. The solution was concentrated by evaporation to 1 L to eliminate alcohol traces. The whole protocol starting with ethanol precipitation was repeated twice. Finally, the residual salts present in the solution were removed by dialysis. The salt-free solution was filtered with 3-μm membrane and freeze-dried. This fraction (1200 mg) would contain non-cellulosic polysaccharides.
The most acidic polysaccharides present in the non-cellulosic fraction were removed using diethylaminoethanol (DEAE)-Sepharose fast flow gel in batch. After pre-equilibration using 20 mM sodium acetate pH 4.5, the sample (400 mg in 40 mL H2O) was loaded onto the chromatography gel (150 mL). The matrix was manually stirred over 20 min to allow the most acidic polysaccharides to fix. A RG-I enriched fraction that did not bind to the gel was recovered by rinsing the gel with 4 × 100 mL of 20 mM sodium acetate buffer pH 4.5. The most acidic polysaccharides were then eluted by 4 × 100 mL of 50 mM acetate pH 4.5 + 0.6 M NaCl and discarded. The whole procedure was repeated three times and the three RG-I-enriched fractions pooled, dialysed and freeze-dried. The RG-I enriched sample (650 mg) was then treated with an endo-1,4-β-galactanase (from Aspergillus niger, Megazyme International, 1 mL at 2.35 U/mL) in 50 mM sodium acetate buffer pH 4.5 (130 mL) for 150 min at 40 °C, followed by an endo-arabinanase digestion (Novozyme, cloned from Aspergillus aculeatus in Aspergillus oryzae, batch PPJ4381, repurified on a Superose column using 5 mM succinate buffer pH 4, to eliminate any non-specific activity) (2.3 mL at 3.42 U/mL) in 50 mM sodium acetate pH 4.5, for 24 h at 30 °C. In both cases, the reaction was stopped by boiling the sample in order to avoid full digestion and preserve the beginning of the side chains. The sample was then dialysed for 48 h to eliminate the generated oligosaccharides and freeze-dried. This was designated as a low-branched RG-I-enriched fraction.
A sample aliquot (116 mg) was digested with a family GH28 rhamnogalacturonase (Novo Nordish Batch PPJ4478, 25 g recovery pilot plant 09/12/1993) (300 µL at 1 mg/mL) in 10 mM sodium acetate pH 4.0, for 180 min at 40 °C. The undigested polysaccharides were precipitated using 50 % EtOH at 4 °C overnight, centrifuged for 10 min at 15000 g and rinsed 3 times with 50 % EtOH. The EtOH-soluble fraction was concentrated by rotary evaporation at 40 °C, desalted using a column (100 × 1.6 cm) of Sephadex G-10 at 1 mL/min eluted by deionised water and freeze-dried. This fraction (designated RG-I oligosaccharides-enriched fraction or RUP (R, rhamnose; U, uronic acid; P, potato), 78 mg) was coupled to bovine serum albumin (BSA) to prepare the immunogen.
Uronic acids were measured by the automated m-hydroxybiphenyl method (Thibault 1979). The difference in response of glucuronic acid (GlcA) and galacturonic acid (GalA) in the presence and absence of Na-tetraborate (11.9 g of Na-tetraborate decahydrate in 2.5 L of sulphuric acid (97–98 %) (Filisetti-Cozzi and Carpita 1991) was used to quantify them individually in purified fractions. Total neutral sugars were measured by the orcinol method with correction for the interference by uronic acids (Tollier and Robin 1979). Individual neutral sugars were analysed as their alditol acetate derivatives (Blakeney et al. 1983) by gas liquid chromatography (GLC) after hydrolysis with 2 M trifluoroacetic acid at 121 °C for 2.5 h. Myo-inositol was used as an internal standard.
Carbohydrate microarrays of oligo- and polysaccharides
Carbohydrate microarrays printed on nitrocellulose were produced and quantified as described (Pedersen et al. 2012). In brief, the printed microarrays were probed with appropriate rat MAbs (10-fold dilution in phosphate-buffered saline (PBS) containing 5 % w/v milk powder (MPBS). Secondary anti-rat antibodies conjugated to alkaline phosphatase (Sigma) were diluted in MPBS to 1/5000. Developed microarrays were scanned at 2400 dpi (CanoScan 8800F), converted to TIFFs, and signals were measured using Array-Pro Analyzer 6.3, Media Cybernetics software. The mean spot signals obtained from four experiments are presented in heat maps in which colour intensity is correlated to signal. The highest signal in each dataset was set to 100, and all other values were normalised accordingly as indicated by the colour scale bar.
Immunisation procedures and generation of rat MAbs
The RUP oligosaccharides (10 mg) were coupled to BSA in order to enhance their immunogenicity following the procedure from Lees et al. (1996). The coupling efficiency was checked by the phenol sulphuric acid assay.
Rat immunisation, preparation of hybridomas and cell cloning were performed as described (Willats et al. 1998). Two male Wistar rats were injected with 25 μg of RG-I oligosaccharides coupled to BSA in complete Freund’s adjuvant administered sub-cutaneously on day 0, with the same amount administered with incomplete Freund’s adjuvant on days 28 and 63. On day 133, the rat was given a pre-fusion boost of 1 mg immunogen in PBS by intraperitoneal injection. Spleen lymphocytes were isolated 3 days later and fused with rat myeloma cell line IR983F (Bazin 1982). Hybridoma cells were selected by enzyme-linked immunosorbent assay (ELISA) using the coupled oligosaccharides as the immobilised antigen and this led to the isolation of rat MAbs LM27 and LM28. The determination of the immunoglobulin isotypes revealed that both antibodies are IgM.
Established MAbs used included rat MAbs LM2 (Smallwood et al. 1996), LM11 (McCartney et al. 2005), LM19 (Verhertbruggen et al. 2009; Marcus et al. 2010) and mouse MAb INRA-RU2 (Ralet et al. 2010).
Plant-derived oligo- and polysaccharides and materials for immunochemical assays
Various sources of plant cell wall polysaccharides were used for the characterisation of LM27 and LM28 using ELISAs. These included tamarind xyloglucan (100403, Megazyme International), maize xylan (McCartney et al. 2005), birchwood xylan (X0502, Sigma-Aldrich), rye arabinoxylan (20601b, Megazyme International) and oat spelt xylan (95590, Sigma-Aldrich). Individual xylan-derived aldouronic acid oligosaccharides were a kind gift from Sanna Koutaniemi (Koutaniemi et al. 2012), and a mixture of aldouronic acids (tri:tetra:penta—2:2:1) was obtained from Megazyme.
The Arabidopsis thaliana triple mutant in gxm1gxm2gxm3 was generated by crossing single mutants prepared in Li et al. (2013). The insertion lines are SALK_087114 (gxm1, At1g33800), SALK_084669 (gxm2, At4g09990) and SALK_050883 (gxm3, At1g09610). Plants from the triple mutant line were grown for 6 weeks, and 5-cm of basal inflorescence stem was harvested. The alcohol-insoluble residue (AIR) was obtained and pre-treated with alkali and digested with a GH11 xylanase as described (Mortimer et al. 2010). After GH11 digestion, resulting sugars were derivatised by 9-aminopyrene-1, 4, 6-trisulfonate (APTS) and analysed by DNA sequencer-Assisted Saccharide analysis in high throughput, DASH (Li et al. (2013). GH11 products of stem AIR digestion were deuteropermethylated and analysed by MALDI-TOF-Mass Spectrometry as described (Tryfona et al. 2010).
Enzyme-Linked Immunosorbent Assays (ELISAs) were performed as described (Cornuault et al. 2014). For isolated polysaccharides, 100 µL of polymers at the indicated concentrations in PBS (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) was coated overnight at 4 °C on to microtitre plates. In some cases, enzymes (a family GH11 xylanase (Megazyme International) and a family GH115 xylan glucuronidase (kind gift of Harry Gilbert, Newcastle University)) were used to pretreat samples prior to ELISAs.
Primary MAbs (LM2, LM11, LM27 and LM28) were coated in 1:5 dilution in PBS at 4 °C overnight, 100 µL/well. After incubation, the plates were washed thoroughly with tap water and then blocked using 200 µL/well with MPBS for 2 h at room temperature (RT). As a control of the blocking efficiency, some wells were directly blocked with MPBS without any MAb pre-coating. The plates were washed with tap water and incubated with 100 µL of 50 µg/mL solution of polysaccharides in MPBS. Another wash step was performed before the incubation with directly coupled LM28-horseradish peroxidase (HRP) antibody. The LM28-HRP antibody was diluted 1/25 in MPBS and incubated at 100 µL/well for 1 h at RT.
LM28 antibody purification and coupling to horseradish peroxidase (HRP)
LM28 was purified using the euglobulin precipitation protocol which involved hybridoma cell culture supernatant (250 mL) being dialysed for 3 days at 4 °C with 2 mM sodium phosphate buffer pH 6.0. The precipitated IgM was then centrifuged at 4 °C, 4000g for 10 min. The pellet was re-suspended and rinsed twice with cold 2 mM sodium phosphate buffer pH 6.0. Finally, the pellet was re-suspended in 10 mL of 1X PBS, centrifuged at 2000g for 10 min at RT. The supernatant was collected and aliquoted for storage. The efficiency of the purification was checked via SDS-PAGE, and the activity of the purified antibody was checked via immuno-dot assay on oat spelt glucuronoarabinoxylan (GAX). The concentration of the purified LM28 antibody was estimated by absorbance reading at 280 nm. The coupling of LM28 to HRP was performed using the EZ-link™ Plus Activated Peroxidase kit (Thermo Scientific) following manufacturer instructions.
Epitope detection and anion-exchange chromatographies
Epitope detection chromatography (EDC) analysis was performed as described (Cornuault et al. 2014) using a 1-mL HiTrap ANX FF column (GE Healthcare, 17-5162-01). As this analysis was performed on pre-purified samples, only 50 µg of oat spelt xylan, birchwood xylan or low-branched RG-I-enriched fraction was injected. For optimised separation, the samples were eluted at 1 mL/min using a two-step gradient starting with 20 mM sodium acetate buffer pH 4.5 from 0 to 25 min with a step change to 50 mM sodium acetate buffer pH 4.5 at 25 min with the onset of a linear gradient of 0–50 % 0.6 M NaCl to 73 min followed by a second step from 73 to 83 min of a linear gradient from 50 to 100 % 0.6 M NaCl. The salt gradient remained at its maximum (50 mM sodium acetate buffer pH 4.5, 0.6 M NaCl) from 83 to 96 min.
For comparison of EDC profiles with chemical assessment of sugar content, a 30 mL anion-exchange column (DEAE-Sepharose Fast Flow 16 × 150 mm) was used. 10 mg of low-branched RG-I-enriched fraction was injected. The sample was eluted at 1 mL/min using a two-step gradient starting with 20 mM sodium acetate buffer pH 4.5 from 0 to 110 min with a step change to 50 mM sodium acetate buffer pH 4.5 at 110 min with the onset of a linear gradient of 0 to 50 % 0.6 M NaCl to 275 min followed by a second step from 275 to 310 min of a linear gradient from 50 to 100 % 0.6 M NaCl. The salt gradient remained at its maximum (50 mM sodium acetate buffer pH 4.5, 0.6 M NaCl) from 310 to 350 min.
Preparation of plant material and immunocytochemistry
Tobacco (Nicotiana tabacum L.) stem sections were obtained as described in Marcus et al. (2008). Brachypodium distachyon (L.) P.Beauv stem sections were obtained from the 5th internode of 50-day-old stem. The Arabidopsis thaliana (L.) Heynh. stem sections were obtained from the first centimetre of inflorescence stem of 1-month-old plant. They were fixed and embedded in resin as described (Lee and Knox 2014). Transverse sections of A. thaliana,
B. distachyon and tobacco stems were incubated for 30 min with MPBS to prevent non-specific binding, and then washed for 5 min with PBS. Primary rat MAbs at 5-fold dilutions of hybridoma cell culture supernatants in MPBS were incubated on sections for 90 min at RT. Sections were then washed three times with PBS for 5 min. The secondary antibodies (anti-rat IgG-FITC (Sigma-Aldrich) at a 100-fold dilution were added in MPBS and incubated for 90 min in the dark. Sections were washed with PBS for three times for 5 min. To diminish sample auto-fluorescence, the sections were incubated with 0.1 % Toluidine Blue O (pH 5.5, 0.2 M sodium phosphate buffer) for 5 min. Following Toluidine Blue O labelling, sections were washed twice with PBS for 5 min, and then mounted in anti-fade reagent Citifluor AF1 (Agar Scientific). After mounting, slides were stored at 4 °C in darkness until use. Immunofluorescence was observed with a fluorescence microscope (Olympus BX61), and images were captured using a Hamamatsu ORCA285 camera (Hamamatsu City, Japan) using PerkinElmer Volocity software (PerKinElmer).