Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls
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- Cornuault, V., Buffetto, F., Rydahl, M.G. et al. Planta (2015) 242: 1321. doi:10.1007/s00425-015-2375-4
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The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls.
Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.
KeywordsArabinogalactan-proteins Cell wall Glucuronoxylan Pectin Polysaccharides Rhamnogalacturonan-I
Epitope detection chromatography
Enzyme-linked immunosorbent assay
Plant cell walls are highly complex composites of macromolecules that are constructed from of a range of structurally varied polysaccharides/glycoconjugates that potentially include cellulose, pectins, xyloglucans, heteroxylans, heteromannans, mixed-linkage glucans and arabinogalactan-protein proteoglycans (AGPs). The biochemistry of these components has been studied extensively, and in broad terms, most of the abundant and widespread structures are well characterised (Burton et al. 2010). However, the overall architectures of plant cell walls, the ways in which these components are organised, and variations in relation to cell types and developmental events largely remain unknown.
A range of models outlining the organisation of cell wall polymers has been presented (Keegstra et al. 1973; McCann and Roberts 1994; Carpita and Gibeaut 1993; Somerville et al. 2004; Baba 2006). Many of these depict cellulose microfibrils interlinked by xyloglucans/xylans and this scaffolding embedded in a pectic matrix. Recent work has allowed the development of a more nuanced view of cell wall matrix glycans including xyloglucans, heteroxylans and pectic supramolecules as all being components of cell wall matrices, and several of which may interact with cellulose microfibrils and several of which can be interconnected (Zykwinska et al. 2007; Burton et al. 2010; Cosgrove 2014). Proposed links between xyloglucan and pectins (Cumming et al. 2005; Popper and Fry 2008), the identification in Arabidopsis cell cultures of a low-abundance polymer, Arabinoxylan Pectin Arabinogalactan Protein1 (APAP1), consisting of AGP, rhamnogalacturonan-I (RG-I) and arabinoxylan domains (Tan et al. 2013) and evidence for enzyme capabilities leading to matrix glycan interlinks (Franková and Fry 2013) all support the idea of highly dynamic sets of matrix glycans in cell walls that can vary in configurations, inter-linkages, and properties. Pectic supramolecules appear to be a highly complex and heterogeneous components of cell wall matrices. The major polysaccharide domains of pectic molecules are homogalacturonan (HG) and the branched heterodomain rhamnogalacturonan-I (RG-I) containing side chains rich in galactan and arabinan motifs with other domains including rhamnogalacturonan-II and xylogalacturonan (Atmodjo et al. 2013). How these domains of supramolecules are ordered and arranged is not clear (Burton et al. 2010: Yapo 2011; Atmodjo et al. 2013). It is known that pectic polysaccharides have capacities to link to or associate with cellulose microfibrils and to matrix components such as xyloglucans as discussed above. This may indicate aspects of the synthesis of the cell wall components or relate to the organisation of cell wall matrix components that impact on cell wall architectures and properties. Several questions arise concerning cell wall matrix glycans including how diverse are the inter-linkages between glycans and what are their functional roles in impacting on overall cell wall structures and associated properties.
New molecular tools and approaches are required to reveal the developmental dynamics of these links as an aid to inform functional understanding. The study of cell wall molecular architectures is a major challenge, and studying individual cell wall components is not straightforward as extraction procedures may alter polymer structures, and all cellular contexts are likely to be lost. Efforts have been made to study cell wall components directly in muro using monoclonal antibodies (MAbs), and these have proven useful to indicate the diversity and dynamics of cell wall architectures. More recently, MAbs are being used to study cell wall components in glycan microarrays/glycomic approaches (Moller et al. 2007; Pedersen et al. 2012; Pattathil et al. 2012) and in chromatographic separations revealing heterogeneities and potential inter-linkages (Verhoef et al. 2008; Cornuault et al. 2014). However, in order to extend the existing libraries of MAbs and to increase probe coverage of potential oligosaccharide features found in cell walls, additional MAbs recognising novel epitopes are required.
Here, we report the isolation of a cell wall fraction from potato tubers enriched in RG-I oligosaccharides with the aim of generating MAbs. Immunisation with this fraction led to the isolation of two heteroxylan-directed MAbs. These probes, LM27 and LM28, have been used in a series of analyses, and their high sensitivity and detection capabilities allow their use to indicate that a small proportion of heteroxylan in potato tuber cell walls appears to be connected to pectic molecules and also that sub-fractions of oat spelt heteroxylan may be associated with AGPs.
Materials and methods
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.
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).
Preparation of a potato RG-I oligosaccharides-enriched fraction for use in an immunogen
Isolation of xylan-directed MAbs LM27 and LM28
LM28 binds to the glucuronosyl substitution of heteroxylan
The potential of other enzyme deconstructions of heteroxylan/GAX involving α-L-arabinofuranosidase (GH51) and endo-/exo-arabinanases (GH43) to disrupt LM27 recognition of oat xylan led to no loss of binding suggesting recognition of a structurally complex substitution of heteroxylan or the presence of an associated non-xylan structure.
A link from RG-I to xylan: Epitope detection chromatography/sandwich-ELISA analysis of the low-branched RG-I-enriched fraction using LM28 reveals a capacity for links between xylan and pectic polysaccharides
The P1 pool is equivalent to the region of the EDC profile that contains most of the LM28 signal and this had the highest level of xylose (Fig. 5c). Together these observations suggest that the low-branched RG-I-enriched fraction isolated from potato tubers contains small amounts of glucuronoxylan, which can be bound by LM28. The potential link between pectin and glucuronoxylan indicated by the co-elution profiles of the LM28 and INRA-RU2 epitopes (Fig. 5a) was explored further. A sandwich-ELISA using LM19 and INRA-RU2 antibodies immobilised to microtitre plates as bait was used. Incubation of these microtitre plate wells with the low-branched RG-I-enriched fraction and subsequent probing with LM28 directly coupled to horseradish peroxidase (HRP) confirmed that some LM28 epitope was present in a complex with pectin (Fig. 5d).
The accumulated observations above indicate that the fraction enriched in RG-I oligosaccharides, used as the immunogen, contained low levels of heteroxylans that led to the isolation of the LM27 and LM28 MAbs and that some of these heteroxylan structures are associated with/linked to pectic polysaccharides.
A link to from xylan to AGPs in oat spelt xylan
To explore the potential for the occurrence of links between sub-populations of heteroxylan and other glycans further, commercial preparations of oat spelt and birchwood xylan were screened for the presence of pectic and also AGP epitopes using ELISAs. The LM2 AGP epitope was abundantly detected in oat spelt xylan (with an antibody signal equivalent to that for LM11, LM28 and LM27) but was absent from birchwood xylan (Fig. S2). LM2 binds to β-linked GlcA in AGPs (Yates et al. 1996). Pre-treatment of oat spelt xylan with α-1,2-glucuronidase resulted in no loss of LM2 signal when binding to this preparation confirming that the LM2 antibody was not binding to GlcA residues in xylan (Fig. 3).
Comparative experiments (EDC and sandwich ELISAs) carried out on birchwood xylan led to a distinct set of observations (Fig. 6). The LM27 and LM2 AGP epitopes were not present confirming earlier observations. The LM11 epitope was associated with a large peak, eluting later (more acidic) than oat spelt xylan. The LM28 glucuronoxylan epitope was detected in this late eluting peak co-incident with LM11 but also in an early eluting peak in a similar position to its elution with the sample of oat spelt xylan. These EDC profiles indicate the presence of two separate glucuronoxylans in the birchwood samples with the later eluting polymer presumably having a higher density of GlcA residues.
In situ detection of the LM27 and LM28 epitopes
The pursuit of MAbs to plant cell wall glycans
The method of choice for the production of MAbs to carbohydrate structures requires the preparation of a neoglycoprotein immunogen using a defined oligosaccharide. However, producing sufficient quantities of structurally defined oligosaccharides by isolation or chemical synthesis routes is a challenging task, and the isolation of mixtures of oligosaccharides from complex biological materials remains a valuable way forward. The approach described here involved the isolation of a fraction enriched in RG-I oligosaccharides with the aim of isolating MAbs to novel RG-I epitopes covering the junction of side chains with the rhamnogalacturonan backbone. It is therefore of interest that one of the isolated MAbs is directed to an epitope of heteroxylan and one is associated with heteroxylan. This is not too surprising as the pool of oligosaccharides that was isolated for immunogen preparation was complex and contained xylose, mannose, fucose and glucuronic acid which are not found in RG-I and is therefore likely to have contained, at low abundance, fragments of heteroxylan, and these polymers may be immunodominant. LM28 is a glucuronoxylan-directed MAb as confirmed by glycan microarray, hapten inhibition and enzyme deconstruction analyses. Regarding the specificity of the LM27 MAb, its epitope is most abundant in grass cell wall xylan preparations/GAXs, and analyses indicate the recognition of a yet unknown side chain substitution of GAX or an associated macromolecule. Invitro, no enzyme treatments were found to alter LM27 binding to oat xylan, indicating that the recognised epitope is possibly a structurally complex GAX structure. As the oligosaccharides used for immunisation were generated using enzyme treatments, their final structure may be different from native RG-I molecules. The procedures used for oligosaccharide isolation may also have increased the relative abundance of uncommon structures that were resistant to enzymatic treatment and rare in the original sample. The discovery that the LM2 AGP epitope is abundant in oat spelt xylan suggests the possibility that the LM27 epitope is an AGP-like epitope in xylan preparations, although LM27 does not bind to the sample of type II arabinogalactan (gum Arabic) that is included on the microarray (Fig. 2a).
Functional significance of inter-linkages between cell wall matrix glycans
The LM28 MAb has allowed a potential link between glucuronoxylan and pectic supramolecules to be identified in potato tuber cell walls. It is also possible that a sub-fraction of the pectic glycans in the potato RG-I preparation is also attached to the LM27 epitope—but that this is below the level of detection using the current approaches. The sandwich-ELISA analysis of an oat spelt xylan preparation suggested that both the LM27 and LM28 epitopes may be linked to AGP molecules.
Xylans are abundant in secondary cell walls of dicotyledonous plants and both primary and secondary cell walls of grasses where heteroxylan is present in the form of GAX. The specific roles of heteroxylan in secondary cell walls are proposed to be cross-linking of cellulose microfibrils to make tough composites that withstand compressive forces. In primary cell wall matrices, functions are far from clear, and heteroxylan or GAX may take on some of the roles in cell wall matrices that are carried out by pectic molecules in dicotyledons. These may relate to the construction of cellulose microfibrils and cell wall assembly, allowing cell expansion, controlling other aspects of matrix properties and cell adhesion. Although not abundant in primary cell walls of dicotyledons, low levels of xylans do occur in primary cell walls (Hervé et al. 2009) and possibly have specific functions.
Although there are numerous reports of pectin links to xyloglucan, a structural linkage of xylan to other matrix molecules has only recently been identified (Tan et al. 2013), and relevant enzymatic activities have been proposed (Franková and Fry 2013). The Arabinoxylan pectin arabinogalactan protein 1 (APAP1) of Arabidopsis cells is a very low-abundance molecule, and the links here observed in potato tuber cell walls involving xylan are also likely to be of low abundance and hence not previously documented. The low abundance of links between subsets of pectic supramolecules attached to subsets of heteroxylan may reflect a remnant of an aspect of the biosynthesis of cell wall molecules or cell wall assembly, an aspect of cell wall architecture that is required at a few locations or a small element of signalling systems that conveys information on the status of the cell wall matrix. Defined MAbs and their use in methods such as EDC will be useful tools to explore these factors further.
Two new MAbs have been isolated. LM27 binds to grass GAX samples and its epitope is proposed to be a complex substitution of heteroxylan or is an epitope carried by as yet unknown attached molecule. LM28 binds to a glucuronosyl-containing epitope of heteroxylan. These probes bind with high avidity to their respective epitopes and are complementary to previously characterised heteroxylan-directed antibodies LM10 and LM11 (McCartney et al. 2005) and INRA-AX1 (Guillon et al. 2004) that bind to the backbone of xylans and also INRA-UX1 that requires alkali treatment for recognition of glucuronoxylan in plant cell walls in situ (Koutaniemi et al. 2012). Using EDC and sandwich-ELISA approaches, we have used LM27 and LM28 to demonstrate the potential attachment of a sub-fraction of potato tuber heteroxylan to pectic supramolecules and a sub-fraction of oat spelt xylan to AGP. LM27 and LM28 are therefore both useful molecular tools to study the significance and developmental dynamics of interlinks between heteroxylans and other cell wall matrix glycan classes.
Author contribution statement
VC, FB, MCR and JPK conceived and designed research. VC, FB, MGR, SEM, TAT, JX, MJC and NFB conducted experiments. VC, MGR, WGTW, PD, MCR and JPK analysed data. VC, MCR and JPK wrote the manuscript. All authors read and approved the manuscript.
This work was supported by the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (Grant Agreement number 263916). (This article reflects the authors’ views only and the European Union is not liable for any use that may be made of the information contained herein). The work was also supported by the United Kingdom Biotechnology and Biological Research Council (BBSRC, Grant BB/K017489/1). JX acknowledges support from the Chinese Scholarship Council, TAT from a BBSRC studentship and MGR from the Danish Strategic Research Council and The Danish Council for Independent Research, Technology and Production Sciences as part of the GlycAct project (FI 10-093465). We acknowledge kind gifts of enzymes from Harry Gilbert and oligosaccharides from Sanna Koutaniemi. We thank Theodora Tryfona for mass spectrometry analysis.
|Funder Name||Grant Number||Funding Note|
|Seventh Framework Programme|
|Seventh Framework Programme|
|Biotechnology and Biological Sciences Research Council|
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