Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls
© Marcus et al. 2008
Received: 07 February 2008
Accepted: 22 May 2008
Published: 22 May 2008
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© Marcus et al. 2008
Received: 07 February 2008
Accepted: 22 May 2008
Published: 22 May 2008
Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.
Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.
These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.
Cell walls are major components of plant cells that impact significantly on the modes of cell development and the growth and the mechanical properties of plant organs. Plant cell walls are also of considerable economic significance in that they are major components of terrestrial biomass and of plant-derived materials that are used for fibre, fuel and food. Primary and secondary cell walls are comprised of sets of polysaccharides of considerable structural complexity and diversity [1–3]. The major polysaccharide classes are cellulose, hemicelluloses (or cross-linking glycans) and pectic polysaccharides with the latter two classes containing a diversity of polymer structures. In order to understand how specific configurations of polysaccharides and their interactions and associations constitute diverse cell wall structures and functions, methodologies are required to assess polymers in-situ throughout organs and within cell walls. Tagged proteins, with the capacity to specifically bind to a structural motif of a polysaccharide, are currently one of the best ways to do this. These proteins are most notably monoclonal antibodies and carbohydrate-binding modules. Cell wall probes, directed to some structural features of polymers of the three major polysaccharide classes have indicated that the occurrence of cell wall polysaccharide structures can be highly regulated in relation to developmental context [4–10]. However, probes are not yet available for all the structural motifs known to occur within cell wall components and thus in-situ locations of all polymer structures cannot yet be determined.
Xyloglucans are one of the most abundant hemicelluloses of the primary cell walls of non-graminaceous species and are proposed to have a functional role in hydrogen bonding to and tethering the cellulose microfibrils together. This load-bearing hemicellulosic network maintains the strength of primary cell walls which is a crucial factor underpinning expansive plant growth [1–3, 11, 12]. The xyloglucan set of hemicelluloses is highly diverse and displays significant taxonomic variation in structure [1, 12–15]. Xyloglucans have a backbone of (1→4)-β-D-glucan and some glucosyl residues are substituted with short side chains. A structure-based nomenclature has been developed for xyloglucan-derived oligosaccharides to indicate the attachments to backbone glucosyl sequences . For example, an unbranched glucosyl residue is designated G, a glucosyl residue bearing a single xylose is designated X and one bearing a disaccharide of β-Gal-(1,2)-α-Xyl is designated L. Xyloglucans are classified as XXXG or XXGG type based on the number of backbone residues that carry side chains with the XXXG type having three consecutive glucosyl residues with xylose attached and a fourth unbranched residue . To date it has not been easy to put this structural complexity into cell biological context as only few probes are available. An antiserum to xyloglucan  and a monoclonal antibody (CCRCM1) that binds to a fucosylated epitope that is carried by xyloglucan  have been developed. These have been used to detect xyloglucan in-situ [4, 7, 20–22].
Here we describe the coupling of a heptasaccharide with 3 xylosyl and 4 glucosyl residues (XXXG in xyloglucan nomenclature) obtained from tamarind seed xyloglucan to a protein carrier to act as an immunogen. Subsequent to immunization we have identified a rat monoclonal antibody, designated LM15, that binds to the XXXG motif of xyloglucan and we have used this antibody to demonstrate the regulation of xyloglucan structure and occurrence within cell walls and in relation to plant anatomy in a range of species.
Polysaccharides do not exist in isolation in plant cell walls and understanding the links and associations between classes of polymers is an important goal to increase our understanding of plant cell wall biology. Biochemical evidence is accumulating that xyloglucan can be attached to pectic polymers in plant cell walls [23–27]. Using enzymatic degradation to remove the pectic homogalacturonan (HG) from cell walls of transverse sections of plant materials in conjunction with the LM15 anti-xyloglucan monoclonal antibody we have demonstrated the existence of developmentally regulated sets of xyloglucan epitopes within cell walls that are masked by the presence of HG. This observation has significant implications for our understanding of the precise developmental patterns of occurrence of xyloglucan in cell walls and of cell wall biology in general.
Xyloglucan is known to be a major structural component of the cell walls of the cotyledon parenchyma of tamarind and nasturtium seeds [29–31]. To explore the binding of the XXXG-directed monoclonal antibody LM15 to plant cell walls, excised cotyledon parenchyma tissue from mature seeds of these two species were fixed and embedded in resin and sectioned prior to indirect immunofluorescence analysis. The mouse monoclonal antibody CCRCM1  that binds to a fucosylated epitope of xyloglucan (not present in tamarind seed xyloglucan but present in nasturtium seed xyloglucan) was used for comparison with LM15.
We show that immunization with a neoglycoprotein has been successful in the generation of a rat monoclonal antibody, LM15, directed to the XXXG motif of xyloglucan that can bind effectively to tamarind xyloglucan (a polymer without fucosylation) and also to cell walls in a range of species. LM15 can bind to pea cell walls (with XXXG xyloglucan) and also to tobacco stem cell walls with XXGG xyloglucan indicating that the LM15 antibody may not require the entire heptasaccharide XXXG for optimal recognition – a facet of LM15 binding to xyloglucan that was also indicated by hapten oligosaccharide inhibition studies in which a mixture of XXLG and XLXG oligosaccharides was observed to effectively inhibit binding.
Polysaccharide-directed probes such as monoclonal antibodies are important tools to gain an understanding of polysaccharide diversity and occurrence in cell walls which is an essential aspect of understanding the molecular basis of cell wall functions. A xyloglucan antiserum  and a monoclonal antibody CCRCM1  have been used to demonstrate developmentally regulated patterns of xyloglucan occurrence in a range of systems [7, 21, 22]. In sections of nasturtium seed monoclonal antibodies LM15 and CCRCM1 bound to distinct regions of cotyledon parenchyma cell walls indicating the spatial regulation of xyloglucan structure within these cell walls. A region of the nasturtium cell walls between the regions bound by these antibodies (revealed by Calcofluor White fluorescence) was not strongly bound by either probe (and pre-treatment with pectate lyase had no impact on epitope occurrence). To know whether xyloglucan is reduced in amount or the xyloglucan is structurally distinct in these regions requires the development of probes for other structural features of xyloglucans.
Binding of LM15 to untreated transverse sections of stem internodes of pea and tobacco indicated recognition of only a few cell types and often weakly. Binding was shown to be increased considerably by the enzymatic removal of pectic HG. In cell wall immunochemistry studies, it is often assumed that a section through an organ/cell and thus across cell wall layers from the plasma membrane to the middle lamella would expose all polymers present in the cell walls. The implication, from many documented occurrences of cell wall epitopes, has been that a restricted occurrence of an epitope reflected the presence or absence of a particular epitope and this has in some cases been supported by physicochemical analysis of isolated polymers. This is the first report of a clear case of substantial epitope masking where most of the copies of an epitope present in a section have been masked by the presence of another cell wall polymer, in this case pectic HG. It is of considerable interest that, in both pea and tobacco stem internodes, some xyloglucan epitopes were detected without enzymatic removal of pectic HG in addition to those revealed by pectic HG removal. This suggests the possibility of two distinct presentations of the LM15 epitope in relation to pectin in cell walls. These observations have important implications for our understanding of cell wall polymer configurations and should be born in mind when considering the developmental regulation of cell wall structures, as evidenced by antibody probes.
The basis of the uncovering of xyloglucan epitopes by pectic HG removal could be a general increase in cell wall porosity allowing increased access to epitopes or alternatively an intimate specific structural association between pectin and xyloglucan in muro that occludes xyloglucan structures. The comparative assessment of the LM6 arabinan epitope in tobacco stem indicated that its detection increased after pectate lyase treatment but not to the extent observed for the LM15 xyloglucan epitope. Moreover, no difference in the pattern of occurrence of the LM6 epitope was revealed. This observation suggests that the pectate lyase pre-treatment can increase cell wall porosity and thus all antibody access to some extent. However, the clear impact on the extent and pattern of LM15 xyloglucan epitope detection indicates a more intimate association between xyloglucan and pectin in this system.
Pectin and xyloglucan are both quantitatively important polymers of plant cell walls comprising approximately a third each of the polysaccharides of primary cell walls of dicotyledons . In terms of cell wall architectures, xyloglucan is known to attach to cellulose microfibrils by means of hydrogen bonds and is proposed to tether adjacent microfibrils providing the mechanical basis of the resisting cell enlargement. The complex pectic network of several polymers (the major one being HG) embeds the cellulose-xyloglucan network and imposes cell wall properties including cell wall porosity [2, 36, 37]. An early model of cell wall structure proposed a glycosidic link between xyloglucan and a neutral side chain of pectin . Recent evidence has confirmed a link between xyloglucan and acidic pectic polymers but specifically through the rhamnogalacturonan-I domains of the pectic molecules [23–27]. These studies indicate that such links are widespread and, in the case of cultured cells of arabidopsis, evidence has been reported that up to 50% of xyloglucan is synthesized on a pectic primer prior to cell wall deposition and that the interpolymer bonds are stable in the cell wall [26, 27]. The significance of glycosidic links between pectic polymers and xyloglucan and the observations reported here are not yet clear.
In cell walls of pea and tobacco stem internodes the pectic HG and the LM15 xyloglucan epitope do not co-localize precisely. It is of interest that the uncovered LM15 xyloglucan epitopes occurred in diverse complex patterns in relation to cell wall features and microstructures in a range of cell types such as parenchyma, metaxylem and collenchyma cells. In many cases the LM15 epitope was not evenly distributed throughout cell walls. At the inner face of transverse walls in the pith parenchyma of tobacco stem the LM15 epitope did not co-localize with Calcofluor White fluorescence (likely to be indicative of cellulose). Other approaches have indicated that cellulose microfibrils may not be evenly coated with xyloglucan, that xyloglucan can occur in distinct cell wall domains and may be minimal in some cell walls [39, 40]. It has also been demonstrated that xyloglucan structure changes during cell growth in pea  and it is possible that structural changes are spatially regulated within cell walls during cell development. The detection of abundant LM15 xyloglucan epitope at corners of intercellular spaces, shown most strikingly in this study for tobacco pith parenchyma cell walls, is a pattern of occurrence that has been observed for the LM7 pectic HG epitope in a wide range of parenchyma systems . This may indicate, for this tissue, a role for xyloglucan in intercellular space formation or stabilisation, possibly through an association with pectin.
A novel xyloglucan binding rat monoclonal antibody LM15 has been developed for xyloglucan analysis in planta. The demonstration that, in certain organs, large sets of xyloglucan epitopes are masked by the presence of pectic HG has implications for understanding xyloglucan function in primary cell walls and cell wall biology in general. The use of enzymic degradation in conjunction with cell wall probes is likely to be an important analytical tool for the study of the developmental regulation of links between pectic polymers and xyloglucan. Further work will be required to dissect the extent of cell wall epitope masking occurring between other sets of cell wall polysaccharides.
A neoglycoprotein (XXXG-BSA) was prepared by coupling a heptasaccharide containing 3 xylosyl and 4 glucosyl residues (XXXG, Megazyme, Bray, Ireland) to BSA by reductive amination . XXXG (30 mg) was dissolved in 1.0 ml of 0.2 M sodium borate buffer pH 9.0. This was followed by the addition of 20 mg BSA and then 30 mg of sodium cyanoborohydride. The mixture was maintained in a water bath at 50°C with occasional mixing. After 24 h the pH was adjusted to pH 4.0 by the addition of 45 μl of 80% (v/v) acetic acid. The solution was then dialysed extensively against distilled water with several changes over 4 days.
Rat immunization, hybridoma preparation and cloning procedures were performed as described previously . Two male Wistar rats were injected with 100 μg XXXG-BSA in complete Freund's adjuvant administered subcutaneously on day 0, with the same amount administered with incomplete Freund's adjuvant on days 33 and 71. On day 145, a selected rat was given a prefusion boost of 100 μg XXXG-BSA in 1 ml PBS by intraperitoneal injection. The spleen was isolated three days later for isolation of lymphocytes and fusion with rat myeloma cell line IR983F . Antibodies were selected by ELISA using tamarind xyloglucan as antigen. Subsequent characterization was by means of a glycan microarray of cell wall polymers  and competitive inhibition ELISAs using the xyloglucan XXXG heptasaccharide from tamarind xyloglucan and a series of related xyloglucan oligosaccharides. A mixture of the XXLG and XLXG octasaccharide isomers and the XLLG nonasaccharide were derived from tamarind xyloglucan as described  and purified by HPLC using Tosoh TSK Gel Amide column (21.5 × 300 mm) eluted with 65% aqueous acetonitrile. Cellotetraose GGGG was prepared by acetolysis of cellulose  and separated from the mixture of deacetylated oligosaccharides by HPLC as above. The sample of pea xyloglucan was a gift from Marie-Christine Ralet (INRA, Nantes, France). ELISAs were carried out as described previously  and in all cases immobilised antigens were coated at 50 μg/ml. Mannan, tamarind xyloglucan polymers, isoprimeverose and xylose disaccharide were obtained from Megazyme, Bray, Ireland. The selected antibody, an IgG2c, was designated LM15.
Tamarind (Tamarindus indica L.) seeds were obtained from Jungle Seeds, Watlington, UK) and nasturtium (Tropaeolum majus L. cv Tom Thumb) seeds from Mr. Fothergill's Seeds Ltd., Newmarket, UK. Tamarind and nasturtium seeds were imbibed for 24 h and then pieces of cotyledon parenchyma were excised, fixed and prepared for embedding in LR White resin with subsequent sectioning for indirect immunofluorescence analysis as described previously . Tobacco (Nicotiana tabacum L.) and pea (Pisum sativum L.) plants were grown in a greenhouse with 16 h days and maintained between 19 and 23°C. Regions of second internodes from the top of six-week old plants were fixed, embedded in wax and sectioned as described previously .
In addition to LM15, three further monoclonal antibodies were used in this study using indirect immunofluorescence: CCRCM1, a mouse monoclonal antibody to a fucosylated epitope of xyloglucan , a gift from Dr. Michael Hahn (CCRC, University of Georgia, USA), JIM5, a rat monoclonal antibody to methyl-esterified and unesterified epitopes of HG  and LM6, a rat monoclonal antibody to arabinan . Section pre-treatment to remove HG from cell walls involved incubation of sections with a recombinant microbial pectate lyase 10A  (a gift from Prof. Harry Gilbert, University of Newcastle-upon-Tyne) at 10 μg/mL for 2 h at room temperature in 50 mM N-cyclohexyl-3-aminopropane sulfonic acid (CAPS), 2 mM CaCl2 buffer at pH 10 as described . The high pH of the enzyme buffer removes HG methyl esters in cell walls and results in HG being susceptible to pectate lyase degradation and also suitable for recognition by JIM5. Sections not treated with the pectate lyase were incubated for an equivalent time with the high pH buffer without enzyme and imaged as untreated controls. After enzyme or buffer treatment, sections were incubated in phosphate-buffered saline (PBS) containing 5% (w/v) milk protein (MP/PBS) and a 5-fold dilution of antibody hybridoma supernatant for 1.5 h. Samples were then washed in PBS at least 3 times and incubated with a 100-fold dilution of anti-rat IgG (whole molecule), or anti-mouse IgG, linked to fluorescein isothiocyanate (FITC, Sigma, UK) in MP/PBS for 1.5 h in darkness. The samples were washed in PBS at least 3 times and incubated with Calcofluor White (0.2 μg/mL) (Fluorescent Brightner 28, Sigma, UK) for 5 min in darkness. Samples were washed at least 3 times and then mounted in a glycerol-based anti-fade solution (Citifluor AF1, Agar Scientific, UK). Immunofluorescence was observed with a microscope equipped with epifluorescence irradiation and DIC optics (Olympus BX-61). Images were captured with a Hamamatsu ORCA285 camera and Improvision Volocity software.
We acknowledge financial support from the UK Biotechnology & Biological Sciences Research Council and the EU FP6 Wallnet Research Training Network (MRTN-CT-2004-512265). Partial support was obtained from the Slovak Grant Agency for Science (VEGA).
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