Restricted access of proteins to mannan polysaccharides in intact plant cell walls

Summary

How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

Introduction

The robust cell walls of land plants are complex biomaterials that are both abundant and structurally diverse. They are important cell components that underpin cell defence, organ growth and the mechanical properties of plants. Plant cell walls are largely constructed from a diverse range of about a dozen polysaccharides that are grouped into cellulose, hemicelluloses and pectic polysaccharides (O’Neill and York, 2003). Cellulose forms load-bearing microfibrils that are cross-linked by, or are co-extensive with, some polymers of the hemicellulose polysaccharide group (including xyloglucans, xylans, mixed-linkage glucans and mannans) and the pectic polysaccharide group (Cosgrove, 2005). Pectic polysaccharides, which are abundant in primary cell walls, include homogalacturonans, rhamnogalacturonans, xylogalacturonans, galactans, arabinans and arabinogalactans. Secondary cell wall polysaccharides are predominantly cellulose and xylan and/or mannan. Most polysaccharides of the hemicellulose and pectic groups display extensive variants in terms of both glycosyl structure or polysaccharide modifications, such as methyl esterification or acetylation. How these structural modulations within polysaccharides influence polymer properties and functions is not yet clear. The relative abundances of polysaccharides vary with cell wall type, cell and tissue type, developmental status and taxonomy (Harris, 2005). Current studies indicate that cell walls are a structurally diverse set of cellular components, and this chemical heterogeneity is evident within single cell walls (Knox, 2008). The biochemical understanding of the structures of cell wall polysaccharides is generally well advanced, and we have a broad understanding of how a major polymer such as cellulose functions (Cosgrove, 2005). What is less well understood are details of polysaccharide occurrence and cell wall heterogeneity for the hemicellulosic and pectic polymers, their role in cell wall assembly, and their contribution to the properties displayed by cell walls.

Mannans are a complex set of hemicellulosic heteroglycans that are currently considered to have both storage and structural functions in cell walls (Ebringerováet al., 2005; Moreira and Filho, 2008; Schröder et al., 2009; Scheller and Ulskov, 2010). Mannan polysaccharides have been proposed to cross-link cellulose by means of hydrogen bonds, and to act in a similar way to the xyloglucans, xylans and 1,3-1,4-glucans that are the other main hemicelluloses. Studies incorporating structurally distinct mannans into synthetic composites have shown that they have this ability in vitro (Whitney et al., 1998). Mannans have a core feature of 1,4-β-linked mannosyl residues, and homomannans are structural polymers that occur in microfibrillar form in some algae (Mackie and Preston, 1968) and as storage polymers in some seeds (Buckeridge et al., 2000). In some cases, glucosyl residues are incorporated into mannan backbones, and such glucomannans are widely present in the primary cell walls of plants, including the dicotyledons. In some cases, 1,6-α-linked galactosyl residues are substituents of mannan backbones and galactomannan polymers are particularly abundant as storage polymers in legume seeds (Buckeridge et al., 2000; Srivastava and Kapoor, 2005). Another class, the galactoglucomannan polymers, containing both backbone glucosyl residues and galactosyl substituents, are abundant in the secondary cell walls of gymnosperms (Whistler and Chen, 1991), and may be widely distributed at low levels in primary cell walls (Harris, 2005; Schröder et al., 2009). The mannan polysaccharides are widespread in land plants, and vary with cell wall types and taxa. They are abundant in the model bryophyte Physcomitrella patens (Moller et al., 2007; Liepman et al., 2007), in pteridophytes, in which their abundance may vary taxonomically (Bailey and Pain, 1971; Harris, 2005), and in gymnosperms (Whistler and Chen, 1991). Mannan polysaccharides appear to be less abundant in angiosperm primary cell walls but are still clearly present (Popper and Fry, 2003; Nothnagel and Nothnagel, 2007; Vogel, 2008; Goubet et al., 2009; Scheller and Ulskov, 2010). The persistence of these varied sets of hemicelluloses through land plant evolution may indicate diversification of functions (Liepman et al., 2007).

Genes encoding enzymes capable of cleaving the backbone of mannan polysaccharides (endo-β-mannanases) are widespread in plant genomes and are implicated in diverse processes such as seed germination and fruit ripening (Yuan et al., 2007). Recent analysis of the endo-β-mannanase of kiwi fruit has indicated that they may also have biosynthetic activity, and it is thus possible that this endo-β-mannanase represents an enzyme class equivalent to the well-studied xyloglucan transglycosylase/hydrolase (XTH) group of cell wall enzymes (Schröder et al., 2004, 2006, 2009). The functions of such enzymes in cell wall modifications and cell function are not well understood, but could contribute to assembly processes and influence cell wall mechanical properties. Analysis of genes/proteins involved in the synthesis of mannans has indicated important roles for these polysaccharides in cell development, but again mechanistic details are not clear (Liepman et al., 2007; Goubet et al., 2009). Evidence has also been presented that galactoglucomannan oligosaccharides can exert a variety of physiological effects indicating a developmental role, particularly in vascular cell differentiation (Auxtováet al., 1995; Beňová-Kákošováet al., 2006). Taken together, these observations indicate that specific roles of the mannan group of polysaccharides (mannans, glucomannans, galactomannans and galactoglucomannans) may extend beyond simple structural or microfibril cross-linking roles in plant cell walls.

Bulk chemical analyses of cell wall polysaccharides obtained from homogenized cells/tissues are important in obtaining compositional and linkage information. However, these approaches can only provide broad overviews of polysaccharide structural features in cell walls, and cannot provide detailed information relating to cellular and subcellular levels, for which other complementary approaches are required. Sets of monoclonal antibody and carbohydrate-binding module (CBM) probes are very useful tools to explore polysaccharides in situ in intact cell walls (Knox, 2008). The insights obtained using these sets of probes have reinforced the view that cell walls are highly dynamic, structurally heterogeneous and metabolically active compartments of plant cells. In terms of current methodologies molecular probes present the only effective method to associate specific polysaccharide structures with precise cell wall contexts in order to develop an understanding the functions of structural polysaccharides within wall architectures. Antibodies to the mannan set of polymers have been developed, and their use has indicated a wide distribution of mannans within plants, with some variation with respect to cell types and cell wall domains (Maeda et al., 2000; Pettolino et al., 2001; Handford et al., 2003).

Recent cell biology studies on other hemicelluloses have shown that antibody recognition of xyloglucan and xylan polymers in some primary cell walls can only occur after the removal of pectic homogalacturonan (HG), which effectively masks these polymers (Marcus et al., 2008; Hervéet al., 2009). These studies have revealed previously unknown aspects of cell wall heterogeneity (Marcus et al., 2008; Hervéet al., 2009; Ordaz-Ortiz et al., 2009). They also indicate that there is an intimate association, and possibly covalent links, between these polymer sets. Here, using two novel mannan-directed antibodies (LM21 and LM22) and a mannan-specific family 27 CBM (CBM27; Boraston et al., 2003), we show that molecular recognition of mannans can be extensively masked by esterification in intact secondary cell walls, and by the presence of pectic HG in primary cell walls. Using a range of plant materials, we show that masking is a widespread phenomenon, and, through use of a CBM27 directly coupled to a fluorescent tag and enzyme treatments, we demonstrate that masking is not an artefact of indirect immunocytochemistry or embedding procedures. These observations indicate that the masking of mannan polymers is a phenomenon of cell biological significance with important consequences for understanding cell wall structures, cell wall heterogeneity, cell wall micro-environments and the functions of mannans and endo-β-mannanases.

Results

Comparative analysis of the binding of mannan-directed probes to cell wall oligo- and polysaccharides

Molecular probes that show specific recognition of the mannan group of polysaccharides were used in this study. Two rat mannan-directed monoclonal antibodies, designated LM21 and LM22, were generated subsequent to immunization with a β-mannopentaose–BSA neoglycoprotein and a digalactosylmannopentaose–BSA neoglycoprotein, respectively. A recombinant His-tagged family 27 mannan-directed carbohydrate-binding module from Thermotoga maritima (Boraston et al., 2003) was designated CBM27. The binding specificity of these probes towards a series of oligosaccharides found in plant cell wall polysaccharides was assessed by use of carbohydrate microarrays in which oligosaccharides were coupled to BSA prior to immobilization. Probing of equivalent arrays with LM21, LM22, CBM27, and, for comparative purposes, the xylan-directed rat monoclonal antibody LM11 (McCartney et al., 2005), is shown in Figure 1(a). A heatmap showing quantification of mean spot signals for mannosyl- and xylosyl-containing samples is shown in Figure 1(b), and a full list of the samples used on the arrays is provided in Table S1. All three anti-mannan probes displayed binding specificity towards BSA-conjugated mannan and related oligosaccharides, and showed no binding to pectin-, xylan- or glucan-derived BSA-coupled oligosaccharides. However, there were subtle differences between the three probes. LM21 bound most effectively to manno-oligomers with degrees of polymerization (DPs) of 4 and 5, but also to lower-DP manno-oligomers, a konjac glucomannan oligosaccharide (konjac Glc₂Man₂, sample D7) and to a lesser extent to galactomannan-derived oligosaccharides. In contrast, LM22 bound strongly to carob galactomannan oligosaccharides and to lower-DP manno-oligomers and also bound to konjac Glc₂Man₂. CBM27 displayed the most restricted specificity, binding only to mannoligosaccharides with a DP >2, although weak recognition of digalactosylated mannotetraose was also observed (Figure 1b). The reason for the lack of recognition of a barley glucomannan-derived oligosaccharide (Man₃Glc₂, sample D8) by all three probes is uncertain.

Figure 1.  Cell wall oligosaccharide microarray analysis of LM21, LM22 and CBM27 binding specificity. (a) Direct views of completed microarrays containing pectic, glucan, xyloglucan and xylan oligosaccharides in addition to mannan-derived oliogsaccharides. Xylan-directed monoclonal antibody LM11 is included for comparison. (b) Derived quantitative heatmap of LM21, LM22, CBM27 and LM11 binding to mannosyl- and xylosyl-containing oligosaccharides.

The recognition by these three probes of isolated mannan polysaccharides was studied using ELISAs with a series of mannan and related polysaccharides including ivory nut mannan (99% Man), konjac glucomannan (Glc:Man, 2:3), carob galactomannan (Gal:Man, 22:78), guar galactomannan (Gal:Man, 38:62) and guar galactose-depleted galactomannan (Gal:Man, 21:79), as shown in Figure 2. LM21 and CBM27 bound in an approximately equivalent manner to all six polysaccharides as assessed by ELISA, and therefore displayed clear recognition of most polymers of the galactoglucomannan group of polysaccharides. Surprisingly, LM22 did not bind effectively to galactomannans, although it was generated subsequent to immunization with a BSA-conjugated galactomannan-derived oligosaccharide and recognized this and related conjugates (Figure 1). The lack of recognition of this subset of polymers does not seem to directly involve galactosylation, as binding was not increased in galactosidase-treated guar galactomannan, in which the proportion of the galactose in the polymer is reduced from 38 to 21% by α-galactosidase action (Figure 2a). The LM22 mannan-directed probe therefore appears to bind to novel features of mannan polymers that have yet to be identified. It is possible that polysaccharide conformational issues come into play for recognition of polysaccharides as opposed to oligosaccharides.

Figure 2.  Analysis of probe binding to mannan polysaccharides. (a) ELISA analysis of LM21, LM22 and CBM27 binding to isolated mannan-related polysaccharides. All polymers were coated on to microtitre plates at 50 μg ml⁻¹, and two absorbance values for each probe are shown. These correspond to 25- and 125-fold dilutions of hybridoma supernatant for LM21 and LM22, and 10 and 2 μg ml⁻¹ concentrations for CBM27. (b) Indirect immunofluorescence analysis of Calcofluor, LM21, LM22 and CBM27 binding to equivalent sections of L. corniculata seeds, indicating LM21 recognition of all cells and the lack of recognition by LM22. CBM27 bound only to endosperm and outer regions of the seed coat (sc). Scale bar = 100 μm.

LM21, LM22 and CBM27 recognition of intact plant cell walls using immunocytochemistry

To explore the ability of antibodies LM21 and LM22 and CBM27 to recognize mannan polymers in the context of cell walls, the probes were used for indirect immunofluorescence labelling of sections of Lotus corniculatus seeds as shown in Figure 2(b). L. corniculatus is a legume, the seeds of which contain abundant highly branched galactomannan in which >50% of the backbone residues have α-galactosyl substitution, which functions as a seed storage polysaccharide (Srivastava and Kapoor, 2005). Immunofluorescence analysis of sections across seed coat, endosperm and cotyledon indicated that the LM21 epitope was readily detected in all three organs but the LM22 epitope was not, reflecting the differential recognition of related polymers by these two probes in ELISA (Figure 2). CBM27 bound to some of the same sets of cells as LM21 but showed a more restricted binding to the outer cell walls of seed coat cells and the endosperm, with no recognition of the embryo (Figure 2b).

Gymnosperm secondary cell walls are known to contain galactoglucomannan (in which approximately 5% of the backbone residues have α-galactosyl substitution) as the major hemicellulose (Whistler and Chen, 1991). In sections of resin-embedded Pinus radiata compression wood, both LM21 and LM22 bound strongly to secondary cell walls but with clear differences: LM21 bound to the S1 secondary cell wall layer and weakly to the inner S2 layer, whereas LM22 bound equally to the S1 layer and the inner S2 layer (Figure 3). CBM27 bound in a manner similar to LM21 but less strongly (results not shown).

Figure 3.  Indirect immunofluorescence analysis of LM21and LM22 binding to resin-embedded sections of Pinus radiata compression wood. Arrows indicate the S1 secondary cell wall layer and asterisks indicate the inner region of the S2 layer. Scale bar = 20 μm.

These initial analyses indicated that the three probes could all bind to epitopes in the context of intact cell walls. The data demonstrate heterogeneity in the detection of mannan epitopes, and also differential binding profiles for the three probes, indicating recognition of distinct structures or epitopes.

Blocked recognition of mannans in secondary cell walls

Initial analysis of transverse sections of Arabidopsis, tobacco and pea stems indicated very weak or no binding by any of the mannan-directed probes. Given the known potential for masking of xyloglucan and xylan hemicellulose polymers in these organs, this limited binding was explored further. One possibility was that pre-treatment of sections with alkali would solubilize sets of polysaccharides, resulting in the exposure of hidden epitopes. Therefore, sections were pre-treated with 1 m KOH for 1 h prior to immunolabelling procedures. After this treatment, LM21 and CBM27 mannan probes bound strongly and evenly to the secondary cell walls in these three species, as shown in Figure 4. In the case of Arabidopsis stem xylem secondary cell walls, there was some binding before pre-treatment, but this was variable. However, after KOH treatment of the sections, the probes bound equally and evenly across all secondary cell walls, as shown for CBM27 in Figure 4(b,c). In the case of tobacco stem secondary cell walls, KOH treatment greatly increased probe recognition, as shown for LM21 in Figure 4(d,e). This suggests that KOH possibly acts as a saponifiying agent, removing ester groups, presumably acetyl substitutions, from mannans. This was confirmed by the pre-incubation of sections with the esterase CjCE2C from Cevibrio japonicus, which has the capacity to deacetylate hemicelluloses (Montanier et al., 2009), resulting in strong LM21 recognition of secondary cell walls of both xylem cells and phloem sclerenchyma fibres, as shown in Figure 4(f,j).

Figure 4.  Indirect immunofluorescence analysis of LM21and CBM27 binding to secondary cell walls in transverse sections of A. thaliana and tobacco stems. (a) Calcofluor staining of all cell walls in an A. thaliana stem showing the vascular bundle. (b) Equivalent region of stem to (a) showing immunolabelling with CBM27. (c) Equivalent region of stem to (a) showing CBM27 labelling after pre-treatment with 1 m KOH. (d) Weak LM21 binding to xylem cells of tobacco stem. (e) Equivalent section to (d) treated with 1 m KOH prior to immunolabelling with LM21. (f) Equivalent section to (d) pre-treated with CjCE2C esterase prior to immunolabelling with LM21. (g) Calcofluor staining of tobacco stem region with phloem fibres. (h) Same section as (g) immunolabelled with LM21, which binds weakly. (i) Equivalent section to (g) treated with 1 m KOH prior to immunolabelling with LM21. (j) Equivalent section to (c) pre-treated with CjCE2C esterase prior to immunolabelling with LM21. Scale bars = 100 μm (a–f) and 10 μm (g–j).

These observations indicate that mannan-specific probe recognition in the secondary cell walls of these dicotyledon species is restricted by esterification, presumably acetylation, and, in combination with esterase pre-treatment, can provide information on acetylation heterogeneity across the secondary cell walls. The use of KOH or esterase pre-treatments on equivalent sections of P. radiata compression wood cells did not result in substantially altered binding, indicating that, although gymnosperm galactoglucomannans are generally regarded as acetylated, this may be at a level that does not interfere with probe binding.

Mannan polysaccharides in primary plant cell walls are masked by pectic homogalacturonan

It was noted that use of KOH on sections resulted in slightly increased binding to some regions of primary cell walls, possibly due to solublization of pectic polymers from the sections. It has previously been shown that recognition of xyloglucan and xylan epitopes in primary cell walls can be effectively masked by the presence of pectic HG (Marcus et al., 2008; Hervéet al., 2009). In these cases, the pectic HG was enzymatically removed from cell walls in sections prior to antibody immunolabelling procedures using a pectate lyase (Pel10A). Pel10A, and other pectate lyases, act preferentially on de-esterified HG, and the optimum pH for these enzymes is 10. In vitro ELISA analysis of the occurrence of HG epitopes in a sample of methyl-esterified pectin after a range of pH pre-treatments indicated that HG methyl ester groups were not always effectively removed at pH 10 (Figure 5). The widely used JIM5 and JIM7 pectic HG monoclonal antibodies (Knox et al., 1990; Clausen et al., 2003) were used alongside the more recently generated pectic HG monoclonal antibodies LM19 and LM20 (Verhertbruggen et al., 2009). ELISA analysis indicated that loss of the LM20 and JIM7 epitopes (both requiring some methyl esters for recognition) was maximized by pre-treatment at pH 11 or above, and that binding of LM19, which is the most effective probe for recognition of de-esterified HG, was also maximized by an identical pre-treatment. To explore the effect of pectate lyase (PL) on monoclonal antibody and CBM recognition of mannans in primary cell walls, sections were pre-treated for 2 h with 0.1 m sodium carbonate (pH 11.4) to maximize pectic HG degradation by the subsequent use of PL. In all cases, the effects attributed to PL were confirmed by studying the sections after sodium carbonate pre-treatment alone.

Figure 5.  ELISA analysis of the impact of alkaline pH pre-treatments on the binding of four pectic homogalacturonan-directed monoclonal antibodies, JIM5, JIM7, LM19 and LM20 to a high methylester pectin. E96 pectin was coated onto the plate at 50 μg ml⁻¹, and all antibodies used at 10-fold dilution. Pre-treatments using CAPS-buffered solutions of pH 7–12 for 2 h before immunolabelling were performed. The pH of a 0.1 m sodium carbonate solution, used to pre-treat sections prior to application of pectate lyase, is shown for comparison as a vertical line.

Immunofluorescence analysis of LM21, LM22 and CBM27 binding to sections of Arabidopsis thaliana stems indicated weak recognition of some primary and some secondary cell walls, as reported for a mannan-directed antiserum (Handford et al., 2003), and as shown in Figure 6. LM21 and CBM27 bound weakly to epidermal and cortical primary cell walls, and LM22 binding was restricted to developing xylem vessel elements. The enzymatic removal of pectic HG from equivalent transverse sections resulted in increased binding of all three probes to all primary cell walls, and this was particularly strong in the epidermis. The LM21 epitope was also strongly unmasked in the pith parenchyma cell walls. To confirm that the binding, subsequent to PL action, was to mannan polysaccharides (and not some artefact of enzyme action or section treatments), equivalent sections were treated with an endo-β-mannanase prior to immunolabelling, which resulted in loss of most of the immunofluorescence (Figure 6).

Figure 6.  Indirect immunofluorescence micrographs of binding of monoclonal antibodies LM21 and LM22 and CBM27 to transverse sections of the base of an Arabidopsis thaliana stem. Top row: no pre-treatment of sections. Middle row: Sections pre-treated with pectate lyase (PL). Bottom row: Sections pre-treated with PL followed by 2 h treatment with endo-β-mannanase (Mnase). e, epidermis; pp, pith parenchyma. Scale bar = 100 μm.

The capacity of PL to uncover mannan epitopes in primary cell walls was also confirmed for tobacco and pea cell walls (Figure 7). In these cases, the binding of probes to untreated sections was very low, or not detectable, but the target epitopes for all three probes were clearly unmasked by PL. In most cases, the probes bound to all primary cell walls, although preferential recognition of cell junctions and cell walls lining intercellular spaces of the parenchyma was apparent in some cases. Further analysis of the pith parenchyma of tobacco stems using LM21, LM22 and CBM27 indicated variable recognition of cell walls in relation to cell junctions and intercellular spaces, and effective recognition of pit fields when exposed on the inner face of cell walls, as seen at the end of cells in transverse sections (Figure 8). In this case, there was clear recognition of pit fields by LM21 as indicated by comparison with Calcofluor binding, indicating an aspect of cell wall heterogeneity that is revealed by enzymatic removal of pectic HG (Figure 8).

Figure 7.  Indirect immunofluorescence analysis of mannan probe binding to transverse sections of pea and tobacco stem cell walls. (a) Calcofluor binding showing all cell walls in a transverse section of pea stem. (b) Same section as (a) immunolabelled with LM21. (c) Equivalent section to (a) pre-treated with PL and labelled with LM21. (d) Equivalent section to (a) pre-treated with PL and immunolabelled with LM22. (e) Calcofluor imaging of cell walls in a region of cortical cells of tobacco stem. (f) Same section as (e) labelled with CBM27. (g) Equivalent section to (e) pre-treated with PL and labelled with CBM27. (h) Equivalent section to (e) pre-treated with PL and endo-β-mannanase (Mnase) prior to CBM27 labelling. Scale bars = 100 μm.

Figure 8.  Indirect immunofluorescence analysis of mannan probe binding to tobacco stem pith parenchyma cell walls. (a) Calcofluor staining of a region of pith parenchyma. (b) Same section labelled with LM21. (c) Equivalent section pre-treated with PL and labelled with LM21. (d) Equivalent section pre-treated with PL and labelled with LM22. (e) Equivalent section pre-treated with PL and labelled with CBM27. (f) Region of pith parenchyma showing inner face of primary cell walls pre-treated with PL and immunolabelled with LM21. (g) Same section as (f) showing Calcofluor staining. (h) Images (f) and (g) combined. Arrows indicate pit fields that have weaker Calcofluor and strong LM21 labelling. Scale bars = 50 μm.

To study the extent of masking of mannan polysaccharides in parenchyma systems of a wider diversity of land plants in which mannans are known to be relatively abundant, PL pre-treatment was used on transverse sections of petioles obtained from several fern species. The data for Hymenasplenium obscurum (Figure 9) show that PL pre-treatment resulted in LM21 detection of mannan epitopes in parenchyma cell walls that were previously undetected by the immunofluorescence procedure. In the model bryophyte Physcomitrella patens, mannans have been shown to be abundant and also extensively developmentally regulated (Liepman et al., 2007). In this case, LM21 bound effectively to a resin-embedded section of the leafy gametophores, and PL pre-treatment had no effect on the intensity of LM21 fluorescence, as shown in Figure 9. At the surface of intact P. patens protonema filaments, LM21 and CBM27 (but not LM22) bound specifically to cell walls at the base of branching cells, and PL pre-treatment had little impact on this restricted pattern of binding (data not shown).

Figure 9.  Immunofluorescence analysis of LM21 mannan binding to pteridophyte and bryophyte parenchyma systems. (a) Calcofluor staining of a region of parenchyma a in transverse section of a petiole of the fern Hymenasplenium obscurum. (b) Same section as (a) immunolabelled with LM21. (c) Equivalent section to (a) pre-treated with PL and immunolabelled with LM21. (d) Transverse section of leafy gametophore of bryophyte Physcomitrella patens immunolabelled with LM21. (e) Equivalent section to (d) pre-treated with PL and immunolabelled with LM21. Scale bars = 50 μm.

Cell biological significance of restricted access to primary cell wall mannan polysaccharides by pectic HG

It is possible that the action of PL on hemicellulose probe recognition of primary cell walls is an artefact due to the ethanol dehydration steps during embedding procedures, the restricted access of large immunoglobulin molecules to cell walls during indirect immunocytochemistry procedures, or the reduced antigenicity of embedded plant materials. To explore these possibilities, and to demonstrate that pectic HG masking of mannan epitopes is a phenomenon of cell biological significance, two strategies were employed.

CBM27 is the smallest mannan probe, with a molecular mass of approximately 25 kDa (Boraston et al., 2003), and this was coupled directly to FITC to produce a probe for direct fluorescence imaging procedures. This CBM:FITC probe was used with hand-cut sections of pea stems that had not been through any dehydration procedures. The directly labelled probe bound only weakly to primary cell walls, as shown for a transverse section of pea stem in Figure 10. Pre-treatment of an equivalent section with PL resulted in greatly increased binding to all primary cell walls (Figure 10). These investigations also explored whether an endo-β-mannanase had restricted access to its substrate in primary cell walls. In this case, immunofluorescence detection of the LM21 epitope was carried out after sequential treatments of equivalent sections of tobacco stem with first PL and then mannanase and vice versa. Treatment with PL followed by mannanase, prior to LM21 labelling, resulted in greatly reduced immunofluorescence detection of the LM21 epitope relative to PL treatment alone. In contrast, treatment with mannanase followed by PL prior to LM21 immunolabelling resulted in an equivalent level of LM21 epitope detection in primary cell walls as in the PL treatment alone (Figure 10). It was noted that, in both cases in which mannanase was a component of the pre-treatment, the enzyme was effective in removing most of the LM21 epitope from the secondary cell walls of xylem cells (Figure 10).

Figure 10.  Cell biological significance of masking of mannans by pectic HG in primary cell walls. (a) Hand-cut section of pea stem stained with CBM27 directly coupled to FITC (CBM27:FITC). (b) Equivalent section to (a) pre-treated with PL and stained with CBM27:FITC. (c) Region of a transverse section of tobacco stem, showing vascular tissues, immunolabelled with LM21. (d) Equivalent section to (c) pre-treated with PL and labelled with LM21. (e) Equivalent section to (c) pre-treated with PL followed by endo-β-mannanase (Mnase) prior to LM21 labelling. (f) Equivalent section to (c) pre-treated with endo-β-mannanase followed by PL prior to LM21 labelling. Arrows indicate epidermis. sb, sclerenchyma bundle; cp, cortical parenchyma; x, xylem; pp, pith parenchyma. The LM21 epitope in the xylem secondary cell walls is removed by both mannanase treatments. Scale bars = 100 μm.

Discussion

Recognition of mannans in plant cell walls

This study demonstrates that mannan polysaccharides are not always readily accessible to antibody and CBM probes in intact plant cell walls. In some cases, notably the secondary cell walls of the dicotyledons shown here, esterification (probably acetylation) prevents recognition of mannans. The impacts of alkali or esterase pre-treatments on patterns of mannan detection indicate the heterogeneity of mannan polysaccharide structures in these cell types. Such approaches enable assessment of spatial aspects of mannan acetylation in cell walls. In the case of primary cell walls, recognition was greatly increased by enzymatic removal of pectic HG, and this was observed in both angiosperms and pteridophytes. In the case of A. thaliana stems, there was recognition of some primary and secondary cell walls in untreated sections as reported previously (Handford et al., 2003; Goubet et al., 2009), indicating developmental regulation of detectable mannan epitopes. The increased detection of mannan epitopes after PL treatment demonstrates that mannan masking by pectic HG is also developmentally regulated, indicating that the masking is an intrinsic aspect of cell wall heterogeneity. In the case of tobacco and pea stem sections, mannan recognition was very weak, but mannans in both primary and secondary cell walls were made accessible to probes by specific pre-treatments. The clear unmasking of mannan polymers in primary cell walls by enzymatic removal of pectic HG means that mannans can be added to xyloglucan and xylans as hemicellulose polymers that can be masked in primary cell walls by pectic HG. These observations extend our understanding of the phenomenon of masking, in that unmasking of mannan is specific to enzymatic removal of pectic HG (and cannot be effected by KOH pre-treatment, which is presumed to solubilize some pectic polymers). Moreover, it is not an artefact due to the preparation of embedded sections or restricted access of large immunocytochemistry detection molecules, as a directly coupled fluorescent CBM was used in conjunction with a hand-cut section (to preserve maximum antigenicity), and a clear effect of PL pre-treatment on mannan detection was still apparent.

This is the first time that investigations on epitope unmasking have included use of a CBM. CBMs are thought to have evolved for the specific recognition of cellulose and hemicellulose polysaccharides in cell walls, and thus potentiate the action of attached catalytic modules (Boraston et al., 2004; Guillén et al., 2010). Recombinant CBMs have been shown to be effective probes for polymer recognition in both primary and secondary cell walls (McCartney et al., 2006; Blake et al., 2006; Hervéet al., 2009), and a family 27 CBM has been used to detect mannans in the red alga Bangia atropurpurea (Umemoto and Araki, 2010). Although in the context of cell wall deconstruction, it would perhaps be expected that a CBM would display wide recognition of a polymer class, it appears that CBM27 from T. maritima is most effective towards non-acetylated mannan in secondary cell walls and PL-unmasked mannan in primary cell walls. A minor caveat to this interpretation, however, is that the esterase used displays activity for both acetylated xylans and glucomannans (Montanier et al., 2009), and thus it is possible that the positive effects of the enzyme on probe recognition of secondary cell walls may not be restricted solely to modification of the mannan-based polymers. However, CBM action in cell wall deconstruction or modification processes is as a component of mannanases, which act in concert with other enzyme systems such as esterases and pectinases (Gilbert et al., 2008). Enzyme action against hemicelluloses has been found to be enhanced in plants that have been engineered to have altered/reduced pectic HG (Lionetti et al., 2010).

Implications for mannans in plant cell biology

The enzymatic removal of HG from primary cell walls has revealed structural heterogeneity of these composite structures. In pea and tobacco stem parenchyma, mannan epitopes were specifically detected in cell walls at cell junctions lining the intercellular spaces of parenchyma systems, as has also been observed for unmasked xyloglucan and xylan epitopes (Marcus et al., 2008; Hervéet al., 2009). In the tobacco pith parenchyma, the mannan epitopes were specifically associated with pit fields, as seen at the inner surface of transverse cell walls. This is in clear contrast to unmasked xyloglucan, which did not have a specific pit-field location but was more widely distributed in equivalent cell walls (Marcus et al., 2008). The pattern of the LM21 mannan epitope on this inner cell wall to some extent reflects the pattern of the LM6 arabinan epitope and callose on the inner surfaces of tomato fruit parenchyma cell walls (Orfila and Knox, 2000). This observation adds to our understanding of the distinctive cell wall structure associated with pit fields, as well as the structural heterogeneity within cell walls in general.

What is the significance of the potential coating of mannan polysaccharides with pectins in terms of cell wall assembly and cell wall functions? Cell wall polymers need to be interlinked to generate insoluble cell walls. Although there is no evidence for links between pectins and mannans, an association indicated by the masking may have a structural role in maintaining primary cell wall integrity. Recent evidence that treatment with a mannanase can result in cell separation in tomato fruit parenchyma (Ordaz-Ortiz et al., 2009) may indicate that mannans are part of a mechanism that links intercellular pectins to primary cell walls. In some cases, pectic HG may coat mannans and other hemicelluloses, and thus restrict or control the access of enzymes (and probes), and this modulation of cell wall micro-environments may be a feature of cell wall modification. The potential ability of mannanases to release active galactoglucomannan oligosaccharides (Beňová-Kákošováet al., 2006) could also be relevant here, as masking of sets of mannans may be part of a regulatory mechanism to control the release of signals from cell wall polysaccharides. Whether the observations reported here indicate a specific link between mannan and pectic polymers remains an open question. Currently, there appears to be no evidence indicating the cross-linking of mannans with cell wall polymers other than cellulose (Fry, 1986; Iiyama et al., 1994; Whitney et al., 1998; Obel et al., 2006). It is now clear that some endo-β-mannanases also have transglycosylase activity (Schröder et al., 2006, 2009), and it is possible that some members of the large families of mannanases, which seem to have wide roles in development (Yuan et al., 2007), have the ability to link mannan to other cell wall polymers, and that such links have structural roles. The observations reported here may also explain the inability to detect mannan epitopes in A. thaliana embryo cell walls, in which genetic evidence has strongly indicated a functional role (Goubet et al., 2009). Could cryptic mannan epitopes have a specific role in embryogenesis?

In summary, these observations confirm that masking of hemicellulose polymers by pectic HG is a real phenomenon, with significance for understanding cell wall assembly, properties and metabolism. They confirm an important central role for pectin in the primary cell wall matrix, and how it may generate micro-environments that can regulate the access of enzymes and other proteins to sites of action. The work also indicates that immunocytochemistry directed towards hemicelluloses, and possibly other polysaccharides, will require consideration of the possible presence of cryptic epitopes.

Experimental Procedures

Preparation of mannan-directed probes

Two rat monoclonal antibodies, designated LM21 and LM22, were derived subsequent to immunization of rats with two neoglycoprotein immunogens, prepared by coupling of mannopentaose (Man5) and digalactosylmannopentaose (Gal2Man5) oligosaccharides (Megazyme International, http://www.megazyme.com) to bovine serum albumin (BSA) by reductive amination (Roy et al., 1993). Briefly, 30 mg of oligosaccharide were dissolved in 1.0 ml sodium borate buffer, pH 8.5, followed by addition of 20 mg BSA and then 30 mg sodium cyanoborohydride. The mixture was maintained at 50°C for 24 h, and then the pH was adjusted to 4.0 using acetic acid. The materials were then dialysed against distilled water. The resulting neoglycoproteins were designated Man5–BSA and Gal2Man5–BSA. Rat immunization, hybridoma preparation and cloning procedures were performed as described previously (Willats et al., 1998). For each conjugate, two male Wistar rats were injected with 100 μg neoglycoprotein in complete Freund’s adjuvant administered subcutaneously on day 0, and the same amount was administered with incomplete Freund’s adjuvant on two subsequent occasions for Man5–BSA and three for Gal2Man5–BSA. Pre-fusion boosts of 100 μg neoglycoprotein in 1 ml PBS were given by intraperitoneal injection 3 days before splenectomies leading to lymphocyte isolation and fusion with rat myeloma cell line IR983F (Bazin, 1982). Antibodies were selected by ELISA using the neoglycoproteins as antigens. Subsequent characterization was by means of immunochemical assays, oligosaccharide microarrays and immunofluorescence analysis of binding to plant materials. The LM21 and LM22 hybridoma cell lines both secrete IgM immunoglobulins. Other rat monoclonal antibodies used in this study include the LM11 xylan probe (McCartney et al., 2005) and the JIM5, JIM7, LM19 and LM20 pectic HG probes (Clausen et al., 2003; Verhertbruggen et al., 2009). A mannan-directed CBM27 from a Thermotoga martima mannanase was obtained as a recombinant His-tagged protein as described previously (Boraston et al., 2003).

All use of animals was in accordance with UK Home Office guidelines and procedures.

Production of carbohydrate microarrays

Oligosaccharides were conjugated to BSA by reductive amination as described previously (Roy et al., 1993). Samples were spotted onto nitrocellulose membrane (pore size 0.45 μm, Schleicher & Schuell, http://www.schleicher-schuell.com) using a microarray robot (MicroGrid II, Genomic Solutions/Digilab Inc., http://www.digilabglobal.com) equipped with split pins (MicroSpot 2500, Genomic Solutions/Digilab Inc.) as described previously (Moller et al., 2008). Samples were printed in two concentrations (1.0 and 0.5 mg ml⁻¹), with two replicates per concentration. Consequently, each sample was represented by a total of four spots arranged in a 2 × 2 grid on the membrane. Each individual microarray contained 63 oligosaccharides conjugated to BSA (for a complete sample list, see Table S1).

Probing and quantification of oligosaccharide microarrays

Arrays were probed with LM21, LM22, LM11 and CBM27 as described previously (McCartney et al., 2004; Moller et al., 2008). Briefly, arrays were blocked in 5% fat-free milk powder in PBS prior to incubation for 2 h in primary antibody or CBM27. Rat monoclonal antibodies were used at a 10-fold dilution of hybridoma supernatants, and CBM27 was used at a concentration of 10 μg ml⁻¹. After extensive washing in PBS, arrays were probed with secondary antibodies conjugated to alkaline phosphatase. Anti-rat and anti-His secondary antibodies were used for the primary antibodies and CBM27, respectively. After further washing in PBS, arrays were developed in 5-bromo-4-chloro-3′-indolylphosphate/nitroblue tetrazolium chloride-based substrate. Arrays were scanned and quantified as described previously (Moller et al., 2008). Briefly, for each array, mean signal values were derived from the four individual spots representing each sample. The final mean signal values are the average of the mean signal values from three independent experiments. In the final datasets, the highest value for antibody binding was set to 100, and the remaining signals adjusted accordingly. The normalized datasets were converted into heatmaps using online heatmapper software (http://bar.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi).

Enzyme-linked immunosorbent assays

ELISAs were performed as described previously (Willats et al., 1998). Mannan polysaccharides (ivory nut mannan, konjac glucomannan, carob galactomannan, guar galactomannan and galactose-depleted guar galactomannan) were obtained from Megazyme International. Pectin with a degree of methyl esterification of 0.96 was a gift from Danisco (http://www.danisco.com), and was used to coat plates at 50 μg ml⁻¹ for analysis of the impact of pre-treatment at alkaline pH (adjusted between 7 and 12 using N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)-based buffers for 2 h) on pectic HG antibody binding.

Plant materials and preparation for microscopy

In all cases, plant materials were fixed in formaldehyde-containing buffer as described previously (McCartney et al., 2006) and were prepared as follows. Lotus corniculatus L. seeds were imbibed and wax-embedded using a standard protocol (Marcus et al., 2008). Pinus radiata D. Don compression wood samples were prepared by embedding in LR White resin. Pisum sativum, Nicotiana tabacum and Arabidopsis thaliana stems were grown and wax-embedded sections prepared as described previously (Marcus et al., 2008; Verhertbruggen et al., 2009). In some cases, hand-cut sections of these stems were prepared directly into fixative. Hymenasplenium obscurum and other fern petiole sections were cut by hand. Physcomitrella patens gametophores were embedded in LR White resin as described previously (Orfila and Knox, 2000).

Immunofluorescence imaging procedures

Plant section pre-treatments included 1 m potassium hydroxide for 1 h, 0.1 m sodium carbonate for 2 h, 10 μg ml⁻¹ pectate lyase (Pel10A, Brown et al., 2001) in 50 mm CAPS, 2 mm CaCl₂ buffer, pH 10, for 2 h at room temperature, 100 μg ml⁻¹Bacillus spps. endo-β-mannanase (Megazyme International) in 0.1 m glycine, pH 8.8, for 2 h at 37°C, and Cevibrio japonicus esterase CE2C (Montanier et al., 2009) at 1 μm in PBS. LM21 and LM22 were used as unpurified hybridoma cell culture supernatants in indirect immunofluorescence procedures with anti-rat FITC as described for other rat monoclonal probes (Marcus et al., 2008). For compression wood samples, anti-rat Alexa 568 (Invitrogen, http://www.invitrogen.com) was used to allow imaging at longer wavelengths to avoid lignin autofluorescence. In most cases, His-tagged CBM27 was used with a triple indirect immunofluorescence labelling procedure as described previously (McCartney et al., 2006). In some cases, CBM27 was coupled directly to FITC by means of a Lightning-Link conjugation protocol (Innova Biosciences Ltd, http://www.innovabiosciences.com) with CBM27 protein at 1 mg ml⁻¹ to produce a directly fluorescent probe designated CBM27:FITC. CBM27:FITC was incubated with plant materials at 10 μg ml⁻¹ for 2 h prior to washing and preparation of slides for viewing. In most cases, Calcofluor was used as a counter-stain for visualization of all cell walls. Immunofluorescence imaging was performed using an Olympus BX61 microscope (http://www.olympus-global.com/) equipped with epifluorescence irradiation. Micrographs were obtained using a Hamamatsu ORCA285 camera (Hamamastu, http://www.hamamatsu.com) and Improvision Volocity software (Improvision, http://www.improvision.com). For compression wood, imaging was performed using a Leica TCSNT confocal laser scanning microscope (http://www.leica.com/). All related and comparative micrographs were captured using equivalent settings, and relevant micrographs were processed in equivalent ways for generation of datasets.

Acknowledgements

We acknowledge funding from the UK Biotechnology and Biological Sciences Research Council (grant numbers BB/E014364/1 and BB/G024898/1 and a studentship to T.A.S.B.).