Developmental complexity of arabinan polysaccharides and their processing in plant cell walls

Authors


*(fax +44 113 3433144; e-mail J.P.Knox@leeds.ac.uk).

Summary

Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.

Introduction

Plant cell walls are robust polysaccharide-based materials at the surfaces of plant cells that function in the generation of cell morphology, and the maintenance of cell integrity, cell adhesion and tissue cohesion. Cell walls also generate and impact upon the mechanical properties of tissues and organs. Diverse cell wall structures and morphologies underpin many aspects of plant growth and development. It is becoming increasingly apparent that the polysaccharides that comprise cell walls are diverse in structural terms, that these structures are developmentally regulated, and that not all cell walls in an organ are comprised of the same configurations of polysaccharides (Knox, 2008).

Both primary and secondary cell walls have a fibrous-composite type construction, with cellulose microfibrils as the fibrous components involved in generating resistance to both tensile and compressive forces. In primary cell walls, the cellulose microfibrils are currently viewed as being tethered together by hemicellulose components, and that the cellulose–hemicellulose network is embedded in a matrix of pectic polysaccharides (Cosgrove, 2005). There is an increasing body of evidence to suggest that hemicellulose polymers can be linked to pectins, and also that some pectic polymers can directly associate with cellulose microfibrils in a manner similar to hemicelluloses (Zykwinska et al., 2007a,b; Popper and Fry, 2008). This is suggestive of complex mechanisms for the tethering and spacing of microfibrils, which are likely to be important factors underpinning cell wall properties and extensibility (Cosgrove, 2005; Thompson, 2005). The lack of an extensive pectic network in thick secondary cell walls is associated with a lack of a capacity for cell wall stretching and extension.

The pectic polysaccharides, abundant in extendable primary cell walls, are structurally diverse, with three main polysaccharides: homogalacturonan (HG), rhamnogalacturonan-II (RGII) and rhamnogalacturonan-I (RGI) (Ridley et al., 2001; Willats et al., 2001; Mohnen, 2008). HG is a linear chain of galacturonosyl residues that can be variably methyl-esterified and/or acetylated: both factors that influence its capacity to form ionic cross links with cations, and to be processed by cell wall enzymes such as pectin methylesterases. RGII is composed of a backbone of HG that is substituted by four conserved extreme heteropolymeric side chains, and acts as a point of HG cross linking through its dimerization in the presence of boron. RGI is a diverse and heterogeneous set of polymers. A core feature of RGI is a backbone consisting of a repeating unit of a galacturonosyl–rhamnosyl disaccharide, in which the rhamnose residues can be substituted with side chains rich in arabinose and/or galactose. The degree of polymerization of arabinan, galactan and arabinogalactan side chains may vary from 1 up to ∼50 residues (Ridley et al., 2001; Mohnen, 2008). The covalent links between these pectic polymers within the primary cell wall matrix, i.e. whether HG is exclusively associated in a linear way with RGI, or whether the RGI backbone acts as a basis for the attachment of HG as a side chain in a manner equivalent to neutral side chains, is still a matter of debate (Vincken et al., 2003; Coenen et al., 2007; Verhertbruggen and Knox, 2007; Mohnen, 2008).

Pectic arabinogalactans are split into two types. Arabinogalactan I (AGI) consists of a 1,4-β-linked d-galactan backbone with occasional interspersed 1,3-β-links (Hinz et al., 2005), and can be substituted at O-3 or O-2 with α-l-arabinose residues, and may contain other sugars such as fucose and glucuronic acid (Ridley et al., 2001; Øbro et al., 2004). Arabinogalactan II (AGII) has a 1,3-β-linked d-galactan backbone substituted by numerous short side chains consisting of 1,6-β-linked d-galactan, and also arabinosyl and uronosyl residues. Pectic arabinan chains are composed of 1,5-α-linked l-arabinofuranosyl residues, and these can be branched at O-2 or O-3 by single arabinosyl residues or short side chains (Beldman et al., 1997; Ridley et al., 2001; Mohnen, 2008). 1,5-Linked arabinan structures may also exist as free polymers unattached to pectic domains (Beldman et al., 1997; Ridley et al., 2001).

The neutral sugar side chains associated with RGI are of considerable complexity and diversity, and their functions remain obscure. Within the context of cell walls and other cell wall polymers, arabinans and (arabino)galactans have been identified as highly flexible and mobile polymers that may maintain ‘pores’ in cell walls through interactions with water (Fenwick et al., 1999; Renard and Jarvis, 1999; Ha et al., 2005). In contrast, other work has indicated that in certain cell walls arabinan can be found in close association with cellulose (Vignon et al., 2004), and that pectin neutral side-chains may enable cross links to cellulose microfibrils through hydrogen bonding (Zykwinska et al., 2005, 2007a,b). Arabinans, in certain species of the Amaranthaceae, which includes sugar beet, can be cross linked through ferulic acid links (Levigne et al., 2004a,b; Ralet et al., 2005). In these diverse ways arabinans have the potential to contribute to cell wall properties in terms of matrix porosity as well as mechanical properties. A range of experimental approaches have implicated pectic arabinan in mechanical properties, and their presence has been shown to be required to maintain the flexibility of guard cell walls (Jones et al., 2003, 2005). The genetic manipulation of arabinan levels in RGI has been shown to have severe impacts upon plant development (Oomen et al., 2002; Skjøt et al., 2002). In addition, pectic arabinans are implicated in cell adhesion events in a range of plant systems (Iwai et al., 2001; Orfila et al., 2001; Leboeuf et al., 2004; Peña and Carpita, 2004; Devaux et al., 2005).

There is some understanding of the molecular machinery involved in the synthesis of arabinans (Harholt et al., 2006; Konishi et al., 2006; Egelund et al., 2007), and some elements of the enzymatic machinery involved in arabinan dynamics or processing in cell walls are currently being characterized (Chávez Montes et al., 2008). To understand the functional implication of the presence of a specific polymer in a cell wall, methods are required to detect the polymer in situ in relation to cell status, cell development or cell type. One of the best current ways to detect polysaccharides is by using molecular probes, such as antibodies and carbohydrate-binding modules (CBMs). LM6 is a monoclonal antibody that was generated using a neoglycoprotein immunogen, in which a heptasaccharide of 1,5-arabinan was coupled to BSA (Willats et al.,1998). This antibody has been used to detect arabinans in cell walls in a range of systems: it recognizes arabinan in pectins, and, in certain cases, recognizes 1,5-linked arabinosyl residues in arabinogalactan-proteins (AGPs; e.g. Willats et al., 1999; Bush et al., 2001; Lee et al., 2005; Chávez Montes et al., 2008). Here, we describe the generation and characterization of three further monoclonal antibodies that bind effectively to isolated samples of arabinan. We show that two of these monoclonal antibodies (LM13 and LM16) identify novel epitopes present in pectic arabinan samples that are quite distinct from the LM6 arabinan epitope. The presence of the epitopes in arabinan samples can be modulated in vitro using arabinofuranosidases. The distinct epitopes have diverse developmental occurrences in the cell walls of organs in a range of species. The use of this panel of antibodies provides a more nuanced understanding of arabinan occurrence and processing in cell walls, and also provides insight into the wider functional roles of arabinans in the context of cell wall biology.

Results

Comparative immunochemical analysis of four rat monoclonal antibodies directed to pectic arabinans

Four rat monoclonal antibodies that bind to samples of pectic 1,5-α-linked l-arabinan were assembled for this study. The isolation of LM6 subsequent to immunization with a 1,5-α-l-arabinofurano-heptasaccharide-BSA conjugate has been described previously (Willats et al., 1998). Monoclonal antibody LM13 was isolated subsequent to immunization with a cation-chelator-soluble extract of Arabidopsis seedlings, and the preliminary characterization of this probe using glycan microarrays has indicated the recognition of a pectic arabinan epitope (Moller et al., 2007). Monoclonal antibodies LM16 and LM17 were isolated subsequent to immunization with an isolate of RGI derived from apple fruit, designated MHRB, which has been described in detail by Verhoef et al. (2008). Using ELISAs, LM16 and LM17 were found to bind strongly to samples of pectic arabinan and related polymers.

Three samples of arabinan polysaccharides derived from sugar beet pectin were available for this study: branched arabinan, de-branched arabinan and a more purified linear arabinan sample (97.5% arabinose). All four monoclonal antibodies generated a signal with the de-branched/linear arabinan samples when hybridoma supernatants were diluted by more than 100-fold. At this dilution, no binding to a control polysaccharide, larch arabinogalactan, was detected. Figure 1(a) shows ELISAs of LM6, LM13, LM16 and LM17 binding to branched and de-branched arabinans. LM6, LM13 and LM17 bound to both samples in a similar way, whereas LM16 binding was distinct in that it had a strong preference for the de-branched sample. To explore the recognition of the arabinan samples further, the sensitivity of antibody binding to these antigens after treatment with arabinan-degrading enzymes was studied (Figure 1a). Immobilized branched and de-branched arabinans were treated with either 10 μg ml−1 of an endo-arabinanase (pH 4.0) (Proctor et al., 2005) or 50 μg ml−1 of an Aspergillus nigerα-l-arabinofuranosidase (pH 4.0) (Beldman et al., 1993). The binding of LM6, LM13 and LM17 was reduced by the action of these enzymes, with antibody signals being <50% of the no-enzyme control in all cases. The LM13 epitope was particularly sensitive to enzyme action (Figure 1a). In contrast, the binding of LM16 to a sample of the branched arabinan was increased considerably by the action of arabinofuranosidase, but not by the endo-arabinanase.

Figure 1.

 Monoclonal antibody binding to arabinan samples and sensitivity to enzyme action.
(a) ELISAs of monoclonal antibodies LM6, LM13, LM16 and LM17 binding to branched and de-branched samples of sugar beet arabinan, and effect of pre-treatments with an endo-arabinanase and an arabinofuranosidase. Antibodies were used to provide 90% of the maximal binding to untreated samples. Error bars indicate the SEM of three replicates.
(b) Monoclonal antibodies LM6, LM13 and LM16 binding to a branched arabinan after titration of arabinofuranosidase action. Lines indicate means of three replicates (SEM < 0.1 absorbance units in all cases).

Arabinofuranosidase had no impact on the occurrence of the LM16 epitope in debranched arabinan, but the endo-arabinanase did reduce binding to this sample, although by <50% of the control. These observations confirm that LM6, LM13 and LM17 bind to arabinan, and that the binding is sensitive to arabinan-modifying enzymes to varying extents. In contrast, the LM16 epitope can be revealed in a sample of branched arabinan by the action of arabinofuranosidase. It was noted that in these assays that the arabinofuranosidase action that resulted in the appearance of the LM16 epitope resulted in the the reduction/loss of the LM6, LM13 and LM17 epitopes in the same sample. To further explore the relationships between the epitopes and arabinofuranosidase action, the enzyme concentration was titrated for a 1-h incubation period with branched arabinan, and the epitope occurrence was subsequently determined by ELISA, as shown in Figure 1(b). The LM16 epitope appeared concurrently with the loss of the LM13 and LM6 epitopes. It was also noted that mid-range concentrations of arabinofuranosidase (∼0.1–1 μg ml−1) increased the detection of the LM13 and LM6 epitopes in the branched arabinan sample prior to the loss of binding effected by higher levels. Up to ∼1 ug ml−1, the arabinofuranosidase is presumably optimally hydrolysing the α1,3-linked and α1,2-linked arabinosyl side chains that are present in sugar beet arabinan, and at higher concentrations has impact on the 1,5-linked backbone. The A. niger arabinofuranosidase is ∼50-fold less active against α1,5-linked arabinosyl residues than α1,2- or α1,3-linked arabinosyl residues.

As the titration of arabinofuranosidase concentration provided insight into the potential for epitope formation/loss within arabinan samples, this strategy was extended. The branched arabinan and linear arabinan samples were subject to similar titration experiments with two, more selective, arabinofuranosidases that have amino acid sequences placing them in the glycosyl hydrolase family 43 (GH43) and GH51 of the CAZy data base (Henrissat, 1991; Cantarel et al., 2009). The GH51 arabinofuranosidase from Cellvibrio japonicus (formerly Pseudomonas cellulosa) is ∼300-fold less active against α1,5-linked arabinosyl residues than α1,2- or α1,3-linked arabinosyl residues (Beylot et al., 2001). The GH43 arabinofuranosidase from Bacteroides thetaiotaomicron displays ∼1000-fold less activity towards the 1,5-backbone than the side chains (LM and HJG, unpublished observations). The results, shown in Figure 2, indicate that the LM6 and LM17 epitopes have similar patterns of epitope dynamics in these in vitro assays, in that GH51 leads to an increase in epitope abundance in branched arabinan, and then leads to their loss at the highest concentrations. GH51 also led to the loss of the epitopes from linear arabinan samples. By contrast, the action of the GH43 enzyme elevated the epitope levels in the branched arabinan sample to a level seen for linear arabinan, with no loss of epitopes at the higher concentrations. The LM13 epitope was increased in occurrence in branched arabinan samples by both GH43 and GH51 to the level seen in linear arabinan or above, with minimal loss at the highest concentrations. The LM16 epitope was effectively generated in branched arabinan, and levels were elevated in linear arabinan by GH51, but not by GH43.

Figure 2.

 Monoclonal antibody binding to arabinan samples, and sensitivity to action of glycosyl hydrolase family 43 (GH43) and GH51 arabinofuranosidases. Monoclonal antibodies LM6, LM13, LM16 and LM17 binding to branched arabinan (BA, black lines) and linear arabinan (LA, blue lines) after pre-treatment incubation with GH43 (solid lines) and GH51 (dashed lines) arabinofuranosidases (with a 10-fold dilution titration of enzyme concentrations from 0.1 ng ml−1 to 100 μg ml−1). Lines indicate the means of three replicates (SEM < 0.1, absorbance units in all cases).

Competitive inhibition ELISAs, using a range of linear 1,5-α-l-arabinofurano-oligosaccharides with degrees of polymerization (DP) up to 11 were used for a comparative analysis of the LM6, LM13, LM16 and LM17 epitopes. Hybridoma supernatants were used at dilutions generating 90% or less of maximal binding, and de-branched sugar beet arabinan was used as the immobilized antigen. The capacities of 1,5-α-l-arabinofurano-oligosaccharides to inhibit antibody binding are shown in Table 1. For LM6 and LM17, the 1,5-α-l-arabinofurano-hexasaccharide was the most effective inhibitor, but smaller DP oligosaccharides were also effective, with the dimer being about 24 times less effective than the hexamer for LM6. For LM17, the dimer was over 140 times less effective as an inhibitor than the hexamer oligosaccharide (Table 1). In the case of LM13, all oligosaccharides from DP5 up to DP11 were similarly effective in inhibiting binding, with oligosaccharides with four or fewer residues not being effective inhibitors at a concentration of 1 mg ml−1. None of the tested 1,5-α-l-arabinofurano-oligosaccharides up to DP11 had any impact on LM16 binding to debranched arabinan at a concentration of 1 mg ml−1. A comparison of the quantity of linear arabinan required to inhibit binding by 50% in competitive inhibition assays (Table 1) indicated that for LM6 and LM17 the concentration required was in the region of 100-fold more than the hexamer, i.e. 32 and 345 μg ml−1, respectively. Linear arabinan at 1 mg ml−1 had no impact on the binding of LM13, whereas LM16 binding was effectively inhibited by 72 μg ml−1 linear arabinan (Table 1).

Table 1.   The capacity of arabino-1,5-saccharide haptens and a linear arabinan polymer to inhibit the binding of arabinan-directed monoclonal antibodies LM6, LM13, LM16 and LM17 using competitive-inhibition ELISAs
Hapten/polymer (molecular weight)LM6LM13LM16LM17
  1. The concentrations (μg ml−1) of arabino-saccharides/arabinan required to inhibit binding by 50% are presented.

Ara2 (282)6.77>1000>1000542.0
Ara3 (414)0.84>1000>100010.2
Ara4 (546)0.76>1000>10005.8
Ara5 (678)0.51544>10004.5
Ara6 (810)0.28418>10003.8
Ara7 (942)0.54458>10004.9
Ara8 (1074)0.70483>10006.6
Ara9 (1206)0.73419>10004.9
Ara10 (1338)0.95553>10007.1
Ara11 (1470)0.55405>10007.7
Linear arabinan (∼18 000)32.40>100072345.0

In summary, LM6 and LM17 bind to similar 1,5-α-linked l-arabinan epitopes, with LM6 having a greater relative recognition of the 1,5-α-l-arabinofurano-disaccharide. Observations with the more selective arabinofuranosidases indicate that the LM6 and LM17 epitopes are likely to be present in the 1,5-linked arabinan backbone (and possibly the short side chains) removed by GH51 (and not by GH43), thereby leading to the reduction in epitope abundance. LM13 binds to a longer highly arabinanase-sensitive epitope of 1,5-α-linked l-arabinan, encompassing more residues than LM6 or LM17, and may be a conformational epitope arising from the de-branching of arabinan and the specific removal of side chains. Higher levels of GH51 did not lead to a significant loss of the epitope. LM16 binds to a feature of the linear arabinan sample that is not found in 1,5-α-l-arabinofuranosides, and appears to recognize a structural feature of arabinan/RGI arising from arabinofuranosidase action. The LM16 epitope can be generated by arabinofuranosidases that are likely, at high concentrations, to degrade the 1,5-linked backbone. Sugar beet arabinan chains can be attached to RGI backbones through a single galactosyl residue or short galactan sequences (Sakamoto and Sakai, 1995; Ralet et al., 2009). To test whether this feature contributed to LM16 recognition, linear arabinan was treated with a β-galactosidase, and this was found to specifically reduce the occurrence of the LM16 epitope, and to have no impact on the LM6/17 and LM13 epitopes, as shown in Figure 3. LM16 therefore appears to bind to the galactosyl stub on the RGI backbone that is revealed by the action of certain arabinofuranosidases.

Figure 3.

 LM16 monoclonal antibody binding to linear arabinan is sensitive to β-galactosidase action. ELISA detection of monoclonal antibodies LM6, LM13 and LM16 binding to linear arabinan after a 1-h pre-treatment with β-galactosidase. Assay absorbance values for untreated antigen were set to 1.0 for all antibodies. Values indicate means of three replicates (SEM < 0.03 units in all cases).

Comparative analysis of the presence of the LM6, LM13, LM16 and LM17 epitopes in plant cell walls

To study the occurrence of the LM6, LM13, LM16 and LM17 epitopes in cell walls, the monoclonal antibodies were used in the immunofluorescence detection assays of a range of plant materials. Arabidopsis thaliana is a useful model system for the study of cell wall biology, and its seedling roots are amenable to a direct immersion immunofluorescence technique. The occurrence of the LM5 galactan epitope at the root surface varies in response to cell development, and to genetic and hormonal factors (McCartney et al., 2003). The LM6 arabinan epitope has previously been shown to occur abundantly in meristems relative to more mature tissues (Willats et al., 1999; Bush et al., 2001). The immunolabelling of intact arabidopsis seedling roots grown for 7 days indicated that the four epitopes had distinct locations at the root surface, as shown in Figure 4. The LM6 epitope was abundant at the root apex, with a decrease in binding in proximal regions. The LM13 epitope was restricted to the root cap cells, but only in ∼35% of root apices, and was absent from the others. The LM16 epitope was detected over the most distal region of all root apices, although its distribution was not as extensive as that of LM6. In all cases, at a region ∼430 μm from the root apex, the LM16 epitope was detected with low abundance in a transient band that related to the onset of cell elongation (McCartney et al., 2003). The LM17 epitope was detected in a pattern similar to LM6, although it showed less recognition of the root cap cells (Figure 4).

Figure 4.

 Indirect immunofluorescence detection of LM6, LM13, LM16 and LM17 arabinan-related epitopes at the surface of intact Arabidopsis seedling roots. Arrowheads indicate the distal extent of root apices. The boxed area in the LM16 micrograph indicates a region of immunofluorescence revealed by longer exposure time. Scale bars = 100 μm.

The binding of the antibodies to transverse sections of inflorescence stems of Arabidopsis seedlings also indicated differences in epitope occurrence (Figure 5). The LM6 and LM17 epitopes were abundant in all cells, and were particularly abundant in epidermal cell walls, and were also associated with the ring of vascular cells/interfascicular fibres, and often appeared to fill the lumen of these cells. The LM13 epitope was generally specific to epidermal cell walls, but was, in some cases, detected to be present at low levels in cell walls surrounding intercellular spaces in the cortical and pith parenchymas, as shown in Figure 5(e). The LM16 epitope was present in most cells, but in a distinct pattern. It was abundant in the thick cell walls of trichomes, and was associated with inner regions of xylem and fibre cells. In the pith parenchyma, the epitope was consistently associated with adhered regions of parenchyma cell walls, and was absent from cell wall regions around the intercellular spaces, and was thus present in a complementary pattern to the LM13 epitope (Figure 5d,f).

Figure 5.

 Indirect immunofluorescence detection of the LM6, LM13, LM16 and LM17 arabinan-related epitopes in transverse sections of an Arabidopsis inflorescence stem.
(a) LM6 binds to most cell walls.
(b) LM13 binding was restricted to epidermal cell walls.
(c) LM16 binds to most cell walls, including the lumen of fibres.
(d) LM17 binds in a similar manner to LM6, although it binds more weakly to the fibre cells.
(e) In certain cases, higher magnification analysis revealed that LM13 binds weakly to cell walls lining the intercellular spaces of cortical and pith parenchyma cells.
(f) In the pith parenchyma, the LM16 epitope was restricted to regions of adhered cell walls, and was absent from cell wall regions surrounding intercellular air spaces.
(g) In fibre cells the LM16 epitope was abundant in the inner regions of secondary cell walls and often in the lumen. Arrows indicate the outer surface of the epidermis. Doubleheaded arrows indicate epitopes present in the fibre lumen. Paired, facing arrows indicate the LM16 epitope in regions of adhered cell walls. Asterisks indicate parenchyma intercellular spaces, and the presence of the LM13 epitope in surrounding cell walls. Calcofluor binding to all cell walls shown in blue (e–g). Scale bars: (a–d), 100 μm; (e–g), 20 μm).

Comparative analysis of the four arabinan-related epitopes, in transverse sections of tobacco stems, indicated patterns of occurrence in relation to tissues and cell types that were distinct from those observed in the Arabidopsis stem (Figure 6). In this case, LM6 bound strongly to primary cell walls of the cortical and pith parenchymas, and LM17 and LM13 did the same, only generally more weakly. The LM13 epitope was abundant at intercellular junctions in cortical parenchyma (Figure 6c). In contrast, the LM16 epitope was not detected in cortical or pith parenchyma, but was restricted to two major subsets of cells. This epitope was detected in files of cells of the xylem tissue with secondary cell walls that were generally adjacent to the larger xylem vessels (Figure 6d,g). LM6 also bound weakly to similar subsets of cells, with secondary cell walls in the xylem tissue (Figure 6f). In addition, the LM16 epitope was detected in specific cells, likely to be sieve elements, of the external and internal phloem tissues. These cells were distinct from the phloem fibres, as detected by the LM11 xylan monoclonal antibody (that binds to xylan in all secondary cell walls) in an equivalent section (Figure 6e).

Figure 6.

 Indirect immunofluorescence detection of the LM6, LM13, LM16 and LM17 arabinan-related epitopes in transverse sections of the tobacco stem.
(a) LM6 binds to all primary cell walls of the epidermis, and cortical and pith parenchymas, and also weakly binds to xylem tissue.
(b) LM17 is bound in a similar pattern to LM6, but more weakly.
(c) LM13 is bound in a similar pattern to LM6, but the epitope was abundant at cell junctions in the cortical parenchyma.
(d) The LM16 epitope was restricted to two sets of cells: subsets of xylem fibres (asterisks) and phloem cells (arrows).
(e) The LM11 xylan epitope was bound to all xylem vessels/fibres and phloem fibres (doubleheaded arrows).
(f) At higher magnification, LM6 was also observed to bind to the files of xylem fibre cells between xylem vessels, in the manner observed for LM16 (g). Abbreviations: cp, cortical parenchyma; e, epidermis; pp, pith parenchyma; x, xylem. Calcofluor binding to all cell walls shown in blue (f, g). Scale bars: 50 μm.

The LM16 epitope was generated in vitro by arabinofuranosidase action. To study the capacity for enzymatic generation of the LM16 epitope in cell walls, tobacco stem sections were incubated with the A. niger arabinofuranosidase prior to immunolabelling procedures. Arabinofuranosidase pre-treatment resulted in the appearance of the LM16 epitope in primary cell walls, and this was most notable in the cortical region of the stem sections (Figure 7). The LM16 epitope was not evenly distributed across cell walls, but was particularly abundant in inner cell wall regions surrounding collenchyma thickenings, and also in regions of cell walls that were adhered to adjacent cells. This pattern of in muro-generated LM16 epitope occurrence reflected the LM16 epitope occurrence in the pith parenchyma of the Arabidopsis stem (Figure 5).

Figure 7.

 Indirect immunofluorescence detection of the LM16 epitope in transverse sections of the tobacco stem after pre-treatment with arabinofuranosidase.
(a) No enzyme pre-treatment. LM16 binds to xylem fibres and phloem cells.
(b) After arabinofuranosidase pre-treatment, LM16 binds to primary cell walls of the cortical parenchyma (cp), and is often found in abundance in adhered regions between adjacent cells (paired facing arrows).
(c) At a higher magnification of the arabinofuranosidase pre-treated sections, the LM16 epitope, highlighted by the Calcofluor staining of all the cell walls (blue), was abundant in the inner regions of cell walls around collenchyma thickenings (arrowheads), and in regions of adjacent adhered cell walls (paired facing arrows). Abbreviations: p, phloem; x, xylem. Scale bars: 20 μm.

The varying patterns of epitope occurrence in Arabidopsis and tobacco led us to study the occurrence of these epitopes in the taxonomically distinct, early evolving fern Equisetum ramosissimum (Figure 8). In transverse sections of the stem of this species, the LM6 epitope was abundant in most cell walls, and the LM17 epitope had a similar distribution, although the epitope was notably less abundant in the cell walls of the sclerenchyma fibre bundles forming the stem ridges (Figure 8c). The LM13 epitope was restricted to two locations: the outer surface of epidermal cell walls of the stem ridges proximal to the sclerenchyma bundles (Figure 8d), and cell walls of the stomatal complexes (Figure 8d,g, see below). The LM16 epitope also had a restricted distribution, and was detected in cells of the vascular bundles distal to the carinal canals, and also in certain cell walls of the stomatal complex (Figure 8e,h). Equisetum species have a complex double guard cell system at stomatal complexes (Pant and Kidwai, 1968; Dayanandan and Kaufman, 1973). Stomatal pores occur in pairs, and, in addition to guard cells, have distinctive distal subsidiary cells with thickened cell walls adjacent to the guard cells. The LM6 epitope (Figure 8f) and the LM17 epitope (not shown) had a similar distribution, and were present in cell walls of both guard cells and subsidiary cells, with a particular abundance in the thickened cell walls of the subsidiary cells. In contrast, the LM13 epitope was restricted to the subsidiary cell walls, and mostly to the cell wall thickening (Figure 8g). The LM16 epitope, when present, had a consistent complementary pattern of occurrence, and was restricted to both guard cells of one of the pair of stomata found at a stomatal complex (Figure 7h).

Figure 8.

 Indirect immunofluorescence detection of the LM6, LM13, LM16 and LM17 arabinan-related epitopes in transverse sections of the stem of Equisetum ramosissimum.
(a) Bright-field image of a region of the Equisetum stem with two sclerenchyma ridges, showing the anatomy. Abbreviations: cc, carinal canal; e, epidermis; p, parenchyma; s, sclerenchyma; sc, stomatal complex; vb, vascular bundle; vc, vallecular canal. The arrowhead indicates the distal edge of the sclerenchyma ridge, and the dotted line indicates the anatomical axis from the ridge through to the vascular bundle.
(b) Equivalent section immunolabelled with LM6, showing binding to the epidermis, cortical parenchyma and vascular bundles. The inset shows a higher magnification micrograph of LM6 binding to epidermal and sclerenchyma fibre cell walls.
(c) The equivalent section showing LM17 binding. The inset shows LM17 binding to epidermal cell walls, but only weak binding to sclerenchyma fibre cell walls.
(d) Stem section with one sclerenchyma ridge, showing LM13 binding to the outer edge of the epidermis at the ridge (inset), and also to the stomatal complexes.
(e) Equivalent section to (d), showing the LM16 epitope restricted to stomatal complexes and the vascular bundle.
(f) LM6 binding to the subsidiary and guard cell walls of the stomatal complex; g, guard cell; sg, subsidiary guard cell. The doubleheaded arrow indicates a thickened cell wall of a subsidiary cell. The arrow indicates the cell wall of a guard cell. The dotted arrows indicate stomatal openings.
(g) Equivalent section to (f), showing the LM13 epitope in the cell wall thickenings of subsidiary cells.
(h) Equivalent section showing the LM16 epitope occurrence in one pair of guard cells at a stomatal complex. Scale bars: (a–e) 150 μm; insets in (b–d) 50 μm; (f–h) 20 μm.

Discussion

Novel antibodies for the detection of 1,5-arabinan and related epitopes

Molecular probes directed to structural features of plant cell wall polysaccharides are extremely important tools for placing polysaccharide structures and structural variants in cell biological contexts. Here, we report on the characterization of three rat monoclonal antibody probes directed to features of 1,5-linked arabinans that complement the known specificity of existing rat monoclonal antibody LM6 (Willats et al., 1998).

LM17.  In most assays, LM17 binds in a similar way to LM6, although it shows less relative binding to the 1,5-α-l-arabinofuranodisaccharide, and shows some indications of subtly altered binding to plant materials, including reduced recognition of the most distal cells of intact Arabidopsis root apices, and the subepidermal sclerenchyma cells forming the stem ridges of E. ramosissimum.

LM13.  This antibody binds to a specific subset of pectic arabinans, and to longer stretches of 1,5-linked arabinosyl residues that are likely to be more abundant in unbranched arabinans. The binding of LM13 is more sensitive to arabinanase action than the LM6/LM17 epitopes. The processing of a branched arabinan by the selective removal of side chains by arabinofuranosidases can lead to the formation of the LM13 epitope. It is possible that the LM13 epitope is conformational, and is a specific feature of extended arabinan chains.

LM16.  The LM16 epitope is shown, both in vitro and in muro, to be a product of arabinofuranosidase action. The sensitivity of LM16 binding to β-galactosidase action suggests that LM16 recognizes a galactosyl residue/galactan stub on the RGI backbone that is unmasked by the enzymatic removal of the arabinan sequences. The presence of a single galactosyl residue or short sequence of galactosyl residues between arabinan chains and the RGI backbone has been identified for sugar beet and other pectins (Lau et al., 1987; Sakamoto and Sakai, 1995; Nakamura et al., 2002; Zheng and Mort, 2008).

The epitopes bound by this panel of antibodies reflects the diverse structural features of the complex sets of cell wall polymers that are arabinans. It is well established from structural analyses that arabinans are structurally complex and diverse (Beldman et al., 1997; Oosterveld et al., 2000; Mohnen, 2008). The LM6/LM17 epitopes are short arabinosyl sequences, and are generally abundant in plant cell walls. These epitopes may appear in 1,5-arabinan backbones, and also in side chains. The LM13 epitope appears to be a conformational state of arabinan that is prevalent in arabinan backbones and influenced by the extent of arabinan branching. The detection of the LM16 epitope is also influenced by branching status, and the epitope can be unmasked by arabinofuranosidase action, and lost by β-galactosidase action. These two new monoclonal antibodies bind to epitopes that therefore reflect two differing aspects of arabinan processing in cell walls: highly branched arabinan structures can be recognized by LM6/LM17, linearized arabinan can be specifically detected by LM13 and degraded arabinans can be detected by LM16. It is also likely that LM16 will recognize galactosyl residue(s) on RGI backbones that are present independently of capping by arabinan chains.

A further question concerns the nature of the arabinan-containing antigens detected by these antibodies when binding to plant materials, and whether the antibodies are binding to pectic arabinan, galactan structures decorated with sequences of 1,5-linked arabinosyl residues or possibly, in some cases, AGPs. AGII structures also form the glycan component of AGPs, which are a complex set of proteoglycans that are abundant at cell surfaces, and are often found in association with plasma membranes (Seifert and Roberts, 2007). There is accumulating evidence that AGPs can covalently attach to RGI (Immerzeel et al. 2006); whether all pectin-associated AGII is likewise attached, by virtue of association with AGPs, is not yet clear. Monoclonal antibody LM6 directed to an epitope of 1,5-arabinan that is found abundantly in pectic arabinans has been found to bind effectively to AGPs from the bryophyte Physcomitrella patens (Lee et al., 2005), thereby indicating the presence of some 1,5-linked arabinosyl residues in this particular set of AGPs, and confirming the possible commonality of epitopes between RGI and AGPs. The discussion of cell binding patterns below indicates that the expression of these epitopes is highly complex in relation to cell development, cell types and taxonomy. In future analyses, antigen identity will need to be determined on an individual basis for each immunocytochemistry study using complementary methodologies. It is possible that the complex patterns relate to structurally diverse antigenic contexts.

Detection of arabinan and related epitopes in plant cell walls

We present data on the occurrence of the LM6, LM13, LM16 and LM17 epitopes in four organ systems (a root and three stems) from three species. This is a limited immunocytochemical study, but clearly demonstrates that the LM6 and LM17 epitopes display similar (although not always identical) patterns of occurrence, which reflects the in vitro analyses of epitope structure. These epitopes are generally abundant in the cell walls of the organs examined, reflecting their potential recognition of short arabinan epitopes. In contrast, the LM13 and LM16 epitopes, reflecting subsets of processed arabinans, show varied and more restricted patterns of occurrence. The important observation from these studies is that the LM13 and LM16 epitope patterns in the three stems, Arabidopsis, tobacco and Equisetum, are distinct in terms of the cell types recognized. The LM13 epitope that is detected in most primary cell walls in the tobacco stem is restricted to epidermal cells in the Arabidopsis stem, and to an epidermal secretion and subsidiary guard cells in the Equisetum stem. The LM16 epitope was detected in a similarly contrasting set of cell locations in the three stems, and this was entirely distinct from the LM13 epitope patterns.

At the level of individual cell walls, two complementary occurrences of this pair of epitopes will be highlighted here. Firstly, in Arabidopsis pith parenchyma, the LM16 epitope was detected specifically in the adhered regions of cell walls, and the LM13 epitope in cell wall regions lining intercellular spaces, i.e. separated cell walls. This in situ modulation of epitopes in relation to cell adhesion supports biochemical evidence for arabinan functioning in this cell process (Iwai et al., 2001; Orfila et al., 2001; Leboeuf et al., 2004; Peña and Carpita, 2004; Devaux et al., 2005). Secondly, in the stomatal complex of Equisetum, the LM13 epitope is present in subsidiary cells, and the LM16 epitope is present in certain guard cells. Again, this reflects work that has demonstrated that pectic arabinans are essential to maintain guard cell flexibility and stomatal opening/closure in a wide range of species (Jones et al., 2003, 2005). The observations reported here indicate that arabinan processing is an aspect of arabinan function(s) in these processes.

Taken together, these observations suggest that arabinans and their processing are highly dynamic features of plant cell walls underpinning diverse aspects of cell wall properties, and that arabinan polysaccharides are not a cell wall feature tightly associated with a single cell differentiation or developmental pathway. Insights with these antibodies indicate that arabinans with abundant levels of the LM13 epitope are linearized, and that an abundance of the LM16 epitope indicates arabinans processed further by arabinofuranosidsase action. Observations reported here indicate that arabinan structure/processing is probably involved in the local control of cell wall mechanical properties, in a range of cell contexts in plants. Diverse lines of evidence increasingly implicate RGI, a major polysaccharide context for arabinans, in the regulation of cell wall mechanical properties (McCartney et al. 2000; Jones et al., 2003; Ulvskov et al., 2005). An integration of the knowledge of cell wall microstructures with a biomechanical understanding of plant cell walls and of plant organs is a significant gap in current plant biology. LM13 and LM16 are therefore important antibody tools that can be used to provide insight into arabinan structure, processing and function in plant cell walls.

Experimental procedures

Immunization, hybridoma preparation and monoclonal antibodies

The isolation of rat monoclonal antibodies LM6 and LM13 has been described previously (Willats et al., 1998; Moller et al., 2007). Rat monoclonal antibodies LM16 and LM17 (immunoglobulin class IgM) were selected after immunization with a modified RGI polymer of apple pectin MHRB, with the following molar percentages of the major sugars: GalA, 37%; Xyl, 18%; Gal, 20%; Rha, 11%; Ara, 11% (Moller et al., 2007;Verhoef et al., 2008). The immunization of rats, and the cell fusion, isolation of MHRB-directed antibodies by ELISAs and cloning of hybridoma cell lines, were carried out as previously described (Willats et al., 1998). Anti-xylan monoclonal antibody LM11 was isolated as described by McCartney et al. (2005). All monoclonal antibodies were used in the form of hybridoma cell culture supernatants.

Polysaccharides, oligosaccharides, enzymes and immunochemical assays

Branched, de-branched and a more purified ‘linear’ arabinans derived from sugar beet pectin and α-(1→5)-l-arabino-oligosaccharides, with degrees of polymerization from 2 to 11, were obtained from Megazyme (http://www.megazyme.com). Microtitre plate-based ELISAs were used for the screening of antibody recognition of apple pectin MHRB, and for the study of the antibody recognition of arabinans and arabino-oligosaccharides, using procedures described by Marcus et al. (2008). Arabinan samples were coated onto microtitre plates for assays at 50 μg ml−1. The enzymes used to explore epitope structures included an α- l-arabinofuranosidase and a β-galactosidase from A. niger (Megazyme), a family 43 endo-arabinanase, Arb43A from C. japonicus (Proctor et al., 2005), a family 51 arabinofuranosidase, Arf51A from C. japonicus (Beylot et al., 2001), and a family 43 arabinofuranosidase, Bt0369 from Bacteroides thetaiotaomicron (KL and HJG, unpublished data). Enzymes were incubated in microtitre plate wells for 1 h at room temperature (RT) (20°C), and then the plates were washed three times in water before starting the antibody detection ELISA protocol. Competitive-inhibition ELISAs were performed by serially diluting potential oligosaccharide haptens by fivefold from 1 mg ml−1 in phosphate-buffered saline containing 5% milk protein (PBS/MP) in microtitre plates coated with sugar beet branched arabinan for LM6, LM13 and LM17, and sugar beet de-branched arabinan for LM16 (50 μg ml−1). Each antibody was used at a dilution to provide 90% of the maximum binding, as determined by antibody-capture ELISA. When determining IC50 values, controls with no added potential inhibitor were taken as 0% inhibition, and no added primary antibody was taken as 100% inhibition.

Plant materials and immunofluorescence microscopy

Arabidopsis thaliana L. (Heynh.), ectoype Col-0, and tobacco (Nicotiana tabacum L) seedlings were grown as described previously (McCartney et al., 2003; Marcus et al., 2008). Samples of E. ramosissimum stems were provided by Olivier Leroux (Ghent University). Intact Arabidopsis seedling roots or sections of plant material were incubated in primary antibody diluted by fivefold in phosphate-buffered saline containing PBS/MP for 1.5 h. After washing with PBS, plant material was incubated with a 100-fold dilution of anti-rat IgG (whole molecule) linked to fluorescein isothiocyanate (FITC; Sigma-Aldrich, http://www.sigmaaldrich.com) in PBS/MP for 1.5 h, in darkness. After the washing material was mounted in a glycerol anti-fade solution (Citifluor AF1; Agar Scientific, http://www.agarscientific.com), an Olympus BX61 microscope (http://www.olympus.com), equipped with epifluorescence irradiation, was used to view the slides. Images were acquired with a Hamamatsu ORCA 285 digital camera using Volocity (Improvision, http://www.improvision.com).

Acknowledgements

We thank Fons Voragen and Peter Ulvskov for useful discussions on arabinan structure and cell walls, and Olivier Leroux for discussions of Equisetum stem anatomy. This work was supported by Marie Curie WallNet (MRTN-CT- 2004-512265) and Pecticoat (FP6-517036) EU Framework 6 research projects, and the UK Biotechnology and Biological Sciences Research Council.

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