• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Mar 21, 2006; 103(12): 4765–4770.
Published online Mar 14, 2006. doi:  10.1073/pnas.0508887103
PMCID: PMC1450244
Plant Biology

Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules


Glycoside hydrolases that degrade plant cell walls have complex molecular architectures in which one or more catalytic modules are appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs promote binding to polysaccharides and potentiate enzymic hydrolysis. Although there are diverse sequence-based families of xylan-binding CBMs, these modules, in general, recognize both decorated and unsubstituted forms of the target polysaccharide, and thus the evolutionary rationale for this diversity is unclear. Using immunohistochemistry to interrogate the specificity of six xylan-binding CBMs for their target polysaccharides in cell walls has revealed considerable differences in the recognition of plant materials between these protein modules. Family 2b and 15 CBMs bind to xylan in secondary cell walls in a range of dicotyledon species, whereas family 4, 6, and 22 CBMs display a more limited capability to bind to secondary cell walls. A family 35 CBM, which displays more restricted ligand specificity against purified xylans than the other five protein modules, reveals a highly distinctive binding pattern to plant material including the recognition of primary cell walls of certain dicotyledons, a feature shared with CBM15. Differences in the specificity of the CBMs toward walls of wheat grain and maize coleoptiles were also evident. The variation in CBM specificity for ligands located in plant cell walls provides a biological rationale for the repertoire of structurally distinct xylan-binding CBMs present in nature, and points to the utility of these modules in probing the molecular architecture of cell walls.

Plant cell walls are highly complex macromolecular structures that consist of repertoires of polysaccharides that interact with each other through extensive networks (1). With the annual recycling of an estimated 100 billion tons of plant biomass, the degradation of cell walls by microbial enzymes is an important biological and industrial process (2). The intimate association of polysaccharides within cell walls limits the access of hydrolytic enzymes that attack these composite structures, and thus the degradative process is relatively slow (3). To reduce the “accessibility problem,” plant cell wall hydrolases have a complex molecular architecture in which catalytic modules are appended to noncatalytic carbohydrate-binding modules (CBMs), which, by binding to specific polysaccharides, potentiate the activity of these enzymes against insoluble substrates such as cellulose, mannan, and xylan (4). CBMs are grouped into sequence-based families, although they have also been classified into “types” based on the topology of the binding site, which reflects the nature of the target ligand (4). The binding site of type A CBMs comprises a planar hydrophobic surface which recognizes crystalline polysaccharides such as cellulose, chitin and unsubstituted mannan. The binding site clefts of type B CBMs accommodate single chains of a variety of polysaccharides, whereas the spatial restriction of the binding site of type C CBMs limits ligand recognition to mono- or disaccharides. Although the ligand specificity of type A CBMs is highly conserved, the sugar polymers recognized by different members of type B families can be very diverse. For example, the CBM4 and CBM6 families both contain members that recognize xylan, β-1,4-glucan and β-1,3-glucan (58), whereas the CBM35 family contains proteins that, currently, have been shown to bind to xylan and mannan (9, 10).

One of the intriguing features of CBMs is the diversity of families that contain members which apparently display the same ligand specificity against purified polysaccharides and oligosaccharides. Thus, CBMs located in families 1, 2a, 3a, 5, and 10 bind to crystalline cellulose, whereas CBMs from families 2b, 4, 6, 15, 22, 35, and 36 all contain protein modules that recognize xylan (4). Recent in vitro studies have started to unravel the possible biological significance for the overlapping specificities displayed by the different CBM families. Carrard et al. (11) showed that Type A CBMs in families 1, 2a, and 3a could target different regions of purified crystalline cellulose, whereas McLean et al. (12), employing fluorescently tagged CBM4, CBM17, and CBM28, showed that these modules recognize distinct substructures within amorphous cellulose. However, these studies have been restricted to defining the specificities of CBMs for purified polysaccharides that have an invariant chemical structure of β-1,4-linked glucose moieties. The specificity of CBMs for polysaccharides embedded within plant cell walls, the substrate available to glycoside hydrolases during the initial rate limiting phase of the degradative process, is currently unknown.

Here we interrogate the ligand specificity of CBMs that recognize xylan, a β-1,4-linked xylose polymer, which can be decorated with glucuronosyl or 4-O-methyl-glucuronosyl residues at O2 and α-arabinofuranosyl or acetyl groups at O2 or O3, with the nature and extent of substitution varying between species and cell type (1, 13). We have selected six CBMs, located in families 2b, 4, 6, 15, 22, and 35, listed in Table 1, which display specificity for purified xylans (6, 10, 1417). The data presented in this study show that there is significant variation in the specificity of the different CBMs for plant cell walls, indicating that the structure and/or context of the xylans embedded in these composite structures is a critical specificity determinant for these protein modules. This variation in specificity for ligands located in cell walls provides a biological rationale for the repertoire of structurally distinct xylan-binding CBMs present in nature.

Table 1.
Origin of the xylan-binding CBMs used in this study

Results and Discussion

Specificity of CBMs for Isolated Polysaccharides.

In vitro binding studies using isothermal titration calorimetry (ITC) and affinity gel electrophoresis have shown that CBMs from several different families bind to purified xylans and xylooligosaccharides (refs. 6, 10, and 1419 and Table 2). For a summary of the composition of the xylans used in this study, see Table 3, which is published as supporting information on the PNAS web site. These xylan-binding CBMs are derived from a range of thermophilic and mesophilic prokaryotic xylanases (Table 1). CBM6, CBM15, and CBM22-2 do not distinguish between the largely undecorated oat spelt xylan (OSX; percentage molar composition: 93% Xyl, 6% Ara), xylohexaose, which contains no side chains, and the decorated xylans methylglucuronoxylan (MGX; 80% Xyl, 18% MeGlcA) and rye arabinoxylan (RAX; 50% Xyl, 50% Ara). However CBM4-2 binds considerably more tightly to xylohexaose than decorated xylans and even OSX, which contains a low density of side chains (Table 2). Although CBM2b-1-2 displays similar affinity for OSX as CBM4-2, it binds ≈5- and ≈10-fold less tightly to RAX and MGX, respectively. CBM35 displays no binding to arabinoxylan or methylglucuronoxylan, exhibiting a preference for an unsubstituted form of the polysaccharide. In addition, this module is unique in displaying no affinity for xylohexaose. The actual affinity of the six CBMs for purified xylans (Table 2) reflects their microbial origin; proteins from thermophilic bacteria bind more tightly to their target ligands (CBM4-2, CBM6, and CBM22-2) (6, 16, 17) than individual CBM2b modules, CBM15 or CBM35, which are derived from mesophilic prokaryotes (10, 15, 17). The relatively high affinity displayed by CBM2b-1-2 is due to the avidity effect of joining two CBMs together that bind to polysaccharides, which contain multiple protein binding sites (15, 20). Because CfXyn11A contains both CBM2b-1 and CBM2b-2, the avidity effect displayed by the CBM2b-1-2 construct also occurs in the multimodular enzyme from which they are derived.

Table 2.
Binding of CBMs to purified ligands as determined by ITC

The binding profile of the CBMs were also screened by using macroarrays in which the polysaccharides are arrayed on nitrocellulose filters. The specificity of the CBMs for the arrayed polysaccharides were similar to the ligand specificities determined by ITC (data not shown). Significantly, however, the macroarray screen revealed that CBM35 recognized a sample of glucuronoarabinoxylan (GAX; 51% Xyl, 30% Ara, 7% GlcA) derived from maize (21). The binding of CBM35 to maize GAX was quantified by ITC which indicated it had an affinity for GAX that was similar to OSX (Table 2).

To summarize, although the binding profile of CBM6, CBM15, and CBM22 to decorated xylans such as RAX and MGX and to the largely unsubstituted OSX and xylohexaose are broadly similar, CBM4, CBM35, and CBM2b-1-2 display significant variation in the specificity for these ligands. Although the binding profile of the CBMs for purified ligands reveals similarities and differences in the specificities of these protein modules, the biological significance of these in vitro data are unclear. To address this issue, we have used an immunohistochemical approach to interrogate the specificity of xylan-binding CBMs toward plant materials. This method exploits the antigenic recognition of the His-tag appended to these proteins as described (22).

Binding of Xylan-Binding CBMs to Dicotyledon Cell Walls.

The capacity of the xylan-binding CBMs to bind to cell walls in sections of plant materials from diverse taxonomic groups was assessed by using immunohistochemistry. In dicotyledons, low substituted xylans and MGX are abundant in the secondary cell walls of specific cell types such as xylem vessels and sclerenchyma fibers (1, 13, 23). The monoclonal antibody LM11, which binds to purified unsubstituted xylans and arabinoxylan, specifically recognizes secondary cell walls in all dicotyledons so far examined and shows no recognition of dicotyledon primary cell walls (23). In a survey of CBMs binding to sections of a range of plant materials CBM2b-1-2 shows a similar binding capability to LM11 and bound specifically to secondary cell walls, including those of xylem vessels and sclerenchyma fibers associated with phloem as shown in Fig. 1.

Fig. 1.
Indirect immunofluorescence microscopy of CBMs and monoclonal antibody LM11 binding to secondary cell walls of vascular tissues in transverse sections of tobacco, pea, and flax stems. Binding to phloem sclerenchyma fibers is indicated by arrows. X, binding ...

In contrast to CBM2b-1-2, however, the other xylan-binding CBMs (except CBM15, see below) showed significant differences in their capacity to bind to cell walls in these same three dicotyledon species when incubated with equivalent sections under the same conditions (Fig. 1). Each CBM bound effectively and specifically to secondary cell walls in at least one species, but was very weak or absent in others. CBM4-2 bound to xylem vessel cell walls of flax but not to the phloem fibers in this species and showed very weak/no recognition of secondary cell walls in tobacco and pea (Fig. 1). CBM6 bound effectively to secondary cell walls of both xylem vessels and phloem fibers in flax and xylem cells in pea, but very weakly to tobacco sections. CBM22-2 displayed the least recognition of secondary cell walls and bound effectively to pea stem but only weakly to walls of xylem vessels of flax and tobacco stems. The inability of certain CBMs to recognize xylan in some species is likely to reflect the context of xylan in different cell walls. Family 2b, 4, 6, and 22 CBMs can readily recognize isolated xylan polymers (although CBM4-2 does display a preference for completely unsubstituted ligands such as xylohexaose) and xylan is clearly present in all of the secondary cell walls in these materials as indicated by the binding of LM11 and CBM2b-1-2 (Fig. 1).

CBM15 and CBM35 were distinct from the other CBMs in that they bound to some dicotyledon primary cell walls. CBM15 bound to all secondary cell walls, but also recognized the primary cell walls in the flax section (Fig. 1) and some primary cell walls in tobacco stem (Fig. 2). CBM35 bound specifically to the secondary cell walls of pea stem, and to both primary and secondary cell walls of flax in a manner similar to CBM15 (Fig. 1). However, in tobacco, CBM35 did not bind to all secondary cell walls of xylem vessels but did interact with the primary cell walls of adjacent parenchyma, indicating clearly contrasting recognition of cell walls by this CBM in the three dicotyledon species. The variation in tobacco stem recognition displayed by CBM2b-1-2, CBM15, and CBM35 is shown for wider areas of tissues in Fig. 2, which demonstrates the secondary cell wall specificity of CBM2b-1-2 and the binding of CBM15 to primary cell walls in cortical parenchyma in addition to secondary cell walls. CBM35 displays a highly distinctive pattern of cell recognition with strong binding to epidermal cell walls and cells associated with regions of internal phloem. Xylans are known to be a taxonomically variable set of polymers, and biochemical analysis have indicated that primary cell walls of dicotyledons have low levels of xylans that are of the GAX type (24). The recognition of primary cell walls by CBM15 and CBM35 in certain species and cell types is likely to reflect low levels of GAX in these walls. This finding is consistent with affinity gel electrophoresis data showing that both CBM35 and CBM15 bind to GAX in vitro (data not shown).

Fig. 2.
Indirect immunofluorescence microscopy of CBM2b-1-2, CBM15, and CBM35 binding to transverse sections of tobacco stem showing cortical parenchyma (cp) and pith parenchyma (pp) in addition to vascular tissues. e, epidermis; p, phloem fibers; x, xylem vessels. ...

Binding of Xylan-Binding CBMs to Cell Walls of Graminaceous Monocotyledons.

Cell walls of the commelinoid monocotyledons, which includes the grasses and cereals, are distinctive in that abundant heavily substituted GAXs are major components of primary cell walls (13, 25). An additional distinctive feature of cereal grains is the abundance of neutral arabinoxylans in the endosperm.

The xylan-binding CBMs displayed differences in their capacity to bind to cell walls when incubated with sections of wheat grain and maize coleoptiles (Fig. 3). CBM2b-1-2, CBM4-2, CBM6, CBM15, and CBM22-2, but not CBM35, bound to wheat endosperm. This binding likely reflects the in vitro recognition of arabinoxylan by the CBMs. The CBMs showed differential recognition of the cell walls of the aleurone layer and the autofluorescent pericarp cell layers. CBM2b-1-2 and CBM15 bound to the aleurone cell walls, whereas the binding of CBM35 was restricted to a specific cell layer of the inner pericarp (Fig. 3).

Fig. 3.
Indirect immunofluorescence microscopy of xylan-binding CBMs interacting with sections of wheat grain and maize coleoptiles. Arrows indicate recognition of aleurone cell walls. Double arrowheads indicates CBM35 recognition of a cell layer of inner pericarp. ...

A transverse section of the maize coleoptile reveals central developing leaves surrounded by a sheath of parenchyma cells. CBM4-2 showed no recognition of cell walls in either of these organs (Fig. 3). CBM2b-1-2 and CBM35 bound to cell walls of developing leaves but not to those of the surrounding coleoptile sheath, whereas CBM6 and CBM22-2 bound to all cell walls in both leaves and sheath, although more effectively to cell walls of the developing leaves (Fig. 3). CBM15 displayed strong recognition of all primary cell walls in both sheath and leaves.

Topology of CBMs.

This study shows that three of the xylan-binding CBMs (CBM6, CBM15, and CBM22), which display similar specificities against purified ligands, exhibit considerable variation in their recognition of plant cell walls. Even more intriguing is the observation that although CBM4-2 exhibits tighter binding to purified xylans than CBM6, CBM15, and CBM22, its capacity to recognize the polysaccharide within plant cell walls is limited. By contrast, CBM2b-1-2, which has a similar binding profile to CBM4-2 for purified undecorated xylan (but binds less tightly to arabinoxylan and MGX), displays more extensive recognition of plant cell walls than the Rhodothermus marinus protein.

The differences in binding profiles of the CBMs in vivo are likely to reflect a combination of the accessibility of the target ligands within the cell wall composites and fine details of xylan structure. The CBMs most suited to bind xylans that are in complex macromolecular structures are predicted to be those that accommodate their respective ligand in shallow clefts, because the exposed binding sites are more likely to be able to interact with xylan chains that are intimately associated with other cell wall components. Inspection of the 3D structures of the five xylan-binding CBMs provides some insight into the influence of binding site topology on the access to xylan within cell walls (Fig. 4). Thus, the tandem CBM2b modules that comprise CBM2b-1-2, which binds effectively to all dicotyledon secondary cell walls, have a very shallow xylan binding site (15, 18), which places the protein modules at the cusp between type A CBMs, in which the ligand binding site is a planar hydrophobic surface, and type B CBMs, where single sugar polymer chains are accommodated in clefts (4). By contrast, the binding clefts of CBM4-2, CBM22-2 and CBM6, which display limited recognition of xylan embedded in plant cell walls, are much deeper than CBM2b-1-2 (6, 16, 26) (Fig. 4).

Fig. 4.
Three-dimensional structures of xylan-binding CBMs CBM2b-1-2, CBM4-2, CBM6, CBM15, and CBM 22-2. (Left) Top views of the binding cleft of each CBM. (Right) The same cleft viewed from the side. Bound oligosaccharide is shown in the clefts of CBM6 and CBM15. ...

However, the hypothesis that the depth of the binding cleft is related to the capacity of the CBM to bind to xylans embedded in the plant cell wall is less tenable when applied to CBM15. Although this protein binds to numerous cell walls, the dimensions of its xylan-binding site (17) are similar to CBM4-2, CBM6, and CBM22, which display limited recognition of secondary cell walls of dicotyledons. Thus, the capacity of CBMs to interact with cell walls is not exclusively dependent on the depth of the cleft. The binding clefts of CBM2b-1-2 and CBM15 have been described as twisted platforms where single aromatic amino acid side chains in the binding subsites stack only against the α faces of sugars in the polymer, in these cases sugars n and n + 2 (4, 15, 17, 18). In contrast, the binding sites of CBM4-2, CBM6, and CBM22 contain a primary binding subsite with two aromatic amino acid side chains that sandwich the same sugar ring, rather like “aromatic tongs,” thus interacting with both the α and β faces of a single sugar (6, 16, 26). Studies have shown that removal of either of these aromatic residues completely abrogates xylan binding in CBM2b, 6, 15, and 22 (6, 19, 27, 28). Within the complex milieu of the plant cell wall, it is likely that at least one face of the xylose residues in the xylan backbone interacts with other polysaccharides providing an explanation for why the CBMs that contain the pair of aromatic tongs do not bind to xylan when it is embedded in the plant cell wall. By contrast, it is likely that cell wall xylans will remain accessible to CBM2b-1-2 and CBM15 as only a single face of the xylose residues stack against the two tryptophans in the binding site of these proteins. Thus, we propose a model in which the capacity of CBMs to bind xylans that are intimately associated with other components of the cell wall depends on the topological arrangement of the aromatic residues that stack against the sugars in the polysaccharide backbone.

However, the structural basis for the different specificities of the CBMs toward cell walls is more subtle than simply the depth of the binding cleft or whether the two key aromatic residues involved in saccharide recognition are adjacent to each other. In CBM22-2 and CBM4-2, which are most restricted in terms of dicotyledon xylan recognition, the binding site is located on the concave face of the jelly-roll fold, whereas in CBM6 the ligand is accommodated between the loops connecting the two β-sheets of the protein, which may impart more flexibility on the binding site (6); CBMs are generally considered to be rigid proteins that do not undergo substantial conformational change upon ligand binding (4). However, CBM6 displays considerable structural flexibility. For example, when bound to xylopentaose, there is a positional shift in residues 25–27, which brings Ser-26 (shifted by 4.1 Å) closer to the sugar at subsite 1 facilitating the interaction between the hydroxy amino acid and O4 of the xylose at this subsite (6). Although such conformational changes are likely to incur an energetic cost, which will lead to a reduction in overall affinity, the movement of the protein may reflect the heterogeneity of the xylan decorations. Thus, although the movement of Ser-26 is optimal for binding unsubstituted xylooligosaccharides, this amino acid may need to be more distant from the binding cleft to accommodate xylans that are decorated or in contact with other polysaccharides. An explanation for the difference in specificity of CBM22-2 and CBM4-2 for pea and flax secondary cell walls and wheat and maize coleoptiles is not readily available because the two proteins display the same fold and the binding site is conserved, as are the key aromatic residues that stack against the bound sugar rings (26, 27). It is apparent that subtle differences in the binding site of CBMs that target xylans can have dramatic effects on their capacity to recognize their ligands within the complex milieu of cell walls of diverse taxonomic origin.

CBM35 displays a significant difference in ligand specificity to the other CBMs evaluated in this study. Although CBM6, CBM4-2, CBM15, and CBM22-2 bind to both unsubstituted xylan and arabinoxylan, CBM35 displays a strong preference for unsubstituted xylan polymers, compared to MGX and RAX (10) but, in addition, shows significant recognition of GAX (the likely target ligand of this module in primary cell walls, see above). Therefore, it is likely that the decorations in GAX are either relatively sparse compared to RAX and MGX, or the substitutions are present in discrete blocks thus presenting significant regions of undecorated xylan, although it is also possible that CBM35 recognizes the specific substitutions in GAX. Although the variation in binding of CBM35 to different cell walls may, in part, reflect the accessibility of the polysaccharide, it also is likely to be a consequence of the unusual ligand specificity displayed by this protein.


Although it is well established that xylan-binding CBMs are located in several different sequence-based families, the biological significance for this structural diversity was unclear. Here we show that CBMs in different families display significant variation in specificity for xylans in both primary and secondary cell walls, providing a biological rationale for this structural diversity. Different CBMs therefore have the capacity to target appended catalytic modules to specific cell walls in diverse species. CBM15 and CBM2b-1-2 confer the widest substrate specificity, whereas the CBMs derived from thermophilic bacteria recognize a more restricted range of cell walls. It is possible that in thermophilic environments the elevated temperature mediates the rapid breakdown of the intricate structure of the plant cell wall increasing the accessibility of the xylan. In view of the variation in specificity displayed by the different CBMs it is interesting that very few xylanases contain multiple xylan-binding CBMs from distinct families. Two xylanases/arabinofuranosidases from Caldicelluorupter contain a CBM6 and CBM22-2 (29), whereas a Paenibacillus xylanase contains a CBM6 and a CBM36 (which also binds to xylan) (30). In contrast, numerous xylanases contain distinct xylan- and cellulose-binding CBMs (10). Thus, in nature, individual xylanases may target specific cell walls. However, as wall degradation progresses, the CBMs will become effective with a wider range of substrates as indicated by their recognition of isolated polymers. From a biotechnological perspective, the portfolio of specificities displayed by CBMs can inform and direct enzyme bioengineering strategies. Finally, these CBMs also represent valuable tools with which to probe the intricate architecture of plant cell walls, because they not only identify xylans but also provide information on the context of these polysaccharides within cell walls.

Materials and Methods

Production of Xylan-Binding CBMs and LM11 Monoclonal Antibody.

The CBMs used in this study, which are listed in Table 1, are as follows: CBM2b-1-2 contains an N-terminal His-10-tag and comprises CBM2b-1 and CBM2b-2 derived from the Cellulomonas fimi xylanase CfXyn11A, which are joined by the enzyme's proline/threonine linker. The construction of CBM2b-1-2, its expression, and its purification were carried out as described previously (15). CBM4-2 contains an N-terminal His-10-tag and the C-terminal family 4 CBM from the R. marinus xylanase RmXyn11A and was produced by the method of Abou Hachem et al. (14). CBM15, a component of the Cellvibrio japonicus xylanase CjXyn10C, contains an N-terminal His-10-tag and was generated by following the protocol of Szabo et al. (17). CBM6, CBM22-2, and CBM35 all contain a C-terminal His-6-tag, are derived from Clostridium thermocellum CtXyn11A, Clostridium thermocellum CtXyn10B, and C. japonicus Xyn10B, respectively, and were produced following the methods of Czjzek et al. (6), Charnock et al. (16), and Bolam et al. (10), respectively. The generation of the anti-xylan monoclonal antibody (LM11), which recognizes both decorated and undecorated xylans, was described (23).


4-O-methyl-d-glucurono-d-xylan and OSX were obtained from Sigma. Soluble OSX was generated by treating the xylan with alkali followed by neutralization using the method of Ghangas et al. (31). Rye arabinoxylan was obtained from Megazyme International (Bray, Ireland). Glucuronoarabinoxylan (GAX) from maize kernels (21) was kindly provided by Luc Saulnier (Institut National de la Recherche Agronomique, Nantes, France).

CBM Assays on Nitrocellulose.

Nitrocellulose-based assays of CBM binding to polysaccharides were carried out as described by McCartney et al. (22). His-tagged CBMs were incubated with nitrocellulose arrays at 2.5 μg/ml in PBS with 5% (wt/vol) milk protein (PBS/MP).


ITC measurements were made at 25°C following standard procedures (10) using a Microcal Omega titration calorimeter. Proteins were dialyzed extensively against 50 mM sodium phosphate buffer (pH 7.0), and the ligand was dissolved in the same buffer to minimize heats of dilution. During a titration experiment, the protein sample (20–200 μM), stirred at 300 rpm in a 1.4331-ml reaction cell maintained at 25°C, was injected with 29 successive 10-μl aliquots of ligand comprising 1–4 mg/ml of polysaccharide or 0.5–10 mM oligosaccharide at 200-s intervals. Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regression using a single site binding model (Microcal Origin, version 7.0).

Preparation of Plant Materials and Labeling for CBM and Immunofluorescence Microscopy.

Pea seedlings (Pisum sativum L.) were grown at 18°C for 3–4 weeks, and tobacco plants (Nicotiana tabacum L.) were grown at 24°C for 6–7 weeks under a regime of 16 h light and 8 h dark. Regions of stem were excised and immediately fixed in PEM buffer (50 mM Pipes/5 mM EGTA/5 mM MgSO4, pH 6.9) containing 4% paraformaldehyde. Maize (Zea mays L.) kernels were soaked overnight in water and germinated on moist filter paper at 27°C in darkness. When coleoptiles were at least 3 cm long, regions were excised and immediately fixed as described above. Wheat grain (Triticum aestivum L.) was imbibed overnight and then cut into small cubes of ≈8 mm3 to include the aleurone layer and immediately fixed as described above. After fixation, plant materials were embedded in wax and sectioned as described (32). Sections of flax hypocotyls embedded in resin as described (33) were a kind gift from Christine Andème-Onzighi (University of Rouen, Mont-Saint-Aignan, France).

Labeling of sections with LM11 was carried out as described (23). For CBM labeling, sections were incubated with CBMs in the range of 2.5–5 μg/ml in PBS/MP for 1.5 h. Samples were then washed in PBS at least three times and incubated with a 100-fold dilution of mouse anti-His monoclonal antibody (Sigma) in PBS/MP for 1.5 h. After washing with PBS, anti-mouse IgG FITC (Sigma) was applied for 1.5 h as a 50-fold dilution in MP/PBS in darkness. The samples were washed at least three times, mounted in a glycerol-based anti-fade solution (Citifluor AF1; Agar Scientific), and observed on an Olympus BH-2 microscope equipped with epifluorescence irradiation. Micrographs shown are representative of observations of three separate plants for each species.

Supplementary Material

Supporting Table:


This work was supported by the U.K. Biotechnology and Biological Sciences Research Council.


carbohydrate-binding module
isothermal titration calorimetry
oat spelt xylan
rye arabinoxylan


Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.


1. O'Neill M. A., York W. S. In: The Plant Cell Wall. Rose J. K. C., editor. London: Blackwell; 2003. pp. 1–54.
2. Coughlan M. P. Biotechnol. Genet. Eng. 1985;3:39–109.
3. Hall J., Black G. W., Ferreira L. M., Millward-Sadler S. J., Ali B. R., Hazlewood G. P., Gilbert H. J. Biochem. J. 1995;309:749–756. [PMC free article] [PubMed]
4. Boraston A. B., Bolam D. N., Gilbert H. J., Davies G. J. Biochem. J. 2004;382:769–781. [PMC free article] [PubMed]
5. Henshaw J. L., Bolam D. N., Pires V. M., Czjzek M., Henrissat B., Ferreira L. M., Fontes C. M., Gilbert H. J. J. Biol. Chem. 2004;279:21552–21559. [PubMed]
6. Czjzek M., Bolam D. N., Mosbah A., Allouch J., Fontes C. M., Ferreira L. M., Bornet O., Zamboni V., Darbon H., Smith N. L., et al. J. Biol. Chem. 2001;276:48580–48587. [PubMed]
7. van Bueren A. L., Morland C., Gilbert H. J., Boraston A. B. J. Biol. Chem. 2005;280:530–537. [PubMed]
8. Boraston A. B., Nurizzo D., Notenboom V., Ducros V., Rose D. R., Kilburn D. G., Davies G. J. J. Mol. Biol. 2002;319:1143–1156. [PubMed]
9. Tunnicliffe R. B., Bolam D. N., Pell G., Gilbert H. J., Williamson M. P. J. Mol. Biol. 2005;347:287–296. [PubMed]
10. Bolam D. N., Xie H., Pell G., Hogg D., Galbraith G., Henrissat B., Gilbert H. J. J. Biol. Chem. 2004;279:22953–22963. [PubMed]
11. Carrard G., Koivula A., Soderlund H., Beguin P. Proc. Natl. Acad. Sci. USA. 2000;97:10432–10437.
12. McLean B. W., Boraston A. B., Brouwer D., Sanaie N., Fyfe C. A., Warren R. A., Kilburn D. G., Haynes C. A. J. Biol. Chem. 2002;277:50245–50254. [PubMed]
13. Ebringerová A., Heinze T. Macromol. Rapid Commun. 2000;21:542–556.
14. Abou Hachem M., Nordberg Karlsson E., Bartonek-Roxa E., Raghothama S., Simpson P. J., Gilbert H. J., Williamson M. P., Holst O. Biochem. J. 2000;345:53–60. [PMC free article] [PubMed]
15. Bolam D. N., Xie H., White P., Simpson P. J., Hancock S. M., Williamson M. P., Gilbert H. J. Biochemistry. 2001;40:2468–2477. [PubMed]
16. Charnock S. J., Bolam D. N., Turkenburg J. P., Gilbert H. J., Ferreira L. M., Davies G. J., Fontes C. M. Biochemistry. 2000;39:5013–5021. [PubMed]
17. Szabo L., Jamal S., Xie H., Charnock S. J., Bolam D. N., Gilbert H. J., Davies G. J. J. Biol. Chem. 2001;276:49061–49065. [PubMed]
18. Simpson P. J., Bolam D. N., Cooper A., Ciruela A., Hazlewood G. P., Gilbert H. J., Williamson M. P. Structure (London) 1999;7:853–864. [PubMed]
19. Pell G., Williamson M. P., Walters C., Du H., Gilbert H. J., Bolam D. N. Biochemistry. 2003;42:9316–9323. [PubMed]
20. Freelove A. C., Bolam D. N., White P., Hazlewood G. P., Gilbert H. J. J. Biol. Chem. 2001;276:43010–43017. [PubMed]
21. Saulnier L., Marot C., Chanliaud E., Thibault J.-F. Carbohydrate Polymers. 1995;26:279–287.
22. McCartney L., Gilbert H. J., Bolam D. N., Boraston A. B., Knox J. P. Anal. Biochem. 2004;326:49–54. [PubMed]
23. McCartney L., Marcus S. E., Knox J. P. J. Histochem. Cytochem. 2005;53:543–546. [PubMed]
24. Darvill J. E., McNeil M., Darvill A. G., Albersheim P. Plant Physiol. 1980;66:1135–1139. [PMC free article] [PubMed]
25. Carpita N. C. Ann. Rev. Plant Physiol. Plant Mol. Biol. 1996;47:445–476. [PubMed]
26. Simpson P. J., Jamieson S. J., Abou-Hachem M., Karlsson E. N., Gilbert H. J., Holst O., Williamson M. P. Biochemistry. 2002;41:5712–5719. [PubMed]
27. Xie H., Bolam D. N., Nagy T., Szabo L., Cooper A., Simpson P. J., Lakey J. H., Williamson M. P., Gilbert H. J. Biochemistry. 2001;40:5700–5707. [PubMed]
28. Xie H., Gilbert H. J., Charnock S. J., Davies G. J., Williamson M. P., Simpson P. J., Raghothama S., Fontes C. M., Dias F. M., Ferreira L. M., Bolam D. N. Biochemistry. 2001;40:9167–9176. [PubMed]
29. Morris D., Gibbs M., Ford M., Thomas J., Bergquist P. Extremophiles. 1999;3:103–111. [PubMed]
30. Jamal-Talabani S., Boraston A. B., Turkenburg J. P., Tarbouriech N., Ducros V. M., Davies G. J. Structure (London) 2004;12:1177–1187. [PubMed]
31. Ghangas G. S., Hu Y. J., Wilson D. B. J. Bacteriol. 1989;171:2963–2969. [PMC free article] [PubMed]
32. McCartney L., Steele-King C. G., Jordan E., Knox J. P. Plant J. 2003;33:447–454. [PubMed]
33. His I., Andème–Onzighi C., Morvan C., Driouich A. J. Histochem. Cytochem. 2001;49:1525–1536. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...