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Keywords:

  • plant cell walls;
  • xylan;
  • carbohydrate-binding module;
  • pectate lyase;
  • xylanase;
  • fluorescence quantification

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The capacity of four xylan-directed probes (carbohydrate-binding modules CfCBM2b-1-2 and CjCBM15; monoclonal antibodies LM10 and LM11) to recognize xylan polysaccharides in primary and secondary cell walls of tobacco stem sections has been determined. Enzymatic removal of pectic homogalacturonan revealed differential recognition of xylans in restricted regions of cortical primary cell walls. Monoclonal antibody binding to these exposed xylans was more sensitive to xylanase action than carbohydrate-binding module (CBM) binding. In contrast, the recognition of xylans by CBMs in secondary cell walls of the same organ was more sensitive to xylanase action than the recognition of xylans by the monoclonal antibodies. A methodology was developed to quantify indirect immunofluorescence intensities, and to evaluate xylanase impacts. The four xylan probes were also used to detect xylan populations in chromatographic separations of solubilized cell wall materials from tobacco stems. Altogether, these observations reveal the heterogeneity of the xylans in plant cell walls. They indicate that although CBM and antibody probes can exhibit similar specificities against solubilized polymers, they can have differential capacities for xylan recognition in muro, and that the access of molecular probes and enzymes to xylan epitopes/ligands also varies between primary and secondary cell walls that are present in the same organ.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant cell walls comprise the major source of organic carbon on earth. These composite structures, together with their component structural polysaccharides, have numerous industrial applications in the paper, textiles and food sectors (Farrokhi et al., 2006; Harris and Bronwen, 2006; Wróbel-Kwiatkowska et al., 2007), whereas their exploitation as a substrate for producing sustainable biofuels is of considerable economic and environmental significance (Ragauskas et al., 2006; Lopez-Casado et al., 2008; Sticklen, 2008). Plant cells synthesize a primary cell wall with flexible properties that accommodates cell enlargement, and functions in cell adhesion within plant organs. In addition, some cell types, notably xylem elements and fibres, produce thicker, structurally and biochemically distinct secondary cell walls that resist compressive forces. Both primary and secondary cell walls are highly complex structures composed predominantly of diverse sets of polysaccharides that vary in structure and abundance. These polysaccharides are conventionally divided into three groups: cellulose, hemicelluloses (e.g. xylans, xyloglucans and mannans) and pectins. Xylans are one of the major hemicelluloses found in plant cell walls, and are the most abundant polysaccharides in nature after cellulose (Carpita and Gibeaut, 1993; Singh et al., 2003). Xylans are based on a linear β-1,4-linked xylose polymer that is generally decorated with side chains that include α-d-glucuronic or α-4-O-methyl-d-glucuronic acid, and α-l-arabinofuranose, with the degree and nature of substitutions varying between tissues and also between species. In dicotyledons, such as tobacco, relatively unsubstituted xylans are abundant components of the secondary cell walls associated with the vascular tissues.

Immunohistochemical techniques with cell wall polymer-directed polyclonal antisera and monoclonal antibodies can be used to probe cell wall architectures. Two monoclonal antibodies against xylan, LM10 and LM11, have been generated using a neoglycoprotein approach (McCartney et al., 2005), and these have been used in immunomicroscopy approaches to reveal the abundance of xylan in secondary cell walls of a range of dicotyledons (McCartney et al., 2005, 2006; Zhou et al., 2006; Peña et al., 2007; Persson et al., 2007). Recently, the repertoire of cell-wall probes has been extended by the use of carbohydrate-binding modules (CBMs) obtained from microbial plant cell wall hydrolases (Boraston et al., 2004; Hashimoto, 2006; Shoseyov et al., 2006). A previous study assessed the capacity of xylan-directed CBMs to bind to cell walls in sections of plant material from diverse taxonomic groups, and revealed that some of these probes can exhibit similar specificities against isolated ligands, but have differing capacities to recognize their target polysaccharides within the context of intact cell walls (McCartney et al., 2006). This variation in ligand recognition in planta likely reflects the interaction of the target polysaccharides with other components of the cell wall. Supporting the idea that the recognition of a specific cell wall polymer in the context of an intact cell wall can be influenced by the presence of other cell wall polymers, is the recent demonstration that the enzymatic removal of pectic homogalacturonan (HG) from transverse sections of tobacco stems can result in increased antibody access to xyloglucan epitopes (Marcus et al., 2008).

Here, we have determined the capacity of two xylan-directed CBMs (CjCBM15 and CfCBM2b-1-2), and two xylan-directed monoclonal antibodies (LM10 and LM11), to recognize xylans in both primary and secondary cell walls of tobacco stems after enzymatic treatments that degrade pectic HG and xylan. CfCBM2b1-2 and CjCBM15 exhibit similar specificities for purified xylans (Bolam et al., 2001, 2004; Szabo et al., 2001), and LM10 and LM11 both recognize unsubstituted β-1,4-xylan, whereas LM11 can also bind to substituted arabinoxylans (McCartney et al., 2005). We show that xylans in primary and secondary cell walls display differential sensitivity to recognition by molecular probes and to degradation by xylanases. This difference in binding ability is not reflected when the probes are used on solubilized/fractionated tobacco stem cell wall polymers, but the occurrence of several xylan populations is observed. These data demonstrate that the context of xylans in primary and secondary walls influences their availability for protein recognition. Such information will inform enzyme design strategies to enhance the efficiency of biofuel generation from plant biomass.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Xylans are abundant components of secondary cell walls in tobacco plants

The capacity of sets of molecular probes and enzymes to recognize diverse cell walls was assessed using sections of 6-week-old tobacco stems. Transverse sections of tobacco stem internodes present epidermal, collenchyma, cortical parenchyma and phloem cells, and associated phloem fibres, xylem tissue and pith parenchyma, as shown in Figure 1. Secondary cell walls are observed in discrete groups of cells in the phloem region, the band of xylem vessels and fibres, and also in groups of cells associated with internal phloem (Figure 1). The matched bright-field and immunofluorescence detection of xylan-directed CfCBM2b-1-2 binding to an untreated section of tobacco stem is shown in Figure 1. CfCBM2b-1-2 binds specifically to all secondary cell walls. CjCBM15 and the monoclonal antibodies LM10 and LM11 also recognize xylans in secondary cell walls. CjCBM15, distinct from the other xylan probes, can also recognize xylan in primary cell walls of some dicotyledons including tobacco, as previously described (McCartney et al., 2006). However, in some tobacco plants no such binding is detected.

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Figure 1.  Transverse sections of tobacco stem, showing cortical and vascular cells. (a, b) Bright field showing anatomy. (c, d) Indirect immunofluorescence detection of CfCBM2b-1-2 binding in the same sections. Abbreviations: cl, collenchyma; cp, cortical parenchyma; e, epidermis; ip, internal phloem; pf, phloem fibres; pp, pith parenchyma; x, xylem vessels. Scale bars: 10 μm.

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Pectate lyase treatment of tobacco stem sections allows recognition of primary cell walls by xylan-directed CBM and antibody probes

Pre-treatment of equivalent sections with pectate lyase CjPel10A (Brown et al., 2001) results in the removal of pectic HG, assessed by the binding of HG-directed monoclonal antibody JIM5, as described previously (Marcus et al., 2008), and as shown in Figure 2. Enzymatic pre-treatment to remove HG led to altered binding to the sections by some of the xylan-directed probes. After HG removal, CjCBM15 bound to regions of epidermal cell walls and cortical collenchyma/parenchyma cell walls (mostly at cell junctions), as shown in Figure 2. Binding to primary cell walls was most abundant in outer cortical cells. In contrast, CfCBM2b-1-2 did not bind to any primary cell walls after treatment with pectate lyase (Figure 2). In equivalent experiments, monoclonal antibody LM11 bound to primary cell walls in a manner similar to CjCBM15. Fluorescence detection at higher magnification revealed that monoclonal antibody LM10 bound to regions of the outer cortical collenchyma/parenchyma cell walls, as shown in Figures 3 and 4. Detailed studies indicated that CjCBM15, LM11 and LM10 bound to distinct regions of the collenchyma cell wall thickenings (Figure 3). CjCBM15 and LM11 bound most intensely to outer regions of cell wall thickenings at cell junctions, in contrast to antibody LM10, which targeted the complementary inner regions of cell wall thickenings (Figure 3). CfCBM2b-1-2 was not observed to bind to cortical cell walls in any of these studies.

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Figure 2.  Indirect immunofluorescence detection of xylan epitopes in transverse sections of tobacco stem before and after treatment of sections with pectate lyase. The binding of the homogalacturonan antibody JIM5 to tobacco stem sections was used to verify pectate lyase action. carbohydrate-binding modules (CBMs) 15 and 2b-1-2, and the monoclonal antibodies LM10 and LM11, bind specifically to secondary cell walls before enzymatic pre-treatment. Binding in the region of cortical parenchyma cells is revealed after the pectate lyase treatment for CjCBM15 and LM11. Abbreviations: cl, collenchyma; e, epidermis; pf, phloem fibres; x, xylem vessels; *Binding not detected at this magnification. Scale bar: 10 μm.

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image

Figure 3.  High-magnification micrographs of xylan probe binding to thickenings of collenchyma cell walls after treatment of sections with pectate lyase. Fluorescence detection at the higher magnification indicates that CjCBM15, LM11 and LM10 bound to distinct regions of the collenchyma cell wall thickenings. The same sections are shown as probe-based fluorescence (green) and, when combined with Calcofluor White fluorescence (blue), show the full extent of cell walls. Abbreviations: cl, collenchyma; e, epidermis. Scale bar: 10 μm.

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Figure 4.  Indirect immunofluorescence detection of xylan probe binding to cortical primary cell walls after xylanase pre-treatment of sections. High-magnification micrographs of tobacco stem sections showing regions of cortical collenchyma/parenchyma are shown. Sections were pre-treated with pectate lyase (PL+/Xyl−) before incubations with the xylanase 11A (PL+/NpXyl11A) or the xylanase 10B (PL+/CjXyl10B). The CjCBM15 binding to primary cell walls of tobacco stem sections revealed by the pectate lyase pre-treatment was only partly abolished by the xylanase treatments, whereas the LM10 and LM11 binding to primary cell walls disappeared entirely. Scale bar: 10 μm.

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The glycosyl hydrolase family 10 (GH10) xylanases and the GH11 xylanases are important enzymes in the degradation of xylans in cell walls. Representative enzymes were used to explore xylan degradation in tobacco cell walls: a GH10 Cellvibrio japonicus xylanase CjXyl10B (DeBoy et al., 2008) and a GH11 Neocallimastix patriciarum xylanase NpXyl11A (Vardakou et al., 2008). Treatment of pectate lyase-treated sections with these enzymes indicated that both enzymes were effective in the complete abolition of LM10 and LM11 binding to the primary cell walls (Figure 4), confirming that the exposed epitopes were xylans. In addition, both CjXyl10B and NpXyl11A treatments resulted in a decrease in, but not the abolition of, CjCBM15 binding to the primary cell walls. Indeed, CjCBM15 bound to xylanase-insensitive structures at the centre of the collenchyma cell wall thickenings (Figure 4).

Development of quantitative 2D spatial mapping of immunofluorescence intensities across secondary cell walls

In tobacco stems secondary cell walls are associated with diverse cell types, including xylem vessels and cortical phloem sclerenchyma fibres. Xylans are especially abundant in these walls as revealed by in situ analysis with xylan probes (Figure 1; McCartney et al., 2006). As shown above, the LM10 and LM11 epitopes, revealed in the primary cell walls by pectate lyase treatments, are highly susceptible to xylanase degradation. In secondary cell walls the xylans are present in a composite polysaccharide context that lacks pectins. The polysaccharides would generally be expected to be more resistant to enzymatic degradation in secondary cell walls, as these structures are more compact than primary cell walls. It is well established that secondary cell walls can be recalcitrant to enzyme action as a result of the tight associations of the component polymers – a context that has led to the proliferation of potentiating CBMs as modular components of cell wall hydrolases (Ali et al., 2001; Bolam et al., 2001; Kittur et al., 2003; Gilbert et al., 2008). Enzyme treatments of sections across secondary cell walls may result in a change in antibody/CBM binding, from a slight decrease to a total loss of cell wall recognition, and therefore it is important to quantify probe binding (fluorescence intensity) to assess the possible subtle impacts of enzyme action upon cell wall architectures and xylan degradation.

A methodology to quantify indirect immunofluorescence intensities captured in micrographs, in terms of both spatial patterns and absolute levels, was developed using Volocity quantitation software, as outlined in Figure 5. Figure 5a shows the immunofluorescence observed for a xylan probe binding, after treatment of equivalent sections (showing equivalent regions of phloem and xylem tissue), with two levels of a xylanase. Using Volocity, different bands of fluorescence intensity were defined and then assigned colours that were overlain on the micrographs, as shown in Figure 5b. Precise intensity bands had to be determined for each set of experiments, but in general the upper 80% of intensities from the control without xylanase treatment were coloured white, the next 15% were coloured orange and the lowest 5% intensities were coloured purple (Figure 5c). Negative control sections from which the xylan probes had been omitted were used to set the fluorescence intensity bands (FIBs). This colour coding of FIBs enabled two things: a visual mapping of reductions in probe binding from coloured FIB overlays in relation to cell wall structures (Figure 5b), and an overall quantification of fluorescence (sum of areas of each FIB) in the equivalent sections (Figure 5c). The quantification indicated that the micrographs derived from the example xylanase-treated section 1 and xylanase-treated section 2 contained 63 and 34% of the level of fluorescence detected in the untreated control section. The corresponding schematic curves and overlain pictures show the size of the white interval decreasing, whereas the orange and purple values increase. These results confirm that the micrographs obtained by indirect immunofluorescence with xylan probes are suitable for quantitative fluorescence microscopy.

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Figure 5.  Quantification of probe-based fluorescence in secondary cell walls of tobacco stem sections. (a) Indirect immunofluorescence detection of a xylan-probe binding (CfCBM2b-1-2). The fluorescein isothiocyanate (FITC) fluorescence is shown as observed. The three pictures feature different intensity levels of immunofluorescence.  (b) Same images, with overlain colours reflecting the fluorescence intensity bands (FIBs). Different colours are used to represent the fluorescence intensity values. The pixels featuring the highest intensity values are shown in white. Less intense fluorescence is shown in orange or purple, with purple indicating the lowest intensity values.  (c) Schematic curves showing the corresponding distribution of intensities. The brightest image on the left shows a maximum fluorescence, with 80% of the values contained in the white interval. The images on the right feature, respectively, 63 and 34% of the total fluorescence observed in the first image. Abbreviations: pf, phloem fibres; x, xylem vessels. Scale bars: 10 μm.

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Impact of xylanase treatments on xylan recognition in secondary cell walls by CBM and antibody probes

The impact of CjXyl10B and NpXyl11A pre-treatments on the capacity of the xylan probes to bind to the secondary cell walls of tobacco stem sections was assessed and quantified in representative sections from at least three separate plants, and the results obtained are summarized in Figure 6. The targeted epitopes/ligands are all susceptible to the enzymatic pre-treatments with xylanases, but display different sensitivities. The impact of each enzyme was similar for both CjCBM15 and CfCBM2b-1-2. NpXyl11A reduced CjCBM15 and CfCBM2b-1-2 binding by approximately 40–50%, whereas CjXyl10B reduced the binding of these CBMs by 80–90% (Figure 6). In contrast, the capacity of LM10 and LM11 to bind to secondary cell walls was less affected by equivalent xylanase treatments (Figure 6). CjXyl10B remained more effective than NpXyl11A, but immunofluorescence was reduced by no more than 40% compared with untreated sections. In all cases, the binding of CBM and antibody probes to the cortical phloem sclerenchyma fibres showed a greater sensitivity to xylanase treatment than the binding to xylem vessels and fibres. The observed patterns and extents of reduced binding were unchanged when sections were incubated with up to 10-fold higher concentrations of xylanase (100 μg ml−1).

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Figure 6.  Effect of the xylanase treatments on xylan epitope/ligand recognition in secondary cell walls of transverse sections of tobacco stems. The results are expressed as percentages of the remaining fluorescence, compared with the corresponding control without enzymatic treatment. The CjCBM15 and CfCBM2b-1-2 epitopes observed in the secondary cell walls of tobacco stem sections were strongly reduced by the xylanase treatments, especially CjXyl10B. The LM10 and LM11 antibody epitopes were less affected. The histograms present the means ± SD of determinations on three different plants.

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CBM and antibody probe recognition of xylans solubilized from tobacco stem cell walls

To further explore CBM and antibody probe recognition of xylans in tobacco stems, alcohol-insoluble residues containing cell wall polymers were prepared and sequentially extracted with trans-cyclohexane-1,2-diamine-N,N,N′,N′-tetraacetate (CDTA), 0.5 m KOH and 4 m KOH, and the solubilized polymers were analysed by anion-exchange chromatography (AEC) using the probes as detection agents. The three fractions represented, respectively, ∼31, 25 and 16% of the alcohol-insoluble starting material, but equal quantities (1 mg) were analysed and collected in 50 AEC fractions, and the binding of CfCBM2b1-2, CjCBM15, LM10 and LM11 was determined by ELISAs (Figure 7). The occurrence of pectic HG epitopes was assessed using the JIM5 monoclonal antibody. Pectins were mainly detected in the CDTA-solubilized fraction with a late elution pattern (Figure 7a), whereas no binding was observed in the 4 m KOH-solubilized fraction (Figure 7b). In the 0.5 m KOH-solubilized fraction, only a slight binding of JIM5 was observed, reflecting the low pectic content of this sample (data not shown).

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Figure 7.  Anion-exchange chromatography elution profiles of the solubilized tobacco cell wall material detected using CjCBM15, CfCBM2b-1-2, and the monoclonal antibodies LM10 and LM11. The antibody JIM5 was used for detection of pectic homogalacturonan (HG). (a) Elution profile for the CDTA-solubilized polymers. This fraction showed significant epitope recognition by JIM5 in the most acidic fractions. The xylan profiles show two retained populations, with population II only recognized by the carbohydrate-binding modules (CBMs). (b) Effect of CjXyl10B treatment on the samples collected from the CDTA fraction. The xylanase treatment was performed on the coated plates before epitope recognition by the xylan probes. The binding was abolished for all xylan probes. No effect was observed on JIM5 binding. (c) Elution profile for the 4 m KOH-solubilized polymers of tobacco stem cell walls. No JIM5 binding was detected. Two xylan populations were detected, with population III being less acidic than population IV.

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From the elution profiles represented in Figure 7 it is clear that both fractions are composed of two different xylan populations, differing in their charge. The CDTA fraction shows two retained populations, with the first and major one (Figure 7a, population I) being recognized by both CBMs and antibodies. The second population is not separated entirely from population I, and shows a late elution profile that overlaps with pectic HG detection (Figure 7a, population II). This population II of xylans seems to be exclusively recognized by the CBMs. Both populations and the binding of all probes were sensitive to treatment with CjXyl10B (Figure 7b).

The AEC of the 4 m KOH-solubilized fraction revealed the presence of two populations, detected by all four probes, and, although not fully resolved by AEC, the larger population (IV) was more acidic (Figure 7b), and was also retained slightly longer by the AEC column than population I of the CDTA-solubilized fraction. In the 0.5 m KOH-solubilized AEC fractions, the xylan probes showed limited binding to one peak, with the same retention time as population IV, indicating similarity to xylans solubilized by the 4 m KOH fraction (data not shown). In all cases, CfCBM2b-1-2 showed the strongest recognition of solubilized xylans, LM10 and LM11 antibodies provided binding profiles that were almost identical, and CjCBM15 displayed the weakest binding to the isolated xylan populations.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Xylans in primary cell walls

The enzymatic removal of pectic HG from tobacco collenchyma/cortical parenchyma primary cell walls revealed xylans that can be bound by antibodies and CBMs, and that this binding is sensitive to xylanase action. Xylans were not thought to be significant components of primary cell walls of dicotyledons (Carpita and Gibeaut, 1993), but this report indicates that the presence of these polymers in these walls has been underreported, as they are shielded by pectic HG. We show here that xylans are detected within epidermal, collenchyma and cortical parenchyma, but not in pith parenchyma cell walls. This observation is supported by the fact that xylan epitopes are relatively abundant in the CDTA-solubilized fractions of tobacco cell walls. Some of these xylans (population II) co-eluted with, and are therefore potentially linked to, pectic HG. Xylans are the second set of hemicellulose polysaccharides to be detected in dicotyledon primary cell walls after the removal of HG, and extend the observations already made for xyloglucan (Marcus et al., 2008). There is no cross-recognition of xyloglucans by the xylan probes (Bolam et al., 2001; Szabo et al., 2001; McCartney et al., 2005), and therefore these probes do not recognize xyloglucan in the cell walls.

Currently, the specific functions of the biochemically distinct hemicellulosic polysaccharides (xyloglucans, xylans and mannans) in primary cell walls are uncertain. It is thought that hemicellulose polymers tether cellulose microfibrils, and are components of networks that underpin cell enlargement mechanisms. The observations here that CjCBM15, LM10 and LM11 can bind to subtly distinct regions of cell wall thickenings of tobacco collenchyma, and not to pith parenchyma cell walls, indicate that xylan structure is highly regulated at the level of individual primary cell walls. The occurrence of xylans in thickened primary cell walls of collenchyma and the epidermis, and not in all primary cell walls, may reflect some commonality with the wall thickening processes of primary and secondary cell walls.

It has been previously shown that CfCBM2b-1-2, from the sets of CBMs tested, displays the widest specificity for plant cell walls, and this was consistent with the open topology of its binding site (McCartney et al., 2006). This CBM also shows the most extensive recognition profiles within the solubilized tobacco cell wall polymers (including xylans contained in both primary and secondary cell walls). It is of interest that CfCBM2b-1-2 did not bind to the primary cell wall xylans under the conditions used in this study, but recognized xylan epitopes co-eluted with pectic HG from the CDTA-solubilized material. This may reflect the length of xylans exposed on the surface of these structures in muro. Thus, although CfCBM2b-1-2 is more likely to recognize xylans that are in contact with other polysaccharides, compared with other probes, such structures must contain more than 10 contiguous xylosyl residues: the protein comprises two related family 2b CBMs, and tight xylan binding only occurs when both protein modules interact with the polysaccharide (Bolam et al., 2001). The lack of CfCBM2b-1-2 binding to primary cell walls on sections may therefore indicate the exposure of only short xylan sequences in primary cell walls (and the recognition of these by CjCBM15, LM10 and LM11). Once xylans have been solubilized and removed from their cell wall context, longer xylan sequences are likely to be available for recognition by CfCBM2b-1-2.

Xylans in secondary cell walls

When used to probe untreated sections, the four probes (CjCBM15, CfCBM2b-1-2, LM10 and LM11) bind to the same sets of secondary cell walls. A question that we have addressed is whether they bind to the same epitopes/ligands or to distinct epitopes/ligands that occur in the same place. It would be expected that antibodies, which are larger than CBMs, should bind to more exposed xylans, and therefore should be more sensitive to xylanase treatment when compared with CBMs. However, the present results show that antibodies still effectively detect xylans in secondary cell walls when the CBMs do not. The binding of CBMs to xylans in secondary cell walls is more sensitive to xylanase action than the binding of monoclonal antibodies LM10 and LM11. The reason for this could be that CBMs bind to exposed xylans, and not to xylans more tightly integrated into the cell wall composites. This is exemplified by the complete disappearance of the CBM and antibody epitopes after a xylanase treatment of the solubilized cell wall material, where all epitopes are expected to be exposed. This may indicate that, when appended to enzymes, CBMs act in the initial stages of cell wall degradation, to initiate xylan degradation, but as levels decline they are unable to direct their appended xylanases to less accessible sites. It is evident that xylanases are linked not only to xylan-specific CBMs, but also to protein modules that recognize crystalline cellulose (Boraston et al., 2004). It is possible, therefore, that the cellulose-specific CBMs direct their cognate xylanases to these ‘inaccessible’ regions.

Differential impacts of xylanase treatments

An intriguing feature of this study is the observation that the xylanase 10B was more effective than the xylanase 11A at degrading xylans that are components of secondary cell walls. This is particularly interesting as GH11 xylanases are generally ∼10-fold more active against isolated xylans than GH10 enzymes (Pell et al., 2004; Collins et al., 2005; Vardakou et al., 2008). The two glycosyl hydrolase families display very different structural topologies, with GH10 xylanases adopting a typical triosephosphateisomerase (TIM) barrel fold, whereas GH11 enzymes display a β-sandwich topology. It is possible that the deeper substrate binding clefts of GH11 xylanases only interact with xylans that are not in tight contact with other polymers in cell wall contexts. In contrast, the active sites of GH10 xylanases are slightly more solvent accessible, which may enable these enzymes to bind productively to xylans that are in closer contact with other polymers. It is also possible that the differential activities displayed by these enzymes may reflect differing capacities to attack decorated substrates. GH10 xylanases can accommodate xylans in which O2-substituted xylose residues are at positions n and n + 3, whereas GH11 xylanases require further separation of these sugars (n and n + 4). A similar slightly longer separation is required for O3-decorated xylans to be bound by GH11 enzymes (n and n + 3) compared with GH10 glycosyl hydrolases (n and n + 2) (Collins et al., 2005).

Populations and heterogeneity of xylan polysaccharides

The elution profiles of solubilized tobacco cell wall material, together with the xylan CBM and antibody detection, indicate that distinct xylan populations (that can be separated with respect to both solubility and charge) exist in cell walls. Broadly, all four probes generated similar profiles, although an indication of a more acidic CDTA-soluble population recognized by the CBMs, and not by the antibodies, was obtained. These observations indicate that although the probes may bind to subtly distinct structures/epitopes, they recognize similar sets (populations) of xylan polymers. However, for xylans in the context of cell walls, any subtle differences recognized in epitopes/ligands can lead to varied binding profiles and differential sensitivities of the xylan probes to xylanase action. Differences between xylan populations are likely to have resulted from structural features not recognized by these probes, including links to other cell wall polymers. Differences in cell wall probe binding to cell walls highlight the heterogeneity of cell walls, the complementary nature of both types of cell wall probe and also validates a CBM/antibody cross-checking for the visualization of polysaccharides and enzymatic degradation processes.

Despite high efficiencies against solubilized polymers, the ability of xylanases to degrade target substrates also seems to be reduced/altered when the substrates are in the context of intact cell walls, indicating that xylanase capabilities are also influenced by polysaccharide accessibility/substrate context. This, of course, is likely to reflect the central role played by the CBMs, ubiquitously appended to the bacterial cell wall hydrolases, in degradation processes (Boraston et al., 2004; Gilbert et al., 2008). The challenge lies in elucidating how these CBMs potentiate the activity of the different catalytic entities that attack cell walls. In the case of xylan degradation, we would predict that CfCBM2b-1-2 would facilitate the activity of GH11 xylanases, as these are always found in association with these catalytic modules, whereas CBM15 is appended to GH10 xylanases (Boraston et al., 2004; DeBoy et al., 2008). The methodologies reported here to quantify and spatially map cell wall probe-based fluorescence intensities, and the complementary chromatographic procedures to map polysaccharide populations by epitope tracking, will enable more in-depth studies on cell wall structures, and also of cell wall degradation – a process of current industrial relevance in terms of biofuel production.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Production of xylan-binding CBMs and monoclonal antibodies

CfCBM2b-1-2 is derived from the Cellulomonas fimi xylanase CfXyn11A. CjCBM15 is a component of the C. japonicus xylanase CjXyn10C (DeBoy et al., 2008). They both contain an N-terminal His-10-tag. Their construction, expression and purification were carried out as described previously (McCartney et al., 2006). The generation of the anti-xylan monoclonal antibodies LM10 and LM11, with LM11 being able to recognize decorated xylans in addition to undecorated xylans, has been described previously (McCartney et al., 2005).

Preparation of plant materials

Tobacco plants (Nicotiana tabacum L.) were grown under a regime of 16-h light (24°C) and 8-h dark (17°C). Regions of the sixth internodes from the top of 6-week-old plant stems were excised and immediately fixed in PEM buffer (50 mm Pipes, 5 mm EGTA, 5 mm MgSO4, pH 6.9) containing 4% paraformaldehyde. After fixation, plant materials were wax-embedded and sectioned as described previously (McCartney et al., 2003).

Enzymatic treatments and labelling for xylan probes

In some cases the cell walls of sectioned plant materials were treated with enzymes prior to CBM or antibody labelling. Removal of pectic HG was carried out as described by Blake et al. (2006), and involved incubating sections with 10 μg ml−1 of the C. japonicus pectate lyase CjPel10A (Brown et al., 2001) for 2 h at room temperature (∼20°C) in 50 mm 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 2 mm CaCl2 buffer, pH 10, prior to labelling. Xylanase treatments involved the C. japonicus xylanase CjXyl10B and the N. patriciarum xylanase NpXyl11A. Treatments were carried out overnight at room temperature at a concentration of 10 μg ml−1 in a 25 mm Na-phosphate/acetate buffer, pH 6.0. The concentration used was shown to be in excess (i.e. no additional effect was observed using larger quantities of xylanases). Sections not treated with the enzymes were incubated for an equivalent time with the corresponding buffers.

Immunolabelling of sections with JIM5, LM10 and LM11 was carried out as described by Knox et al. (1990) and McCartney et al. (2005). The binding of CBMs to plant material preparations was assessed by a three-stage immunolabelling technique, described previously (McCartney et al., 2004). In all cases, sections were incubated with an excess of probe. A 10-fold dilution was used for the antibody hybridoma supernatants. CjCBM15 and CfCBM2b-1-2 were used at 50 and 10 μg ml−1, respectively. The samples were washed at least three times in PBS, incubated with cellulose stain Calcofluor White (Fluorescent Brightner 28; Sigma-Aldrich, http://www.sigmaaldrich.com) for 5 mins in darkness, and mounted after washing in a glycerol-based anti-fade solution (Citifluor AF1; Agar Scientific, http://www.agarscientific.com).

Immunofluorescence microscopy and fluorescence quantification

Immunofluorescence was observed with a microscope equipped with epifluorescence irradiation (BX-61; Olympus, http://www.olympus-global.com). For comparative purposes, equivalent section thicknesses were used for all experiments, and epifluorescence images were used for analyses. Images were captured with an ORCA 285 camera (Hamamatsu, http://www.hamamatsu.com) and Volocity software (Improvision, http://www.improvision.com). For each set of experiments the microscope, light source or camera controls were not changed during data acquisition. The camera gain and offset were both at their minimal settings.

To quantify the immunofluorescence intensities captured in micrographs, Volocity quantitation software was used. To enable comparisons, measurements were performed over equal areas featuring phloem and xylem tissues of the tobacco stem. The Research Objects by Intensity task was used to select all pixels with fluorescence intensity up to the intensities observed in the corresponding negative control section, where the probe was omitted. For each set of experiments the absolute level of fluorescence contained in the micrographs was determined by recording the signal intensity for each pixel. In addition to the quantitation, different bands of immunofluorescence intensity were used to develop a method for spatial mapping. Micrographs obtained for the control without enzymatic treatment were considered to contain 100% of the initial immunofluorescence. Limits for the first band were determined in order to include 80% of the strongest fluorescence intensities captured. A white colour was assigned to this first FIB and overlain on the micrograph. A similar procedure was applied to the following FIBs containing 15% and the last 5% of the intensities, respectively, coloured in orange and purple. The protocol was retained and applied to the other micrographs featuring the enzymatic treatments. This method allowed the visualization of the reduction in probe binding, in addition to changes in the spatial patterns of fluorescence intensities. Analyses used equivalent sections for each set of probes and treatments, and each experiment was carried out for at least three different plants.

Anion-exchange chromatography and ELISA microtitreplate assay of fractions

Similar stem materials as used for immunocytochemistry were used to prepare alcohol-insoluble cell wall material. Briefly, the homogenized samples were washed with hot ethanol (70%), washed three times in cold ethanol (70%), then two times in ethanol (96%), and finally washed in acetone and dried in a ventilated oven. The residue was washed with H2O for 30 mins, and then extracted sequentially according to the method described by Selvendran (1985), with 0.1 m CDTA, pH 6.5, overnight, 0.5 m KOH/20 mm NaBH4 for 1 h, and by 4 m KOH/20 mm NaBH4 for 1 h. After each extraction, the solubilized material was separated from the insoluble residue by centrifugation at 5000 g for 30 mins. The alkaline extracts were neutralized with glacial acetic acid to pH 7.0. All the extracts were filtered and dialysed against H2O before freeze-drying. Weight analysis indicated that the CDTA, 0.5 and 4 m KOH fractions contained, respectively, 31, 25 and 16% of the starting material. The remaining 28% was lost in the washing step or remained in the residue. A total of 1 mg of each fraction was loaded on a 1-ml Hi-Trap Q HP anion-exchange chromatography column (GE Healthcare, http://www.gehealthcare.com), equilibrated previously with 10 mm Na-phosphate buffer, pH 6.0. Elution (1 ml min−1) was performed with a linear gradient of Na-phosphate buffer, pH 6.0, from 10 to 600 mm for 50 min. ELISAs were used to detect pectic and xylan epitopes present within each fraction. Assays were carried out as described previously by Willats et al. (2001) and McCartney et al. (2004), except that the collected samples were used to coat the plates at 50 μl per well. For the detection, a 10-fold dilution was used for the JIM5, LM10 and LM11 cell culture supernatants. The CBMs 15 and 2b-1-2 were applied at a concentration of 50 and 10 μg ml−1, respectively. The impact of a xylanase pre-treatment on the xylan probe binding was assessed by incubating the coated plates with 10 μg ml−1 of CjXyl10B (50 μl per well) in a 25 mm Na-phosphate/acetate buffer, pH 6.0, overnight, and at room temperature before the antibody/CBM detection.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The authors acknowledge funding from the UK Biotechnology and Biological Sciences Research Council. The authors also thank Dr Ash Haeger for his help in the preparation of the tobacco alcohol-insoluble residue, and Dr Anthony Blake for providing a preliminary protocol for the use of xylanases on sections.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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