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Synthetic xylan-binding modules for mapping of pulp fibres and wood sections.

Filonova L, Gunnarsson LC, Daniel G, Ohlin M - BMC Plant Biol. (2007)

Bottom Line: However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties.Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules.Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: WURC, Department of Wood Science, Swedish University of Agricultural Sciences, PO Box 7008, SE-750 07 Uppsala, Sweden. lada.filonova@trv.slu.se

ABSTRACT

Background: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.

Results: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

Conclusion: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

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Related in: MedlinePlus

Binding of FITC-conjugated X-2 to wood samples. Pine and birch sections stained with either FITC-conjugated G-4 (A-D) (negative control) or with FITC-conjugated X-2 (E-L). Sections were either untreated (A-H) or delignified (I-L) prior to staining. Pink arrows show signals in untreated samples. Note that A, C, E, G, I and K are from transverse sections and B, D, F, H, J and L are from longitudinal sections. Pine sections in A and E show both early- (to the left) and latewood (to the right). Scale bar = 50 μm.
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Figure 4: Binding of FITC-conjugated X-2 to wood samples. Pine and birch sections stained with either FITC-conjugated G-4 (A-D) (negative control) or with FITC-conjugated X-2 (E-L). Sections were either untreated (A-H) or delignified (I-L) prior to staining. Pink arrows show signals in untreated samples. Note that A, C, E, G, I and K are from transverse sections and B, D, F, H, J and L are from longitudinal sections. Pine sections in A and E show both early- (to the left) and latewood (to the right). Scale bar = 50 μm.

Mentions: Staining of delignified tissue samples (Figure 4I–L) with FITC-conjugated X-2 resulted in much higher signals in comparison to staining of mature (normal) birch and pine tissues (Figure 4E – H). In normal pine sections, X-2 binding was mainly detected in border pit regions (earlywood) and inner surface of cells (latewood) (Figure 4E, F) whereas in birch section staining was observed mostly in middle lamellae regions (Figure 4G, H). In contrast, signals from X-2 binding were evenly distributed over the surfaces of delignified sections in both wood types (Figure 4I – L). This finding suggests, as expected, that removal of lignin increases the access to the xylan binding sites. Staining of delignified samples by various CBMs may be beneficially preformed to ensure the efficiency of the delignification procedure and to exploit the access of specific carbohydrate epitopes prior to enzyme treatment (high cost procedures) of wood materials. Similarly, hemicellulose removal following a bleaching procedure could be efficiently assessed in pulp fibres (Figure 5) further expanding the area of useful applications of these molecularly engineered modules.


Synthetic xylan-binding modules for mapping of pulp fibres and wood sections.

Filonova L, Gunnarsson LC, Daniel G, Ohlin M - BMC Plant Biol. (2007)

Binding of FITC-conjugated X-2 to wood samples. Pine and birch sections stained with either FITC-conjugated G-4 (A-D) (negative control) or with FITC-conjugated X-2 (E-L). Sections were either untreated (A-H) or delignified (I-L) prior to staining. Pink arrows show signals in untreated samples. Note that A, C, E, G, I and K are from transverse sections and B, D, F, H, J and L are from longitudinal sections. Pine sections in A and E show both early- (to the left) and latewood (to the right). Scale bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099432&req=5

Figure 4: Binding of FITC-conjugated X-2 to wood samples. Pine and birch sections stained with either FITC-conjugated G-4 (A-D) (negative control) or with FITC-conjugated X-2 (E-L). Sections were either untreated (A-H) or delignified (I-L) prior to staining. Pink arrows show signals in untreated samples. Note that A, C, E, G, I and K are from transverse sections and B, D, F, H, J and L are from longitudinal sections. Pine sections in A and E show both early- (to the left) and latewood (to the right). Scale bar = 50 μm.
Mentions: Staining of delignified tissue samples (Figure 4I–L) with FITC-conjugated X-2 resulted in much higher signals in comparison to staining of mature (normal) birch and pine tissues (Figure 4E – H). In normal pine sections, X-2 binding was mainly detected in border pit regions (earlywood) and inner surface of cells (latewood) (Figure 4E, F) whereas in birch section staining was observed mostly in middle lamellae regions (Figure 4G, H). In contrast, signals from X-2 binding were evenly distributed over the surfaces of delignified sections in both wood types (Figure 4I – L). This finding suggests, as expected, that removal of lignin increases the access to the xylan binding sites. Staining of delignified samples by various CBMs may be beneficially preformed to ensure the efficiency of the delignification procedure and to exploit the access of specific carbohydrate epitopes prior to enzyme treatment (high cost procedures) of wood materials. Similarly, hemicellulose removal following a bleaching procedure could be efficiently assessed in pulp fibres (Figure 5) further expanding the area of useful applications of these molecularly engineered modules.

Bottom Line: However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties.Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules.Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: WURC, Department of Wood Science, Swedish University of Agricultural Sciences, PO Box 7008, SE-750 07 Uppsala, Sweden. lada.filonova@trv.slu.se

ABSTRACT

Background: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.

Results: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

Conclusion: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

Show MeSH
Related in: MedlinePlus