Abstract

Wood cell walls represent very complex polysaccharide-lignin biocomposites with a wide diversity of structural and physiological functions. In our work we have used tension wood G-layer as a reference native cellulose substrate for studies on wood fibre architecture with carbohydrate binding modules (CBMs) employed as molecular markers1,2. CBMs represent non-catalytic parts of enzymes involved in carbohydrate degradation and are characterized by high specificity to carbohydrates. CBMs are currently classified into 49 families based on sequence similarity (http://afmb.cnrs-mrs.fr/CAZY). For our studies, well characterized CBMs with high affinity towards crystalline cellulose (CBM1HjCel7A; CBM1PcCel7D) were heterologously expressed in E. coli and fused to detection tag protein (ZZ domain of Staphylococcus aureus protein A). Direct and indirect methods of carbohydrate mapping have been developed. For direct labelling CBM-tag complexes were conjugated to a fluorescence marker (FITC) while with the indirect method, CBM-tag protein complexes were visualised using labelled secondary antibodies for fluorescence and FE-SEM. Both CBM1HjCel7A and CBM1PcCel7D were shown as reliable and highly specific markers of crystalline cellulose in wood sections. Fluorescence signals in samples labelled with ZZ-CBM1HjCel7 were detected mostly within the G-layer that was clearly distinguished from surrounding secondary wall layers (S2, S1). No labelling was detected in tension wood samples after mild delignification due to change of cellulose crystallinity suggesting high specificity of CBM to crystalline cellulose. FE-SEM observations of poplar tension fibres revealed the ultrastructural architecture of the G-layer showing well-defined lamellae of loosely attached cellulose aggregates (i.e. aggregates of cellulose microfibrils) of the order 20-40 nm with a microfibril angle of ~ 0o. Studies on poplar G-layer marked with CBM1HjCel7A followed by Au-labelling and Ag-enhancement complemented fluorescence observations and showed labelling of individual cellulose aggregates . For further characterization of the G-layer aggregates we are currently applying cryo-techniques in conjunction with CBM labelling and TEM

Published in

Conference

Funcfiber 2008; International Symposium on the biology and Biotechnology of wood

SLU Authors

• Daniel, Geoffrey

  • Department of Forest Products, Swedish University of Agricultural Sciences
    

• Stålhandske, Lada

  • Department of Forest Products, Swedish University of Agricultural Sciences
    

• Sandquist, David

  • Department of Forest Products, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Forest Science


Permanent link to this page (URI)

https://res.slu.se/id/publ/17766