Morphological and chemical characterisation of the G-layer in tension wood fibres of Populus tremula and Betula verrucosa: Labelling with cellulose-binding module CBM1(HjCel7A) and fluorescence and FE-SEM microscopy

The gelatinous layer (G-layer) formed on the lumen wall in early- and latewood fibres of poplar and birch tension wood was characterised using a novel molecular marker specific for crystalline cellulose in conjunction with fluorescence and FE-SEM microscopy. Crystalline cellulose was localised using a cloned Cel7A cellulose-binding module (CBM1(HjCel7A)) from the fungus Hypocrea jecorina conjugated directly to FITC/TRITC or indirectly via a secondary antibody conjugated to FITC for fluorescence microscopy or to gold/silver for FE-SEM. With the CBM1(HjCel7A) conjugate, the G-layer was clearly distinguished from other secondary cell-wall layers as a bright green layer visible in fibres of tension wood in fluorescence microscopy. FEM-SEM images revealed the supramolecular architecture of the G-layer of poplar wood, which consists of well-defined, often concentrically orientated, cellulose aggregates of the order of 30-40 nm. The cellulose aggregates typically have a microfibril angle of almost 0 degrees. Studies on cellulose marked with CBM1(HjCel7A) followed by Au labelling and Ag enhancement complemented the fluorescence observations. The studies demonstrate the usefulness of this novel molecular marker for crystalline cellulose in situ, which was previously difficult to localise. Further proof of distinct cellulose aggregates was observed.