Colloidal Gold Cytochemistry of Endo-1,4-β-Glucanase, 1,4-β- D -Glucan Cellobiohydrolase, and Endo-1,4-β-Xylanase: Ultrastructure of Sound and Decayed Birch Wood

Abstract

Colloidal gold coupled to endo-1,4-β-glucanase II (EG II) and 1,4-β- D -glucan cellobiohydrolase I (CBH I), isolated from Trichoderma reesei (QM9414), and endo-1,4-β-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, Phanerochaete chrysosporium, Phellinus pini , and Trametes versicolor , and one brown rot fungus, Fomitopis pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the S 1 layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for the cut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of the S 2 layer, S 1 , and the middle lamella. In contrast, little labeling occurred in the middle lamella with EG II or CBH I. Intercellular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG II- and xylanase-gold were used. Wood decayed by P. chrysosporium or P. pini was delignified, and extensive degradation of the middle lamella was evident. The remaining secondary walls labeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites of erosion labeled with both EG II and CBH I. Erosion troughs that reached the S 1 layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the S 2 layer near cell lumina and corresponded to sites with the most extensive degradation.