Enzyme‐gold affinity labelling of cellulose

Abstract

The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

The Fragile Fiber1 Kinesin Contributes to Cortical Microtubule-Mediated Trafficking of Cell Wall Components

“…Turning to cell wall composition, we first studied the distribution of cellulose in the cell walls of interfascicular fiber cells by staining sections with gold particles coated with cellobiohydrolase 2, a probe that binds crystalline cellulose (Berg et al, 1988). In fiber cells, the mean density of the probe was not particularly different between the genotypes; however, the staining was relatively uniform in wild-type cell walls but appeared uneven or striated in mutant walls (Fig.…”

“…Cell wall thickness was measured using ImageJ for the outermost two layers of interfascicular fiber cells, pith cells in the center of the stem, and cells with thick walls in the protoxylem. Cellulose was labeled with colloidal gold using enzyme-gold affinity cytochemistry as described earlier (Berg et al, 1988) and detailed in Supplemental Methods S1.…”

Localization of polysaccharides in isolated and intact cuticles of eucalypt, poplar and pear leaves by enzyme-gold labelling

“…Hence, the commercial mixtures Celluclast and Pectinex, conjugated with 10 nm citratecoated Au nanoparticles (~0.01%; Sigma-Aldrich) were used. The Au-conjugated enzymes were prepared shortly before tissue labelling according to the methods described by Berg et al [16] and Ferguson et al [17], with some modifications.…”

“…Gold-enzyme labelling has proved to be a reliable tool for the identification and localization of selected polysaccharides in plant cell walls [e.g., 16,17]. Working with spruce leaf transversal sections and Au-enzymatic labelling, Tenberge [18] identified cellulose and hemicelluloses cuticle by, but failed to detect pectins.…”

Immunocytochemical studies of the infection mechanisms of Botrytis fabae II. Host cell wall breakdown

“…In this present study, two probes directed against major components of the plant cell wall were used to investigate the process of cell wall deconstruction in early stages of development of the V. faba-B. fabae interaction, more commonly known as chocolate spot disease of broad bean (Harrison, 1988) : The anti-pectin monoclonal antibody (MAb) JIM 7 was used to label methyl-esterified pectin Vian & Roland, 1991) and the enzyme cellobiohydrolase (CBH1) conjugated to gold was used to label cellulose (Chanzy, Henrissat & Vuong, 1984 ;Berg et al, 1988 ;Vian & Roland, 1991). We previously reported (Cole, Dewey & Hawes, 1998) that both external and internal (i.e.…”