6954 * ApPLIED AND ENVIRONMENTAL MICROBIOLOGY. Sept. 1989. p. 2293-2301 Vol. 55. NO.9 0099·2240/89/092293·09$02.00/0 Copyright © 1989. American Society for Microbiology Colloidal Gold Cytochemistry of Endo-l,4-f3-Glucanase, l,4-f3-D-Glucan Cellobiohydrolase, and Endo-l,4-f3-Xylanase: Ultrastructure of Sound and Decayed Birch Woodt ROBERT A. BLANCHETTE," ANDRE R. ABAD,' KORY R. CEASE,' REX E. LOVRIEN,2 AND TIMOTHY D. LEATHERS' Department ofPlant Pathologl and Department ofBiochemistry,2 University ofMinnesota, St. Paul, Minnesota' 55108. and Northern Regional Research Center, Agricultural Research Service, U.S. Department ofAgricullllre, Peoria, Illinois 616043 Received 7 April 1989/Accepted 26 June 1989 Colloidal gold coupled to endo-l,4-~-glucanase II (EG II) and 1,4-~-D-glucan cellobiohydrolase 1 (CBH I), isolated- from TricIJodenna reesei (QM9414), and endo~1,4~p,~xylanasefrom Aureobasill11J pllllulans (NRRLY­ 2311-1) was used successfully to determine the ultrastructural localization ofcellulose 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, PhaneroclJae/e chrysosporillln, Phellinlls pini, and Trametes versicolor, and one brown rot fungus, F011Jitopt's pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the Sf layer ofthe secondary wall, whereas CBH I labeled ~lIlayers ofthe 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 It whereas only CBH I had a specificity for the cut ends ofmicrofibrHs. Labeling with the xylanase~gold complex occurred primarily in the inner regions of the 5z layer, 51' and the middle lameHa. In contrast, little labeling occurred in the middle lameHa with EG II or CBH I. InterceJJular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG 1I~ and xylanase~gold were used. Wood decayed by P. chrysosporium or P. pint' was delignified, and extensive degradation of the middle lameHa was evident. The remaining secondary walls labeled with EG II and CBH 1, but little labeling was fouud with the xylauase-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 8 1 layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Bro\Vn~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 5z layer near ceU lumina and corresponded to sites with the most extensive degradation. Microbial degradation of wood polysaccharides by fungi major hemicellulose is xylan, constituting approximately 20 involves the combined actions ofseveral enzymes. White rot to 25% of the wood (9, 39). Hcmicellulase activity, including fungi produce endo-l,4-~-glucanase (EO) (EC 3.2.1.4) that endo-l,4-[3-xylanase (EC 3.2.1.8), has been found in culture attacks the cellulose chain at random (13, 16, 44) and filtrates of many white and brown rot fungi (23, 27). exo-l,4-~-glucanase (1,4-~-D-glucan cellobiohydrolase [EC Advances in colloidal gold cytochemistry make it possible 3.2.1.91]) (CBH) that attacks the nonreducing end of the to use enzyme-gold complexes to label specific substrates. crystalline cellulose chain, splitting off glucose or cellobiose Since first used to localize nucleic acids in sectioned material (13, 15). It has been postulated that white rot fungi use both (2). the technique has been used with many plant tissues to enzymes synergistically to degrade a cellulosic substrate (15, label polysaccharides (43, 45), pectins (3), and plant and 25, 42). As white rot fungi gradually degrade cellulose, fungal cell walls (4, 10). In the study of Berg et al. (4), crude degradation products are rapidly utilized (34). In contrast, cellulase preparations and purified EO and CBH I were used brown rot fungi cause a rapid depolymerization of cellulose to show specific labeling of fungal and plant cell walls early in the decay process (14, 24). The only cellulase containing cellulose. Colloidal gold labeling of CBH 1 from enzyme reported from brown rot fungi is EO (21, 22), and it Trichoderma reesei also has been used to visualize specific apparently is a multifunctional enzyme, active on both absorption sites on the surface of microcrystalline cellulose polysaccharides and glycosides (46). The exact mechanism produced by Valollia macrophyso (11). Specificity of this responsible for extensive and rapid depolymerization of enzyme for crystal edges, rather than ends, suggested that cellulose is uncertain; however. a nonenzymatic process has CBH I was acting as an endoglucanase-type enzyme. In been strongly suspected during decay by brown rot fungi (14, addition, CBH I alone has been shown to degrade highly 26, 31, 34). crystalline cellulose (12). In addition to cellulose, hemicelluloses also occur in Colloidal gold-xylanase complexes have been used to various amounts within cell walls of wood. In birch, the identify the presence of xylan in cells of Tilia plalyplyllos (45). In this study the secondary wall layers were labeled with the xylanase-gold. but the middle lamella region and * Corresponding author. cell lumina were free from labeling. Ruel and Joseleau (43) t Published as contribution 16.992 of the series of the Minnesota were able to successfully label mannanase in Picea cell Agricultural Experiment Station. walls. Gold particles were distributed over the entire surface 2293 2294 BLANCHETTE ET AL. ArrL. ENVIRON. MICROBIOL. ofthe wall, with the greatest concentration in the S21ayer of the secondary wall. The present study was done to demonstrate the specificity of EG II, CBH I, and xylanase for cell walls of sound birch (Betula papyri/era) wood and wood decayed by brown or white rot fungi and to determine' the ultrastructurallocaliza­ tion of these enzymes wi.thin woody cell walls. One brown rot fungus, Fomitopis pinicola, and three white rot fungi, Pizanerocizaete chrysosporiufl'l, Phellillus pini, and Trametes versicolor, were used in this study. The white rot fungi represented species responsible for two different forms of decay (5, 38): selective degradation of lignin (P. clzrysas­ porium and P. pinf) and nonselective degradation of all cell wall components (T. versicolor). MATERIALS AND METHODS Wood blocks of sound, freshly cut birch, B. papyri/era, were decayed in the laboratory for 12 weeks as previously described (39). The wood blocks were incubated in the dark at 27'C and a relative humidity of approximately 90%. With these methods a combination of intermediate and advanced stages of decay could be expected within the wood blocks (8). Three white rot fungi, T. (Coriaills) versicolor (MAD 697-R), P. clzrysospori1lln (BKM-F-1767), and P. pilli (RAB­ FlO. 1. Transmission electron micrograph of sound birch wood 83-19), and one brown rot fungus, F. pillicola (RAB-32), fixed in KMnO.;, showing cell wall layers in fibers (F) and vessels were used to inoculate the wood blocks. Noninol;ulated (V). ML, Middle lamella (arrows indicate intercellular region of cell wood blocks served as controls. corner); $1> Sz, and S), layers of secondary walls. No S) is apparent Sound and decayed wood blocks were cut into small in fibers. Bar = 2 j.Lm. segments (approximately 1 by I by 0.4 mm) and fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 after ultracentrifugation with citric phosphate buffer, pH 4.5. for 2 h. Samples were rinsed in buffer three times for 20 min containing 0.6% NaCI and 0.05% Tween 80. The EG II and each and dehydrated through a graded acetone series. Em­ eBB I probes were suspended after ultracentrifugation with bedding procedures with Quetol 651 resin (32) were as citric phosphate buffer, pH 5.4, containing 0.06% NaCI and previously described (1). Samples in resin were cured at 0.05% Tween 80. Sections on grids were floated in citric 74'C for 8 h and cut with a diamond knife. Sections (100 to phosphate buffer, at pH 4.5 or 5.4, for 5 min and then placed 120 nm) were collected on nickel grids. Additional samples in the respective enzyme-gold complexes for 15 min. To were cut from the wood blocks and fixed with 2% KMn04 in determine the effects ofa longer incubation time on labeling. distilled water, followed by dehydration in Quetol 651 and additional sections ofsound wood were also incubated for 30 embedment (1) to observe the extent of lignification within min in EG II- or CBH I-gOld complexes. Grids were rinsed cell walls. with a jet spray of distilled H20 and air dried. The sections Colloidal gold solutions with a gold particle size of13 to 15 were observed and examined with a Hitachi 600 transmis­ nm were produced using the methods of Frens (18). Highly sion electron microscope without additional staining. Cy­ purified EG !l and CBH I were obtained from T. re.esci tochemical controls included labeling with heat-treated EG (QM9414), using procedures described previously (37), and II, CBH I, or xylanase and labeling with nonenzymatic endo-1,4-fj-xylanase was obtained from Aureobasidium pul­ proteins (4). Iliialls (NRRLY-2311-1) by methods of Leathers (33; T. D. Leathers, J. Ind. Microbiol., in press). Each enzyme was combined with the colloidal gold solutions at pH 7.4 by RESULTS methods of Geoghegan and Ackerman (19) and Goodman et Cell walls of fibers and vessels in birch wood.