APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1989, p. 2293-23010099-2240/89/092293-09$02.00/0Copyright © 1989, American Society for Microbiology

Colloidal Gold Cytochemistry of Endo-1,4-13-Glucanase,1,4-r-D-Glucan Cellobiohydrolase, and Endo-1,4-3-Xylanase:

Ultrastructure of Sound and Decayed Birch WoodtROBERT A. BLANCHETTE,'* ANDRE R. ABAD,1 KORY R. CEASE,'

REX E. LOVRIEN,2 AND TIMOTHY D. LEATHERS3

Department of Plant Pathology' and Department of Biochemistry,2 University of Minnesota,St. Paul, Minnesota 55108, and Northern Regional Research Center, Agricultulral

Research Service, U.S. Department of Agricultuire, Peoria, Illinois 616043

Received 7 April 1989/Accepted 26 June 1989

Colloidal gold coupled to endo-1,4-I0-glucanase II (EG II) and 1,4-0-D-glucan cellobiohydrolase I (CBH I),isolated from Trichoderma reesei (QM9414), and endo-1,4-,B-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birchwood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan afterdecay 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 localizedprimarily on the SI layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Obliquesections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of themicrofibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for thecut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of theS2 layer, SI, 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 labeledpositively 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 wallslabeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed byT. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sitesof erosion labeled with both EG II and CBH I. Erosion troughs that reached the S, layer or the middle lamellahad less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levelsof gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowestconcentrations in the S2 layer near cell lumina and corresponded to sites with the most extensive degradation.

Microbial degradation of wood polysaccharides by fungiinvolves the combined actions of several enzymes. White rotfungi produce endo-1,4-,B-glucanase (EG) (EC 3.2.1.4) thatattacks the cellulose chain at random (13, 16, 44) andexo-1,4-3-glucanase (1,4-p-D-glucan cellobiohydrolase [EC3.2.1.911) (CBH) that attacks the nonreducing end of thecrystalline cellulose chain, splitting off glucose or cellobiose(13, 15). It has been postulated that white rot fungi use bothenzymes synergistically to degrade a cellulosic substrate (15,25, 42). As white rot fungi gradually degrade cellulose,degradation products are rapidly utilized (34). In contrast,brown rot fungi cause a rapid depolymerization of celluloseearly in the decay process (14, 24). The only cellulaseenzyme reported from brown rot fungi is EG (21, 22), and itapparently is a multifunctional enzyme, active on bothpolysaccharides and glycosides (46). The exact mechanismresponsible for extensive and rapid depolymerization ofcellulose is uncertain; however, a nonenzymatic process hasbeen strongly suspected during decay by brown rot fungi (14,26, 31, 34).

In addition to cellulose, hemicelluloses also occur invarious amounts within cell walls of wood. In birch, the

* Corresponding author.t Published as contribution 16,992 of the series of the Minnesota

Agricultural Experiment Station.

major hemicellulose is xylan, constituting approximately 20to 25% of the wood (9, 39). Hemicellulase activity, includingendo-1,4-,B-xylanase (EC 3.2.1.8), has been found in culturefiltrates of many white and brown rot fungi (23, 27).Advances in colloidal gold cytochemistry make it possible

to use enzyme-gold complexes to label specific substrates.Since first used to localize nucleic acids in sectioned material(2), the technique has been used with many plant tissues tolabel polysaccharides (43, 45), pectins (3), and plant andfungal cell walls (4, 10). In the study of Berg et al. (4), crudecellulase preparations and purified EG and CBH I were usedto show specific labeling of fungal and plant cell wallscontaining cellulose. Colloidal gold labeling of CBH I fromTrichoderma reesei also has been used to visualize specificabsorption sites on the surface of microcrystalline celluloseproduced by Valonia macrophysa (11). Specificity of thisenzyme for crystal edges, rather than ends, suggested thatCBH I was acting as an endoglucanase-type enzyme. Inaddition, CBH I alone has been shown to degrade highlycrystalline cellulose (12).

Colloidal gold-xylanase complexes have been used toidentify the presence of xylan in cells of Tilia platyplyllos(45). In this study the secondary wall layers were labeledwith the xylanase-gold, but the middle lamella region andcell lumina were free from labeling. Ruel and Joseleau (43)were able to successfully label mannanase in Picea cellwalls. Gold particles were distributed over the entire surface

2293

Vol. 55, No. 9

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2294 BLANCHETTE ET AL.

of the wall, with the greatest concentration in the S2 layer ofthe 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 papyrifera) wood and wood decayed by brown orwhite rot fungi and to determine the ultrastructural localiza-tion of these enzymes within woody cell walls. One brownrot fungus, Fomitopis pinicola, and three white rot fungi,Phanerochaete chiysospor-ium, Phellinus pini, and Trametesversicolor, were used in this study. The white rot fungirepresented species responsible for two different forms ofdecay (5, 38): selective degradation of lignin (P. chrysos-poriirn and P. pini) and nonselective degradation of all cellwall components (T. versicolor).

MATERIALS AND METHODS

Wood blocks of sound, freshly cut birch, B. papyrifera,were decayed in the laboratory for 12 weeks as previouslydescribed (39). The wood blocks were incubated in the darkat 27°C and a relative humidity of approximately 90%. Withthese methods a combination of intermediate and advancedstages of decay could be expected within the wood blocks(8). Three white rot fungi, T. (Coriolus) versicolor (MAD697-R), P. chrysosporium (BKM-F-1767), and P. pini (RAB-83-19), and one brown rot fungus, F. pinicola (RAB-32),were used to inoculate the wood blocks. Noninoculatedwood blocks served as controls.Sound and decayed wood blocks were cut into small

segments (approximately 1 by 1 by 0.4 mm) and fixed in 1%glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4for 2 h. Samples were rinsed in buffer three times for 20 mineach and dehydrated through a graded acetone series. Em-bedding procedures with Quetol 651 resin (32) were aspreviously described (1). Samples in resin were cured at74°C for 8 h and cut with a diamond knife. Sections (100 to120 nm) were collected on nickel grids. Additional sampleswere cut from the wood blocks and fixed with 2% KMnO4 indistilled water, followed by dehydration in Quetol 651 andembedment (1) to observe the extent of lignification withincell walls.

Colloidal gold solutions with a gold particle size of 13 to 15nm were produced using the methods of Frens (18). Highlypurified EG II and CBH I were obtained from T. reesei(QM9414), using procedures described previously (37), andendo-1,4-,-xylanase was obtained from Aureobasidium pial-lulans (NRRLY-2311-1) by methods of Leathers (33; T. D.Leathers, J. Ind. Microbiol., in press). Each enzyme wascombined with the colloidal gold solutions at pH 7.4 bymethods of Geoghegan and Ackerman (19) and Goodman etal. (20) to form the enzyme-gold complex. The amount ofenzyme used to stabilize 0.5 ml of colloidal gold solution was0.01 to 0.02 mg. The concentration of all gold complexes wasdetermined using a spectrophotometer at 560 nm and anabsorbance of 0.27. Xylanase-gold probes were suspended

FIG. 1. Transmission electron micrograph of sound birch woodfixed in KMnO4, showing cell wall layers in fibers (F) and vessels(V). ML, Middle lamella (arrows indicate intercellular region of cellcorner); Sl, S2, and S3, layers of secondary walls. No S3 is apparentin fibers. Bar = 2 ,um.

after ultracentrifugation with citric phosphate buffer, pH 4.5,containing 0.6% NaCl and 0.05% Tween 80. The EG II andCBH I probes were suspended after ultracentrifugation withcitric phosphate buffer, pH 5.4, containing 0.06% NaCl and0.05% Tween 80. Sections on grids were floated in citricphosphate buffer, at pH 4.5 or 5.4, for 5 min and then placedin the respective enzyme-gold complexes for 15 min. Todetermine the effects of a longer incubation time on labeling,additional sections of sound wood were also incubated for 30min in EG II- or CBH I-gold complexes. Grids were rinsedwith a jet spray of distilled H20 and air dried. The sectionswere observed and examined with a Hitachi 600 transmis-sion electron microscope without additional staining. Cy-tochemical controls included labeling with heat-treated EGII, CBH I, or xylanase and labeling with nonenzymaticproteins (4).

RESULTS

Cell walls of fibers and vessels in birch wood, fixed inKMnO4 and prepared for transmission electron microscopy,show the ultrastructure of various cell wall layers (Fig. 1).Transverse thin sections of wood incubated with the gold-EG II complex, using a 30-min incubation, showed that theenzyme had a preference for the S, layer of secondary walls

FIG. 2. Transmission electron micrographs of transverse (a, b, e, and f) and oblique (c and d) sections of sound birch wood incubated withEG Il-gold (a to c) and CBH 1-gold (d to f). (a) Section incubated with EG Il-gold for 30 min, showing intense labeling of the SI layer and somelabeling of other fiber cell wall layers. In vessels, SI and S3 have high concentrations of gold. (b) Incubation of EG II-gold for 15 min, withreduced concentrations of gold throughout the cell wall. The preference of EG II for the S, layer is evident. Low levels of gold labeling areseen in S2, and no labeling is evident in the electron-dense middle lamella. Intercellular areas of cell corner regions, however, were labeled(arrows). (c) Extensive labeling of all cell wall layers of fibers and vessels is observed in an oblique section. (d) CBH I-gold labeling of all cellwall layers in an oblique section, with the greatest concentrations in the S, layer. (e) Incubation of CBH I for 30 min, showing labeling of allsecondary wall layers in fibers and vessels. (f) Fifteen-minute incubation with CBH I, exhibiting moderate labeling of secondary wall layersbut not in boundaries between SI-S2 in fibers and SI-S, or S2-S3 in vessels or in the middle lamella. No labeling was apparent in the intercellularregions of cell corners (arrows). Bar = 2 ,um.

APPL. ENVIRON. MICROBIOL.

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

ULTRASTRUCTURE OF SOUND AND DECAYED BIRCH WOOD 2295

_ :._ ,_ v__ x

^fis-4sso<tA ,,;9.ts -s t i>o s *9s Z_

S 't: ,'7 Fll 11|_nn ,<- E >-1111_

A, ' ,j: s -a -mx g rE-ERt 'tX 00 . S

::f .:: W 8 i-.l 1X Mi:::ks :; 7:. s W.i R F

:,'.*:twi:t:' 4*1:.S Ay': t00: ::S,l :: .Sb t*s * f *00 At X, aRk ., u 1a- s $0. 'Dr f.-fi00 s 2 d

iS 7: 'S Wt: :0E -f ii E <

id- 0dX ,Sj

.t: tiT z ?rL; -ik

.. .. f.g.;;

.

.. ,4it S 4

t --, A-S ;::f -. X-

.& 4?,00 :: .W 0: . i:. .rAv . ;*S> &

C

e

oN.-Msi

00D~, X,

VOL. 55, 1989

,11 ",

" "r

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2296 BLANCHETTE ET AL.

in fibers and vessels (Fig. 2a). The boundary zones betweenS1 and the compound middle lamella and S1S2 layers werelabeled intensely. The S2 layer of fiber walls contained smallamounts of label near the lumen, but concentrations in-creased toward the S1 layer. Vessel cell walls were labeledthroughout all layers, with greatest concentrations in S, andS3, while concentrations of gold particles within the middlelamella remained low. Usually, some labeling occurred inthe cell corner regions but very slight amounts in the middlelamella between cells (Fig. 2a).

Incubation times used for labeling sections with enzyme-gold complexes affected the intensity of gold concentrationsobserved. Longer incubation times of 30 min fully saturatedall available sites for enzyme activity and resulted in anincreased amount of background labeling that was not spe-cific or representative of sites for enzyme activity. Sectionsincubated for a shorter time, 15 min, had considerably lesslabeling, but the location of the labeling and the patterns ofgold particle deposition were similar to those of the longerincubation time (Fig. 2b). A preference of EG II for the SIlayer was evident. In sections of wood, spaces were oftenobserved that were less electron dense in cell corners. Goldparticles were always present in these areas (Fig. 2b, ar-rows). The middle lamella, however, remained relativelyfree from gold particles. When sections of birch wood werecut at oblique angles instead of in a transverse section,moderate amounts of gold labeling were observed through-out the secondary cell wall (Fig. 2c). Results were similarusing the 15- and 30-min-incubation procedures. Otheroblique sections incubated with CBH I also showed labelingthroughout the secondary wall, with the greatest concentra-tions in S, and the least within the middle lamella (Fig. 2d).Transverse sections of wood incubated with CBH I for 30min (Fig. 2e) or 15 min (Fig. 2f) exhibited labeling in allsecondary wall layers of fibers and vessels. The distributionof gold appeared to be relatively uniform throughout thecells. The boundary zones of SI-S, and S2-S3 in vessels andS1-S2 in fibers were distinct and labeled to a lesser extentthan other areas. The middle lamella and the less electron-dense spaces within the cell corner regions were not labeledto any appreciable extent.

All sections of wood decay by brown or white rot fungiwere treated with EG II or CBH I, using the 15-minincubation. Wood decayed by T. versicolor showed anattack on all cell wall layers (Fig. 3a and b). The secondarywall was eroded from cell lumina, where hyphae werelocated toward the middle lamella. Sections incubated withEG II were labeled primarily in the S, layer (Fig. 3a). Inareas of the cell where the secondary wall was eroded toexpose the S, layer, a reduced amount of labeling wasevident (Fig. 3a, arrow). Sections incubated with CBH I hadgold particles distributed throughout the residual secondarywall that remained after degradation (Fig. 3b). Althoughextensive cell wall erosion had taken place in some cells, the

cell wall immediately adjacent to the decayed areas had largeconcentrations of gold particles.Wood decayed by P. pini or P. chrysosporium showed

evidence of selective delignification (Fig. 3c and d). In cellswith advanced decay, the middle lamella regions werecompletely degraded while the S, and S2 layers remained.These cell wall layers appeared slightly swollen, but no cellwall erosion was evident. EG 1I-treated sections of deligni-fied cells showed labeling within the S, layer (Fig. 3c).Sections with CBH I had labeling throughout the secondarywall (Fig. 3d). Even in cells where lignin was removed fromthe secondary wall and the entire middle lamella was re-moved, the secondary wall maintained high concentrationsof gold particles.Wood decayed by the brown rot fungus F. pinicola was

extensively degraded, and secondary wall layers were swol-len and appeared porous. Both secondary wall layers and themiddle lamella remained discernible after the 12 weeks ofdecay. Sections of brown-rotted wood incubated with EG IIhad very low levels of labeling (Fig. 3e). Only a thin outlineof the S, layer was evident by the gold particles. The CBHI-gold treatment displayed high concentrations of gold in thesecondary walls, but in many cells considerably less goldwas present in the S2 layer near cell lumina (Fig. 3f). Nolabeling of the middle lamella was observed for CBH I or EGII.A xylanase-gold probe used on transverse sections of

sound wood showed some labeling in all cell wall layers, butthe greatest concentrations were found within the middlelamella and adjacent secondary wall regions (Fig. 4a and b).The distribution of gold particles within cell walls variedamong different samples of sound wood observed, with somecells exhibiting more labeling of secondary wall layers thanothers (Fig. 4a and b). Sections from wood decayed by T.versicolor and incubated with the xylanase-gold complexshowed gold labeling that was localized primarily in S, andthe middle lamella. Few gold particles were observed inareas where erosion troughs, produced by the fungus,reached S, or the middle lamella (Fig. 4c, arrows). Goldconcentrations in other parts of the cell were similar to thoseobserved in sections of sound wood. Spaces appearing lesselectron dense within the cell corner regions of the middlelamella were labeled with the xylanase-gold probe. Wooddelignified by P. pini or P. chrysosporium had gold labelingonly in cell corner regions (Fig. 4d and e). The middlelamella between cells was in various stages of decomposi-tion. In these areas where the middle lamella was removed,no gold particles were evident, reflecting the lack of xylan.Electron-dense areas of the middle lamella which apparentlywere not delignified were sites for gold labeling. Very fewgold particles were found in sections of wood decayed by F.pinicola, the brown rot fungus (Fig. 4f). Only scatteredparticles of gold occurred randomly over the various cellwall layers, indicating the absence of xylan in the wood.

FIG. 3. Transmission electron micrographs of transverse sections of wood decayed for 12 weeks by T. versicolor (a and b), P. pini (c), P.chrysosporium (d), and F. pinicola (e and f). (a) Erosion of cell wall layers removed portions of the cell wall. Labeling with EG II wasrestricted to the S1 layer. Intercellular regions of cell corners are labeled (arrow). Hyphae (H) are located in cell lumina. (b) Eroded cell walls,showing gold labeling of remaining wall layers with CBH I. Intercellular regions are not labeled (arrow). (c) Delignified fibers after extensivedegradation of the middle lamella show gold labeling by EG primarily in the S1 layer. (d) Partially delignified fibers exhibit areas with acompletely degraded middle lamella (arrowheads) and areas with an intact middle lamella. CBH I labeling occurs in all secondary wall layers.(e) Sections of brown-rotted wood, with cells having a porous, swollen appearance. Little gold labeling of the SI layer is evident with EG II.(f) Section labeled with CBH I, showing moderate to low levels of gold in the secondary wall. Concentrations of gold particles were extremelylow in extensively degraded areas of cell walls. A fiber is seen with small quantities of gold near cell lumina (arrowheads), which is the mostseverely degraded part of the cell. Bar = 2 ,um.

APPL. ENVIRON. MICROBIOL.

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

~~ULTRASTRUCTUJRE OF SOUND AND DECAYED BIRCH WOOD

aVA-

H

S2

IE~~~~~

d~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~el-

f

~VI

2297VOL. 55, 1989

.i,4 Im"

- jli.' -1 X .."

il. I'll4e

t

S

-

W..:.I .4

1 '.

.t

PI

11.. "-'

Z -, I

I

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2298 BLANCHETTE ET AL. APPL. ENVIRON. MICROBIOL.

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

ULTRASTRUCTURE OF SOUND AND DECAYED BIRCH WOOD

All cytochemical controls used in this study for eachenzyme were negative, and no labeling was observed.

DISCUSSION

Colloidal gold cytochemistry. using EG II, CBH I, andxylanase, successfully demonstrated the ultrastructural lo-calization of cellulose and xylan substrates within cell wallsof birch wood. The microfibrillar orientation of cellulosevaries among different layers of the secondary wall (40), andthese differences affect the sites of endo- and exo-typeenzyme activity. In transverse sections of wood, EG II wasspecific for the SI layer rather than the S layer and orien-tations of the cut microfibrils were not sites for EG IIbinding. However, in oblique sections, where the mi-crofibrils are cut differently, exposing their sides, EG II wasable to label intensely the S, layer. These results confirm theaffinity of this enzyme for lateral faces of microfibrils and nottheir cut edges. Labeling of the SI layer in transverse sectionalso suggests an affinity for sides of the crystalline cellulosestructure since the direction of microfibrils is different in thislayer than in the S. layer. Differences in the amount ofcrystalline and amorphous cellulose also may vary betweenthese two layers (40). Most crystalline cellulose is located inthe S, region.

Results with the CBH I-gold complex indicated that largeconcentrations of gold particles were present in transverseand oblique sections. Since specificity for CBH I is consid-ered to take place at the nonreducing ends of the cellulosechains (13, 15), positive labeling of transverse sections wasexpected since large quantities of end chains would occur atthis cut surface. The intense labeling of the oblique sections,where few end chains would be expected, raises the possi-bility that this enzyme has some characteristics of an en-doglucanase. Chanzy et al. (11) also demonstrated that thesides of microcrystals produced by V. inacrophzysi were welllabeled when treated with colloidal gold-CBH I. In anotherstudy (12) it was demonstrated that CBH I alone was able todegrade highly crystalline Valoniia cellulose. The results ofour investigations also show affinity of CBH I for all spatialaspects of cellulose chains within woody cell walls andsupport the possibility that this enzyme recognizes morethan just the nonreducing ends of cellulose.

Micrographs of the xylanase probe demonstrated that theenzyme had endo-type activity and that gold particles weredistributed primarily in the SI layer and the middle lamella.Although some xylan is undoubtedly located throughout thesecondary wall (6), the greatest concentration appears ininner regions of woody cell wall. Other investigations havesuggested that lignin and hemicellulose are closely associ-ated within cell walls (28, 40). Areas of the cell with higherconcentrations of lignin, such as the middle lamella (8),would also be expected to have a higher hemicellulosecontent. The information presented here, using a xylanaseprobe, supports these previous findings.

Within cell corner regions of middle lamellae, less-elec-tron-dense areas that apparently do not stain with KMnO4

and do not appear lignified are frequently observed (Fig. 1).These areas, often referred to as intercellular spaces (17), aresites where EG II-gold and xylanase-gold probes had posi-tive labeling. No labeling, however, occurred with the CBHI-gold probe. These results demonstrate that these areas arenot voids but contain hemicellulose and probably noncrys-talline forms of cellulose. The presence of polysaccharideswithin nonlignified zones of the middle lamella in hardwoodspecies is an interesting phenomenon that has not beenreported previously.

Colloidal gold labeling of sections from wood blocksdecayed by P. cIlrsosporilnm or P. pinii with EG II or CBHI indicated that cellulose was not substantially removed afterextensive delignification. In sections where the middle la-mella had been completely degraded and secondary wallswere free from lignin, patterns of gold labeling by EG II orCBH I were not different from those observed in cell walls ofsound wood. Chemical analyses from previous investiga-tions of wood decayed by P. chrysosporium and P. pini,using the same time period of degradation, demonstratedthat after a weight loss of 17 to 39%, 54 to 73% of the lignin,5 to 15%c of the cellulose, and 30 to 55% of the xylan wereremoved (7, 39). The small percentage of cellulose degradedby these fungi apparently was not sufficient to detect achange in the labeling of EG II or CBH I. Although thesecondary wall was swollen and chemically altered, acces-sibility to EG II was not enhanced. The loss of xylan fromthe wood, however, was detected when the xylanase-goldprobe was used. These results support the view that whenlignin is degraded, hemicellulose is also removed. Theselective loss of lignin and hemicellulose and repression ofcellulase by some white rot fungi make these particularspecies ideally suited for industrial applications where ligninor various phenolic compounds must be altered or removed(29, 36).The degradation of wood by T. i'ersicolor was different

from that caused by white rot fungi that preferentiallydegrade lignin. A nonselective attack removed all layers ofthe cell wall. The remaining cell wall showed no differencesfrom sound wood in gold labeling after EG II- or CBH I-goldtreatment. These results demonstrate that cellulose is notaltered beyond the eroded cell wall region. Evidence fromxylanase-gold-treated sections suggests, however, that thexylan content of cell walls near eroded areas is reduced.Hemicellulases apparently move into the cell wall and de-grade hemicellulose before cellulases. Lignin removal alsohas been postulated to precede cellulose degradation (8).Hemicellulases most likely accompany or follow immedi-ately lignin-degrading enzymes or possibly nonenzymaticprocesses. It is also possible that the lignin degradativeprocesses alone may affect hemicellulose (41).Brown rot fungi remove cellulose and hemicellulose from

wood without degrading large amounts of lignin (30). Thehighly lignified cell wall that remained after decay by F.pili(cola observed in our study had little gold labeling aftersections were incubated with EG II- or xylanase-gold

FIG. 4. Transmission electron micrographs of transverse sections from sound wood (a and b) and wood decayed for 12 weeks by T.i ersicolor (c), P. pinii (d). P. chrysosporiumn (e). and F. pinicola (f) after incubation with a xylanase-gold complex. (a) Gold labeling occurringwithin the inner regions of the secondary wall. SI. and the middle lamella. (b) Some sections, such as this one, exhibit labeling only in partsof the SI layer and the middle lamella. Labeling can also be seen in the intercellular regions of cell corners (arrow). (c) Gold labeling is evidentin some areas of SI and the middle lamella, but absent in the cell wall near eroded areas (arrowheads). (d and e) Fiber cells that were onlypartially delignified show some of the middle lamella still present. Gold labeling is seen only in cell corner regions. (f) Cells from brown-rottedwood show very low levels of gold within the cell walls. Bar = 2 p.m.

VOL 5 5, 1989 2.299

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

2300 BLANCHETTE ET AL.

probes, demonstrating the reduced amount of substrateavailable for these enzymes. Some cellulose remained, how-ever, within secondary walls and gold labeling by the CBH Iprobe was evident. The parts of the cell wall with the mostadvanced decay, exhibiting a swollen appearance, had thelowest levels of gold labeling. These regions apparently haveextremely small amounts of cellulose present. Althoughinitial processes of cellulose depolymerization by brown rotfungi are diffusible and occur extensively throughout thewood (14, 25), removal of cellulose by enzyme actionappears to take place in cell wall layers near the lumen. Asthe brown rot degradation progresses, cellulose is subse-quently removed from the inner portions of the cell wall. Themovement of enzymes into the wood apparently follows theprecelluloytic degradation system (35).

Colloidal gold cytochemistry can provide a unique way tovisualize the precise location of sites for enzyme activity. Insound wood, this provides a mechanism to determine theultrastructural arrangement of cell wall components. It alsohas been shown here to provide important new informationabout how white and brown rot fungi degrade woody sub-strates.

LITERATURE CITED1. Abad, A. R., K. R. Cease, and R. A. Blanchette. 1988. A rapid

technique using epoxy resin Quetol 651 to prepare woody planttissues for ultrastructural study. Can. J. Bot. 66:677-682.

2. Bendayan, M. 1981. Ultrastructural localization of nucleic acidsby the use of enzyme-gold complexes. J. Histochem. Cy-tochem. 29:531-541.

3. Benhamou, N., and G. B. Ouellette. 1986. Use of pectinasecomplexed to colloidal gold for the ultrastructural localization ofpolygalacturonic acids in the cell walls of the fungus Ascocalyxabietina. Histochem. J. 18:95-104.

4. Berg, R. H., G. W. Erdos, M. Gritzali, and R. D. Brown, Jr.1988. Enzyme-gold affinity labelling of cellulose. J. ElectronMicrosc. Tech. 8:371-379.

5. Blanchette, R. A. 1987. New directions in forest productspathology. Can. J. Plant Pathol. 9:261-369.

6. Blanchette, R. A., and A. R. Abad. 1988. Ultrastructural local-ization of hemicellulose in birch wood (Betiula papyrifer-a)decayed by brown and white rot fungi. Holzforschung 42:393-398.

7. Blanchette, R. A., T. A. Burnes, G. F. Leatham, and M. J.Effland. 1988. Selection of white-rot fungi for biopulping. Bio-mass 15:93-101.

8. Blanchette, R. A., L. Otjen, and M. C. Carlson. 1987. Lignindistribution in cell walls of birch wood decayed by white rotbasidiomycetes. Phytopathology 77:684-690.

9. Blanchette, R. A., L. Otjen, W. E. Eslyn, and M. J. Effland.1985. Changes in structural and chemical components of wooddelignified by fungi. Wood Sci. Technol. 19:35-46.

10. Chamerland, H., P. M. Charest, G. B. Ouellette, F. J. Pauze.1985. Chitinase-gold complex used to localize chitin ultrastruc-turally in tomato root cells infected by Fusarium oxysporum

f.sp. radieis-lycopersici, compared with a chitin specific gold-conjugated lectin. Histochem. J. 17:313-321.

11. Chanzy, H., B. Henrissat, and R. Vuong. 1984. Colloidal goldlabeling of 1,4-p-D-glucan cellobiohydrolase adsorbed on cellu-lose substrates. FEBS Lett. 172:193-197.

12. Chanzy H., B. Henrissat, R. Vuong, and M. Schulein. 1983. Theaction of 1,4-p-D-glucan cellobiohydrolase on Valonia cellulosemicrocrystals. FEBS Lett. 153:113-118.

13. Coughland, M. P. 1985. The properties of fungal and bacterialcellulases with comment on their production and application.Biotechnol. Genet. Eng. Rev. 3:39-109.

14. Cowling, E. B. 1961. Comparative biochemistry of the decay ofsweetgum sapwood by white-rot and brown-rot fungi. U.S.Dept. Agric. Tech. Bull. 1259. U.S. Department of Agriculture,Washington, D.C.

15. Eriksson, K. E. 1981. Cellulases of fungi, p. 19-32. In A.

Hollaender (ed.), Trends in the biology of fermentations forfuels and chemicals. Plenum Publishing Corp., New York.

16. Eriksson, K. E., and B. Pettersson. 1975. Extracellular enzymesystem utilized by the fungus Sporotrichum pulverulentum(Chrysosporiui'n lignoruim) for the breakdown of cellulose. l.separation, purification and physio-chemical characterization offive endo-1,4-p-glucanases. Eur. J. Biochem. 51:193-206.

17. Esau, K. 1977. Anatomy of seed plants. John Wiley & Sons,Inc., New York.

18. Frens, G. 1973. Controlled nucleation for regulation of theparticle size in mono dispersed gold suspensions. Nature Phys.Sci. 241:20-22.

19. Geoghegan, W. D., and G. A. Ackerman. 1977. Absorption ofhorseradish peroxidase, ovomucoid and antiimmunoglobulin tocolloidal gold for the indirect detection of concanavalin A,wheat germ agglutinin and goat anti-human immunoglobulin Gon cell surfaces at the electron microscopic level: a newmethod, theory and application. J. Histochem. Cytochem. 25:1187-1200.

20. Goodman, S. L., G. M. Hodges, and D. C. Livingston. 1980. Areview of the colloidal gold marker system, p. 133-146. In R. P.Becker and 0. Johari (ed.), Scanning electron microscopy, partII. SEM Inc. AMF O'Hare, Chicago.

21. Herr, D., F. Baumer, and H. Dellweg. 1978. Purification andproperties of an extracellular endo-1,4-,3-glucanase from Lenzi-ties trabea. Arch. Microbiol. 117:287-292.

22. Highley, T. L. 1975. Properties of cellulases of two brown-rotfungi and two white-rot fungi. Wood Fiber 6:275-281.

23. Highley, T. L. 1976. Hemicellulases of white- and brown-rotfungi in relation to host preferences. Mater. Org. 11:33-46.

24. Highley, T. L. 1988. Cellulolytic activity of brown-rot andwhite-rot fungi on solid media. Holzforschung 42:211-216.

25. Highley, T. L., and T. K. Kirk. 1979. Mechanisms of wooddecay and the unique features of heartrots. Phytopathology69:1151-1157.

26. Highley, T. L., L. L. Murmanis, and J. G. Palmer. 1983.Electron microscopy of cellulose decomposition by brown-rotfungi. Holzforschung 37:271-278.

27. Keilich, G., P. Bailey, and W. Liese. 1970. Enzymatic degrada-tion of cellulose derivatives and hemicelluloses in relation to thefungal decay of wood. Wood Sci. Technol. 4:273-283.

28. Kerr, A. J., and D. A. I. Goring. 1975. The ultrastructuralarrangement of the wood cell wall. Cellulose Chem. Technol.9:563-573.

29. Kirk, T. K., and R. L. Farrell. 1987. Enzymatic "combustion":the microbial degradation of lignin. Annu. Rev. Microbiol.41:465-505.

30. Kirk, T. K., and T. L. Highley. 1973. Quantitative changes instructural components of conifer woods during decay by white-and brown-rot fungi. Phytopathology 63:1338-1342.

31. Koenigs, J. W. 1974. Hydrogen peroxide and iron: a proposedsystem for decomposition of wood by brown-rot basidio-mycetes. Wood Fiber 6:66-79.

32. Kushida, H. 1974. A new method for embedding with a lowviscosity epoxy resin "Quetol 651." J. Electron Microsc. 23:197.

33. Leathers, T. D. 1986. Color variants ofAureobasidium pullulansoverproduce xylanase with extremely high specific activity.AppI. Environ. Microbiol. 52:1026-1030.

34. Ljungdahl, L. G., and K. E. Eriksson. 1985. Ecology of micro-bial cellulose degradation. Adv. Microb. Ecol. 8:237-299.

35. Murmanis, L., T. L. Highley, and J. G. Palmer. 1987. Cy-tochemical localization of cellulases in decayed and nondecayedwood. Wood Sci. Technol. 2:101-109.

36. Myers, G. C., G. F. Leatham, T. H. Wegner, and R. A.Blanchette. 1988. Fungal pretreatment of aspen chips improvesstrength of refiner mechanical pulp. TAPPI J. 71:105-108.

37. Odegaard, G. H., P. C. Anderson, and R. E. Lovrien. 1984.Resolution of the multienzyme cellulose complex of Tricho-derma reesei QM 9414. J. Appl. Biochem. 6:156-183.

38. Otjen, L., and R. A. Blanchette. 1986. A discussion of micro-structural changes in wood during wood decomposition bywhite rot basidiomycetes. Can. J. Bot. 64:905-911.

APPL. ENVIRON. MICROBIOL.

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

ULTRASTRUCTURE OF SOUND AND DECAYED BIRCH WOOD

39. Otjen, L., R. A. Blanchette, M. J. Effand, and G. F. Leatham.1987. Assessment of 30 white rot basidiomycetes for selectivelignin degradation. Holzforschung 41:343-349.

40. Parameswaran, N., and W. Liese. 1982. Ultrastructural localiza-tion of wall components in wood cells. Holzforschung 40:145-155.

41. Paszczynski, A., R. L. Crawford, and R. A. Blanchette. 1988.Delignification of wood chips and pulps by using natural andsynthetic porphyrins: models of fungal decay. Appl. Environ.Microbiol. 54:62-68.

42. Reese, E. T. 1977. Degradation of polymeric carbohydrates bymicrobial enzymes. Recent Adv. Phytochem. 11:311-367.

43. Ruel, K., and J. P. Joseleau. 1984. Use of enzyme-gold com-

plexes for the ultrastructural localization of hemicelluloses inthe plant cell wall. Histochemistry 81:573-580.

44. Shoemaker, S. P., and R. D. Brown, Jr. 1978. Characterizationof endo-1,4-f3-D-glucanases purified from Trichodernma i'iride.Biochim. Biophys. Acta 523:147-161.

45. Vian, B., J. M. Brillouet, and B. Satiat-Jeunemaitre. 1983.Ultrastructural visualization of xylans in cell walls of hard-woods by means of xylanase-gold complex. Biol. Cell 49:179-182.

46. Wolter, K. E., T. L. Highley, and F. J. Evans. 1980. A uniquepolysaccharide- and glycoside-degrading enzyme complex fromthe wood-decay fungus Po)ria plac enta. Biochem. Biophys. Res.Commun. 97:1499-1504.

VOL. 55, 1989 2301

on June 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from