Ultrastructural localization of polysaccharides in the wall and septum of the basidiomycete Schizophyllum commune

E=~PERIMENT.4_L MYCOLOGY 1, 6 9 - 8 2 (1977)

Ultrastructural Localization of Polysaccharides in the Wall and Septum of the Basidiomycete Schizophyllum commune P I E T E R VAN D E R VALK~ I~OGER ~/[ARCHANT, 1 AND JOSEPH G. H. W E S S E L S

Department of Developmental Plant Biology, Biological Centre, University of Groningen, Haren, The Netherlands Received June 28, 1976 VANDER VALK, P., MARCHANT,R., AND WESSELS, J. G. It. 1977. Ultrastructural localization in polysaccharides in the wall and septum of the basidiomycete Schizophyllum commune. Experimental Mycology 1, 69-82. The location of polysaccharides in the wall and the septum cf monokaryotic myeelium of Schizophyllum commune was determined using thin sections and shadowed preparations. Extraction procedures and staining, to detect periodate-sensitive polysaccharides, were used to localize wall polymers that had previously been identified by chemical analysis. Apart from a water-soluble f~-l,3-fi-l,6-1inked glucan (mucilage) that surrounds the hyphae, the lateral wall has an outer layer consisting of relatively pure a-l,3-glucan (S-glucan). The inner layer consists of R-glucan, an alkali-insoluble polymer containing a large proportion of f~-l,3-fi-l,6-1inked glucan, in which chitin microfibrils are embedded. Adjacent to the plasmalemma, these microfibrils are clearly visible but at the interface with the S-glucan layer they are completely embedded in R-gluean. The septum has a central plate mainly composed of randomly oriented chitin microfibrils, except at the septal swelling, where they are arranged circularly around the septal pore. The central plate is covered at both sides by a layer of R-glucan in which chitin microfibrils are embedded. R-Glucan and chitin only occur in the central part of the septal swelling. The main body of this structure is made up of a mucilaginous substance that may be similar to the water-soluble ~-l,3-~-l,6-glucan that surrounds the hyphae. S-Glucan appears to be absent from the septum. At the junction of lateral wall and septum, chitin and R-gluean appear to be resistant to enzymatic treatment, which dissolves most of the other wall material, resulting in the appearance of septal rings. INDEX DESCRIPTORS:cell wall; chitin; glucan (a-l,3) ; glucan (fl-l,3-fl-l,6) ; septum; Schizophyllum commune; ultrastructnre. N u c l e a r m i g r a t i o n as p a r t of the sexual i n t e r a c t i o n in b a s i d i o m y c e t e s is a c c o m p a n i e d b y d e g r a d a t i o n of the complex septal a p p a r a t u s t h a t n o r m a l l y p r e v e n t s the m o v e m e n t of nuclei (Giesy and D a y , 1965; Jersild et al., 1967, K o l t i n a n d Flexer, 1969; N i e d e r p r u e m and Wessels, 1969; R a u d a s k o s k i , 1972, 1973; M a r c h a n t and Wessels, 1974). I n Sehizophyllum c o m m u n e t h e b r e a k d o w n of t h e s e p t u m has been

related t o an increase in R-glueanase, an e n z y m e t h a t solubilizes one of the m a j o r wall c o m p o n e n t s (Wessels a n d Niederp r u e m , 1967; Wessels, 1969a). C h e m i c a l analysis of t h e wall of S. c o m m u n e has revealed the presence of four p o l y s a c c h a rides (Wessels et al., 1972; Wessels and de Vries, 1973; S i e t s m a a n d Wessels, 1977) : (i) a mucilage p r e s e n t at the o u t e r surface a n d also in t h e culture m e d i u m a n d consisting of ¢~-l,3-1inked glyeosyl units with b r a n c h e s of single glucose units a t t a c h e d b y ~-l,6linkages along the chain, (ii) an alkali-

i Present address: School of Biological and Environmental Studies, New University of Ulster, Coleraine; Northern Ireland. 69 Copyright • 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIGS. 1-6. Thin sections of Glu/OsO4-fixed hyphae stained according to Thi~ry (1967) or treated as specified. CW, cross wall; LW, lateral hyphal wall; P, plasmalemma; PS, plasmalcmmasome; R, rim of septal swelling; S, septal swelling. All scale lines represent 0.3 #m. Fio. 1. Near median section through a septum. The cross wall has three layers, viz. a central unstained layer which is bounded on either side by a stained layer. The lateral hyphal wall is stained except for a thin outer layer. The triangular region at the junction of lateral wall and cross wall is often less densely stained than the adjacent lateral wall (arrow). Note the stained plasmalemma and plasmalemmasome. FiG. 2. Transverse section of a septal swelling showing a discrete rim in the central part of the septal swelling. FIG. 3. Stained fibrous material occurs in an otherwise unstained peripheral part of the septal swelling. Note the stained plasmalemma. FIG. 4. Control for the Thi6ry staining reaction in which water was substituted for the periodic acid solution. Note the absence of staining of the wall and the structure of the central part of the septal swelling as compared to the peripheral part.

WALL AND SEPTUM OF SCHIZOPHYLLUIVI soluble a-l,3-glucan (S-gluean) partly present in a mieroerystalline form (iii) an alkaliinsoluble complex that contains a large proportion of B-1,3-fl-l,6-linked gluean (R-gluean), and (iv) chitin. Studies with isolated walls of a monokaryon of S. commune have shown that the septa, but not the lateral walls, can be completely dissolved by the combined action of R-glucanase and chitinase, suggesting that R-glucan and chitin are the only constituents of the septum (Janszen and Wessels, 1970; Wessels and Marchant, 1974). The present study was undertaken to obtain direct evidence for the location of the various polysaccharides in the wall and the septum of S. commune. The model suggested may be generally applicable to the structure of the wall and septum in basidiomycetes. MATERIALS AND METHODS Organism. The monokaryotic strain 699 of Schizophyllum commune Fr. (collection of Professor J. R. Raper, Harvard University, Cambridge, Mass.) was grown on minimal medium (Wessels, 1965) at 25°C. Wall fragments were prepared from 5-day-old standing cultures. For thin sectioning of whole hyphae the mycelium was grown for 3 days on solid minimal medium overlaid with a cellophane membrane. Shadowing of wall preparations. Clean hyphal walls were prepared as described earlier (Wessels& Marehant, 1974). To increase the incidence of exposed septal surfaces the walls were further fragmented in acetone with a Potter-Elvehjem homogenizer. After washing with water the preparations were treated in various ways to remove wall polymers selectively. Suspen-

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sions in water were then placed on formvar/earbon-coated grids and dried at room temperature, before shadowing with platinum, platinum/carbon, or platinum/iridium (80/20) at an angle of 30 °. Shadows appear white in the photographs. Thin sectioning. Myeelia from cellophane cultures or hyphal-wall preparations after various treatments were fixed in (i) 1.5% aqueous potassium permanganate for 30 min or (ii) in 2% (w/v) glutaraldehyde (Glu) in 0.1 M sodium eaeodylate (pH 7.2) for 2 h, washed in buffer and postfixed in 1% (w/v) OsO4 for 12 h at 4°C. The fixed material was dehydrated in an ethanol series and embedded in Epon 812. Thin sections of glutaraldehyde-OsO4fixed material were mounted on formvar/ carbon-coated copper grids and stained with lead citrate (Reynolds, 1963). Alternatively, the sections were mounted on formvar/earbon-eoated gold grids, treated to remove specific wall components, rinsed with water, and stained to localize periodate-sensitive polysaceharides (Thi6ry, 1967). This included subsequent treatment with 1% (w/v) periodic acid (30 rain), 0.2% (w/v) thioearbohydrazide in 20% (v/v) acetic acid (60 min), and 1% (w/v) silver proteinate (30 min). When viewed within a few hours after staining this method gives consistent results. Estimations of thickness of walls and extracted products were based on 100 measurements of permanganate-fixed preparations. The recorded values are probably overestimates because of the presence of oblique sections. However, the values obtained for the different treatments are comparable. Extraction of wall polymers. Details of extraction procedures are given under

FIG. 5. Section treated with exo-CM,3-glueanase (35°C; 2 h) prior to Thi~ry staining. The staining of the septum and lateral wall is reduced. Fro. 6. As Fig. 5, except that the glucanase treatment was 8 h. The staining of wM1 material is completelyabolished. Small holes are also visible in the lateral wall and in the central part of the septal swelling.

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VAN DER VALK, MARCHANT AND WESSELS

WALL AND SEPTUI~i OF SCHIZOPHYLLUIV[ Results. Periodate-sensitive polysaccharides were degraded according to the proeedure of Goldstein et al. (1965), which involves periodate oxidation, reduction with borohydride, and mild acid hydrolysis (Smith degradation). T h e following enzymes were used: (i) chitinase (NBCo), 1 m g / m l M e I l v a i n buffer (14.2 mM citric acid-35.8 mM Na2HPO4, p H 5.6), (ii) Pronase (B grade, Calbioehem), 1 m g / m l 0.05 M a m m o n i u m acetate buffer ( p i t 7.5), (iii) exo-~q,3-glueanase from basidiomyeete Q M 806 (Reese and Mandels, 1966), 1 m g / m l 0.05 M sodium acetate buffer ( p H 5.6), (iv) crude R-glucanase (Wessels, 1969b), 1 m g / m l McI1vain buffer ( p H 5.6), (v) Trichoderma viride ( = harzianum) enzymes (de Vries and Wessels, 1972), 2 r a g / ml 0.05 M sodium m a l e a t e buffer ( p H 5.8). I n all incubations lasting longer t h a n a few hours toluene was added to p r e v e n t bacterial contamination. T h e a m o u n t of S-glucan, R-gluean, and chitin in walls and extracted products was determined as described b y de Vries and Wessels (1973). RESULTS

Thin Sections Figures 1-6 show the a p p e a r a n c e of the lateral wall and s e p t u m of Sehizophyllum

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commune h y p h a e cultured on solid m e d i u m and stained b y the Thi6ry procedure. T h e lateral wall has two layers (Fig. 1 ; compare also Fig. 5), an inner stained layer and an outer layer t h a t takes up v e r y little stain. T h e cross wall of the s e p t u m contains three distinct layers (Figs. 1, 3, and 5), a central unstained layer bounded on either side b y a stained layer. T h e stained layers of the s e p t u m are continuous with those of the lateral wall. T h e central unstained layer of the s e p t u m ends within the stained inner wall layer, where it can often be seen to widen, forming an unstained triangular structure (arrow in Fig. 1). T h e unstained layer extends at the center to the b o u n d a r y of the septal pore. T h e stained material of the s e p t u m radiates into the septal swelling, often forming a discrete rim (Figs. 1 and 2). A loose network of stained fibrous material is present in the otherwise unstained peripheral p a r t of the septal swelling (Figs. 1 and 3). T h e septal swelling is bounded b y the p l a s m a l e m m a which stains heavily with the Thi6ry stain, as do complex p l a s m a l e m m a invaginations (Figs. 1-3). Other cellular m e m b r a n e s show a low affinity for the Thi6ry stain. T h e specificity of the Thi6ry staining reaction is indicated b y the absence of staining in controls from which either periodic acid or thiocarbohydrazide is

FIGS. 7-12. Shadowed hyphal walls. IS, inner surface; OS, outer surface. All scale lines represent 0.3 #m. Inner and outer surfaces were determined on wall fragments showing broken ends. FIo. 7. Outer surface of nonextracted wall. The hyphal surface is rough. Note indications of thick irregular fibers. FIG. 8. Inner surface of nonextracted wall. Randomly arranged microfibrils are embedded in an amorphous matrix. FIo. 9. Outer surface of the wall after extraction with 1 N KOtt (60°C; 20 rain) showing a smooth surface. XOH treatment removes the coarse material at the outside of the wall. FIG. 10. Inner surface of the wall after extraction with I N KOH. The treatment does not appreciably change the appearance of the inner wall surface. FIo. 11. Outer surface of the wall after successive treatments with 1 N KOtt (60°C; 20 rain) and 0.5 N HC1 (80°C; 1 h), showing randomly arranged microfibrils. The tiC1 treatment almost completely removes the alkali-insoluble matrix embedding the microfibrils. Fro. 12. Outer surface of the wall after successive treatments with 1 N KOH (60~C, 20 rain) and exo-~-l,3-glucanase (35°C, 24 h), showing microfibrils. The glucanase-digestion removes the major part of the matrix.

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VAN D E R VALK, M A R C I I A N T A N D WESSELS

FIGS. 13-17. Wall residues after Iorolonged digestion with Trichoderma enzymes (25°C; 24 h). OS, outer surface; LW, lateral hyphal wall. FIG. 13. Shadowed hyphal wall residue. The outer surface of the wall is rough. Scale line, 0.3 ~m. FIo. 14. Section, showing ring-shaped residues of the cross wall located at the iunction of lateral wall and cross wall. Scale line, 1.0 ~m. FIG. 15. Section stained by the Thi~ry method. The remnant of the cross wall is stained but that of the lateral wall is not. Scale line, 0.3 t~m. Fro. 16. Shadowed septal ring with adhering material (S-glucan) from the lateral wall. Scale line, 0.5 ~m. Fro. 17. Shadowed septal ring after extraction with 1 N K O H (60°C; 20 rain), showing microfibrils embedded in an amorphous matrix. Scale line, 0.5 ~m.

WALL AND SEPTUM OF SCHIZOPttYLLUM omitted during the staining procedure (Fig. 4). In addition, these controls show that the central part of the septal swelling has the same electron density as the cross wall and the lateral wall. In contrast, the electron density of the peripheral part of the septal swelling appears similar to that of the embedding resin, indicating that this part of the septal swelling contains little wall material. Treatment of sections with exo-~-l,3glueanase for 2 h effectively reduces the stainability with the Thi6ry stain of both lateral wall and septum (Fig. 5). Longer treatment (8 h) completely abolishes the stainability and in fact gives rise to small holes in the sections, particularly in the central part of the septal swelling (Fig. 6). Shadowed Preparations

Figures 7-12 show the appearance of the inner and outer surface of the lateral wall after various extraction procedures. The outer surface of the nonextracted wall is rough (Fig. 7) due to the presence of a compact layer of S-glucan (a-l,3-gluean) (Wessels et al., 1972). It should be noted that the layer of mucilage that surrounds these liquid-grown hyphae has been removed during washing of the wall preparations. The inner surface of these walls has long randomly oriented mierofibrils embedded in an amorphous matrix (Fig. 8). Treatment with 1 N K O H (60°C, 20 rain) does not appreciably change the appearance of this inner surface (Fig. 10). However, the outer layer of S-gluean is removed by this treatment, exposing a smooth surface (Fig. 9). Treatment of these alkali-insoluble wall residues with hot 0.5 5r HC1 (Fig. 11), exo-fl-l,3-glueanase (Fig. 12), or R-glucanase, or subjecting these residues to Smith degradation, removes most of the matrix, leaving the mierofibrils apparently unchanged. The inner and outer surfaces are indistinguishable after these treatments. The wall preparations contain a small

75

fraction of thick-walled hyphae. In these hyphae, which possibly derive from aerial myeelium, enzyme treatments fail to remove the major portion of the cementing matrix, disclosing only a few mierofibrils at the outer surface. Incubation of KOI-I/ R-glueanase or KOH/exo-~-l,3-glueanasetreated wall fragments with Pronase (35°C, 24 h) did not change the appearance of the inner and outer surfaces. It has been shown that prolonged digestion of hyphal-wall preparations of S. commune with Trichoderma enzymes removes virtually all R-gluean and chitin and about 70~o of the S-glucan (de Vries and Wessels, 1973). Figures 13 and 14 show that the residual S-gluean retains the shape of the hypha and that the surface has a texture similar to that of the outer surface of the nontreated wall (ef. Figs. 13 and 7). This treatment also removes the septa except for an annulus located at the junction of septa and lateral wall (Figs. 14 and 16). Staining by the Thi6ry method leaves the S-gluean remnant of the lateral wall unstained but stains the septal rings (Fig. 15). Removal of the residual S-gluean with K O H gives a preparation consisting almost exclusively of septal rings. Shadowed preparations of these rings show that they contain mierofibrils embedded in a matrix (Fig. 17). Figures 18 and 19 give a view of the surface of nonextraeted isolated septa. The surface of the cross wall has randomly arranged mierofibrils embedded in an amorphous matrix. This surface appears quite similar to the inner surface of the lateral wall (ef. Figs. 19 and 8). The surface of the septal swelling, however, is smooth. K O H treatment does not appreciably change the surface texture (Figs. 20 and 21) except that the septal swelling often appears less pronounced. In addition, K O H treatment sometimes uncovers a few microfibrils circularly arranged at the site of the septal swelling (Fig. 21). When these alkali-resistant residues of the septa are

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-VAN DER VALK, ~AP~CHANT AND WESSELS

WALL AND SEPTU1V[ OF SCHIZOPHYLLUNI subjected to t r e a t m e n t with hot 0.5 N HC1 (Fig. 22), digestion with R-glucanase (Fig. 23) or exo-fl-l,3-g]ucanase, or to Smith degradation, the m a t r i x is completely rem o v e d and circularly arranged microfibrils are uncovered at the site of the septal swelling. Wall Thickness

H y p h a l walls from liquid-grown mycelium were isolated and subjected to various t r e a t m e n t s to extract specific wall polymers. T h e wMls and extracted residues were fixed in p o t a s s i u m p e r m a n g a n a t e and their thickness was measured on p h o t o g r a p h s t a k e n f r o m sections. T a b l e I shows t h a t the R-glucanase p r e p a r a t i o n contains some chitinase activity and t h a t the combination of R-glucanase and chitinase favors the b r e a k d o w n of chitin b u t does not enhance the degradation of R-glucan. This indicates t h a t chitin is embedded in Rglucan. P r e l i m i n a r y t r e a t m e n t with alkali m a k e s b o t h R-glucan and chitin more susceptible to enzymatic degradation. H o w ever, a p a r t f r o m removing S-glucan, alkali m a y somehow influence the structure of the R-glucan/chitin complex m a k i n g it m o r e susceptible to enzymatic degradation. T a b l e I indicates t h a t R-glucanase or chitinase alone do not reduce the thickness of

77

the wall, b u t t h a t the combination of these two enzymes is effective. I ( O H t r e a t m e n t also reduces the thickness of the wall; subsequent enzymatic t r e a t m e n t s h a v e little effect on the thickness of the residue. This indicates t h a t the R-glucan and chitin occur in i n t i m a t e association and t h a t the alkali-soluble S-glucan layer is relatively pure. A conflicting result in T a b l e 1 is t h a t the combination of R-glucanase and chitinase does not reduce the thickness of the alkali-extracted residue similarly to t h a t of the intact wall. However, the a m o u n t of

material left is so small that the sample measured possibly constitutes a nonrepresentative fraction of the original wall sample. DISCUSSION Vicinal hydroxyl groups, which m a k e polysaccharides susceptible to periodate oxidation, a p p e a r to be present only in the mucilage and in the R-gluean c o m p o n e n t of the wall of Schizophyllum commune. T h a t S-gluean (a-l,3-gluean) consumes v e r y little periodate has been d e m o n s t r a t e d earlier (Wessels et al., 1972). M e t h y l a t i o n analysis has shown t h a t the glueosidie linkages in the mucilage and in R-gluean are (1 ~ 3) and (1--+ 6) exclusively and t h a t the periodate consumption and formic acid

FIGs. 18-23. Shadowed isolated septa. All scale lines represent 0.3 t~m. FIG. 18. Nonextracted septum. The pore in the center of the septum is bounded by an annular rim, the septal swelling. FIG. 19. As Fig. 18 but at higher magnification. The surface of the cross wall has randomly arranged microfibrils which are embedded in an amorphous matrix. The surface of the septal swelling is smooth; microfibrils are not visible. FIG. 20. Septum after extraction with 1 N KOI-I (60°Ci 20 rain). The surface texture of the cross wall and septal swelling is as in nonextracted samples. FIG. 21. Treatment as in Fig. 20. The septal swelling is less pronounced. A few microfibrils at the edge of the swelling are circularly arranged around the septal pore (arrows). The microfihrils of the cross wall are more clearly visible than in nonextracted specimens. FIG. 22. Septum after extraction with 1 N KOII (60°C; 20 rain) followed by treatment with 0.5 N I-IC1 (80°C; 1 h). The matrix embedding the microfibrils has been completely removed. At the site of the septM swelling several circularly arranged microfibrils are visible around the septal pore. FIG. 23. Septum after extraction with 1 N K0H (60°Ci 20 rain) followed by R-glucanase digestion (35°C; 1.5 h). Comments as in Fig. 22,

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VAN DER VALK, MARCHANT AND WESSELS TABLE 1 Effect of Extraction of Wall Polymers on the Thickness of the Wall of Schizophyllum commune~ Treatment

No treatment R-Glucanase Chitinase R-Glueanase/ehitinase KOH KOH/R-glucanase KOH/ehitinase KOIt/R-glueanase/chitinase KOH/R-glueanase/Pronase

Percentage of wail polymer remaining S-Glucan

R-Glucan

Chitin

100.0 94.8 99.6 88.6 0.0 0.0 0.0 0.0 0.0

100.0 58.8 100.0 59.6 100.0 8.3 95.4 2.1 7.9

100.0 78.9 91.4 56.6 100.0 65.0 37.3 1.8 64.8

Wall thickness (nm) 120 4- 29 120 4- 39 117 4- 4 79 4- 4 66 4- 33 58 4- 18 57 4- 23 52 4- 26 55 4- 27

a Extractions were done under the following conditions: 1 N KOI-I at 60°C for 20 rain; R-glueanase, chitinase, and Pronase at 30°C for 24 h. production correspond closely to the number of (1--* 6) linkages and end groups present in these glueans (Sietsma and Wessels, 1977). Because chitin does not consume periodate and the mucilage is nearly absent from the cellophane-grown myeelia presented in Figs. 1-6, the Thi6ry staining reaction can be used to locate specifically the R-gluean in the wall and septum. The results obtained with the Thi6ry staining and the shadowing technique, together with earlier data (Wessels et al., 1972; Wessels and Marehant, 1974) allow the construction of a model of the structure of lateral wall and septum (Fig. 24). It should be noted that unlike S-glucan and chitin, which represent homopolymers of glucose and aeetylglucosamine, respectively, the R-gluean is a complex polymer which, in addition to the major glucan part, contains glueosamine, aeetylglueosamine, and amino acids (Wessels and de Vries, 1973). Small amounts of mannose and xylose also occur in the alkali-soluble fraction of the wall (Wessels and de Vries, 1973) for which both the polymeric organization and location remain obscure. The location of t h e mucilage at the outside of the wall, particularly in liquid-

grown myceliunl, as well as the presence of a discrete outer layer of S-glucan have been demonstrated earlier (Wessels et al., 1972). From the absence of Thi6ry staining and the reduction in thickness after alkali treatment it follows that this layer of S-gluean must be quite pure. The inner layer of the lateral wall consists of chitin microfibrils embedded in R-gluean. These chitin microfibrils are randomly oriented, even at the hyphal tips. This is unlike Polyporus myllitae where the chitin microfibrils appear to have a transverse orientation at the inside and a random orientation at the outside (Seurfield, 1967) or in elongating stipe cells of Coprinus cinereus, where a predominantly transverse orientation of the chitin mierofibrils occurs (Gooday, 1975). T hat chitin m[crofibrils are embedded in R-gluean is also suggested by the fact that chitin becomes more aecessible to ehitinase after removal of R-glucan. It is possible that S-glucan also penetrates the R-glucan/chitin layer because the enzymatic breakdown of this complex is greatly facilitated by previous alkalitreatment but, apart from the removal of S,glucan the alkali may also modify the R-gluean/chitin complex. The architecture of the lateral wall of

WALL AND SEPTUM OF SCHIZOPHYLLUIV[

,,'

79

.-plasmalemma

iI

r

',, ~}i:: 1

~i!~il::i~i;~:: ~::::!::i:i!: _

~

Chitinmicrofibrils R-glucan(G- 1,3-6-1,6-glucan) Mucilage( G- 1.3-G-1.6-glucan)

S-glucan(o~-1,3-gtucan)

FIG. 24. Model of the location of polysaecharides in the septum and lateral wall of hyphae of Schizophyllum commune.

S. commune appears similar to that recently proposed for the wall of Agaricus bisporus (Michalenko et al., 1976) except that in S. commune the chitin mierofibrils are com-

pletely covered with R-gluean at the outside and visible at the inside only. The model is clearly at variance with that given by Hunsley and Burnett (1970). These authors proposed that the chitin mierofibrils of the wall of S. commune are embedded in alkali-resistant protein and that this chitinprotein complex is separated from a layer of R-gluean by a discrete layer of protein. In our experiments, Pronase treatment did not change the appearance of the inner or outer surface of alkali-resistant wall fragments that had been extracted with Rglueanase or exo-~-l,3-glucanase. The diserepaney between the models probably results from the fact that Hunsley and Burnett used whole hyphae instead of isolated walls and only looked at outer surfaces after various treatments. In addition, they assumed that enzymes acted only from the outside even when whole hyphae were first treated with KOtI.

A prominent feature of the septum is the central plate which is made up of an assembly of chitin microfibrils. These microfibrils are randomly oriented except at the site of the septal swelling, where they run circularly around the pore. This has previously been observed in Polyporus myllitae (Seurfield, 1967). In ascomyeetes there are only slight indications of a circular arrangement of mierofibrils in the septum (Maret, 1972 ; I-Iunsley and Gooday, 1974). The central chitin plate is covered on both sides by an R-glucan chitin layer that is continuous with the R-glucan/ehitiu layer of the wall. The inner surface of the lateral wall and the surface of the septum (apart from the septal swelling) look quite similar. Alkali treatment does not change the appearance of either surface, indicating the absence of S-gluean. Wessels and Marehant (1974) inferred that S-gluean is virtually absent from the septum due to the fact that a combination of R-glueanase and ehitinase dissolves the septum except for a septal rim and leaves the lateral wall enriched in S-glucan. In the same study it was

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VAN DER VALK, MARCttANT AND WESSELS

shown that such treatment did not dissolve the septa in preparations derived from a dikaryon of S. commune, indicating a structural difference in the septa of the monokaryon and the dikaryon. The resistance to enzymatic degradation of the septal rim at the junction of the lateral wall is also manifest after digestion of the walls with Trichoderma enzymes, which essentially degrade all wall components except part of the S-glucan (de Vries and Wessels, 1973). Indeed, after subsequent alkali-treatment the lateral walls completely dissolve, leaving septal rims as entire isolated rings. The presence of microfibrils in these rings suggests the presence of chitin, a positive Thi6ry reaction indicates that the remaining matrix is R-gluean. Both substances apparently escaped digestion by ehitinase and glueanase for some unknown reason. One possibility is that S-glucan is present at this site preventing access of the enzymes. Septal rings similarly resistant to enzymatic degradation have also been isolated from Neurospora crassa (Mahadevan and Tatum, 1967; Hunsley and Gooday, 1974). The nature of the septal swelling remains difficult to establish. The circularly arranged chitin microfibrils at this site are completely covered by a massive layer of R-glucan: that occupies the central part of the swelling. In glutaraldehyde/OsO4-fixed material the peripheral part of the swelling reveals a network of fibrils as has been shown in S. commune (Niederpruem et al., 1971; Raudaskoski, 1972; Marchant and Wessels, 1973; Mayfield, 1974) and other basidiomycetes (Thielke, 1972 ; Setliff et al., 1972; Gull, 1976). In potassium permanganate-fixed material the whole septal swelling assumes a more homogeneous appearance (Wells, 1965; Jersild et al., 1967; Niederpruem and Wessels, 1969; Moore and Marchant, 1972; Setliff et al., 1972; Marchant and Wessels, 1973). The present study shows that the fibrous material, as seen in glutaraldehyde/OsO4-

fixed material, is susceptible to periodate oxidation and exo-•-l,3-glucanase digestion. The peripherally located material, and possibly some of the more centrally located material, is also soluble in alkali, as was noted earlier using sectioned material (Janszen & Wessels, 1970; Wessels and Marchant, 1974). The alkali-solubility excludes R-glucan and the positive Thi@ry reaction excludes S~gluean as candidates for the material seen in the peripheral part of the swelling. Conceivably this material could be similar to the ¢~-l,3-¢~-l,6-1inked gluean that constitutes the mucilage around the hyphae. Such a glucan would be highly hydrated, which could explain the difference in appearance after using different fixation methods and dehydration (Hayat, 1970). ACKNOWLEDGMENTS This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). We also thank the latter Organization for providing a visitor grant to R. Marchant.

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