INTRODUCTION

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Wheat-straw treatment with Pleurotus species under solid-state fermentation (SSF) conditions has been considered a way to produce materials with improved properties for animal fodder (32, 46) and paper pulp manufacture (20, 36), such as higher digestibility and partial defibriation, respectively. Pleurotus eryngii seems especially appropriate for straw delignification because of its ability to remove lignin selectively (i.e., with a limited attack to cellulose) (31, 34, 45). Several enzymatic activities, including aryl-alcohol oxidase (AAO), have previously been detected during straw SSF with this and other Pleurotus species (8). Ultrastructural aspects of straw degradation by ligninolytic fungi were described by Barrasa et al. (3). However, no immunolocalization studies, which could provide useful information on enzyme secretion and penetration in the plant cell wall (6, 12), have been carried out during wheat-straw degradation. Thus, we localized AAO and the extracellular polysaccharide produced by P. eryngii in liquid culture and during straw SSF by immunolocalization and enzyme-gold labeling.

	MATERIALS AND METHODS

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Culture conditions. The production of extracellular polysaccharide and enzymes was investigated with cultures of P. eryngii CBS 613.91 (= IJFM A169) incubated at 200 rpm (Gallenkamp orbital incubator) and 28°C (400 ml of medium in 1-liter flasks) in the following medium: 30 g of glucose per liter, 0.6 g of ammonium tartrate per liter, 1 g of KH₂PO₄ per liter, 1 g of yeast extract per liter, 0.5 g of MgSO₄ · 7H₂O per liter, 0.5 g of KCl per liter, and 1 ml of trace element solution [10 mg of Na₂B₄O₇ · 10H₂O, 7 mg of ZnSO₄ · 7H₂O, 5 mg of FeSO₄ · 7H₂O, 1 mg of CuSO₄ · 5H₂O, 1 mg of MnSO₄ · 4H₂O, and 1 mg of (NH₄)₆Mo₇O₂₄ · 4H₂O in 100 ml of water] per liter. The influence of wheat straw was investigated in the same medium supplemented with 10 g of straw (SAICA paper mill; Zaragoza, Spain), which had been milled and sieved (0.4-mm pore size), per liter. Washed mycelia from 15-day stationary cultures in the same medium (1-liter flasks with 100 ml of medium) inoculated from 2% malt extract-agar slants were used as the inoculum. Samples (10 ml) from triplicate cultures were taken aseptically after different incubation periods, and analyses of polysaccharide, reducing sugars, ammonium, and AAO activity were carried out after the removal of mycelia, which were fixed for microscopy observation.

Analytical methods. The concentration of polysaccharides was determined after ethanol precipitation (40% final concentration), dialysis, and freeze-drying. Reducing sugars were estimated by the method of Somogyi (44). The ammonium concentration was quantified with an ammonium electrode. The polysaccharide composition was analyzed by acid hydrolysis with 5 M trifluoroacetic acid (16 h, 100°C), followed by acetylation and gas chromatography analysis (35). Fourier transform infrared (FTIR) spectra of polysaccharide were obtained with 1 mg of sample and 300 mg of KBr.

AAO purification. For enzyme purification, the fungus was grown in the medium discussed above, containing 10 g of glucose per liter and 2 g of ammonium tartrate per liter, for 2 weeks. The culture liquid was ultrafiltered (400-fold concentration) and, after polysaccharide removal in 30% ethanol, chromatographed on Sephacryl S-200 equilibrated in 10 mM sodium tartrate (pH 3) (flow rate, 20 ml/h) and on a Mono-Q column equilibrated in 10 mM sodium phosphate (pH 5.5) with a 20-min 0 to 0.25 M NaCl linear gradient (flow rate, 1 ml/min) (23). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 7.5% polyacrylamide gels with high-M_r standards from Bio-Rad. Protein bands were stained with AgNO₃ by using a Silver Stain Plus kit (Bio-Rad).

Antibody production. Antibodies were obtained from New Zealand White rabbits injected with 200 µg of purified AAO dissolved in phosphate-buffered saline (1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 2.7 mM KCl, and 140 mM NaCl) mixed with an equal volume of complete Freund's adjuvant (Difco). Two additional 200-µg doses of AAO were injected intramuscularly at 2-week intervals with phosphate-buffered saline and, in this case, incomplete Freund's adjuvant. Antiserum titer and specificity were assayed by immunoblotting after SDS-PAGE (as described below) with anti-AAO serum, anti-rabbit immunoglobulin G-peroxidase (Bio-Rad) conjugate as the secondary antibody, and 0.5 mM 3,3'-diaminobenzidine tetrahydrochloride, 0.8 mM 4-chloro-1-naphthol, and 0.1 mM H₂O₂ solutions for final color development (39).

AAO immunolocalization. The immunolocalization of AAO by transmission electron microscopy (TEM) was performed by a modification of the method of Ruel (42). Sections treated with antibody-gold or enzyme-gold complexes were observed with or without staining with uranyl acetate. Samples of wheat straw degraded by P. eryngii and mycelia from stationary and shaken liquid cultures were fixed with 0.3% glutaraldehyde-4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 20°C for 3 h, washed with buffer, and dehydrated in ethanol before being embedded in LR-White hard formulation (London Resin Company; acrylic resin hard grade) and polymerized at 50°C. Ultrathin sections were collected on Formvar-coated gold grids. Sections were incubated in a drop of 0.15 M glycine in Tris-buffered saline (TBS) (0.1 M Tris-phosphate buffer [pH 7.4] containing 0.1 M NaCl). After being washed in TBS, sections were put in a drop of 10% normal goat serum in TBS where the primary antibody, anti-AAO serum, was diluted (1:25) and incubated for 15 h. An anti-rabbit serum conjugated with 10-nm-diameter gold (Immuno Gold Conjugate GAR; BioCell), diluted in TBS containing 0.1% bovine serum albumin and 0.1% gelatin (from fish), was used as the secondary antibody (1-h incubation). The procedure used for fluorescence immunolocalization was basically the same as that used for TEM; however, it was carried out with semithin (0.5- to 1-µm) sections and fluorescein isothiocyanate (FITC)-coupled secondary antibody (F-1262 immunoglobulin G; Sigma) was used. Fluorescence microscopy studies were carried out on an Olympus BX-50 microscope with a U-MWB cube, a BP450-480 excitation filter, and a BA515 barrier filter. A Zeiss EM-10C microscope was used for TEM studies.

Glucan localization. For ultrastructural localization of glucan, an enzyme-gold conjugate was used. Colloidal gold (5-nm diameter) was prepared by the method of Benhamou (4), and the pH was adjusted to 9 with 0.2 M K₂CO₃. One hundred microliters of laminarinase (L9259; Sigma) solution (1 mg/ml) was added to 10 ml of the colloidal gold suspension, shaken for 5 min at room temperature, and centrifuged at 43,000 × g (1 h at 4°C), and the pellet was resuspended in 0.6 ml of water. Ultrathin sections were incubated for 30 min in drops of glucanase-gold conjugate and washed with water (five times for 5 min each) before TEM examination with or without 2.5% uranyl acetate stain on a Zeiss EM-10C microscope.

	RESULTS

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The extracellular polysaccharide levels and AAO activities after 15, 30, and 40 days in liquid cultures of P. eryngii are shown in Fig. 1. The polysaccharide concentration was maintained during the whole incubation period because a C source was available, as deduced from levels of reducing sugars (data not shown). The addition of straw stimulated polysaccharide production (attaining near 150 to 200 mg/liter). In the absence of straw supplementation, AAO attained its highest levels at the end of the incubation period (Fig. 1A). Straw addition resulted in rapid ammonium exhaustion after 9 days (data not shown) and earlier production of the maximal AAO level.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Effects of wheat straw on AAO (A) and polysaccharide (B) production by P. eryngii. Dashed bars indicate straw addition. Data are means ± standard deviations.

AAO was purified to homogeneity by Sephacryl S-200 and Mono-Q chromatography (50-fold purification factor [from around 1.5 U of specific activity per mg in culture filtrate to near 80 U of specific activity per mg after Mono-Q chromatography]). Moreover, a high purification yield (around 75%) was attained by taking advantage of the low adsorption and stability of the enzyme on Sephacryl S-200 at an acidic pH (6). The purity of the enzyme preparation was checked by SDS-PAGE, and a single band (M_r around 73,000) was found (Fig. 2). Polyclonal antibodies against AAO were produced and used for AAO immunolocalization by TEM and fluorescence microscopy with gold (10-nm diameter) and FITC-coupled secondary antibody, respectively. The specificity of the antibody against AAO was confirmed by immunoblotting of concentrated culture liquids and purified enzyme (results not shown); in all cases, there was a unique band with the same electrophoretic mobility as that shown in Fig. 2.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Estimation of the homogeneity and molecular mass of AAO from P. eryngii. SDS-PAGE of purified AAO (left lane) and Bio-Rad standards (right lane) was performed on 7.5% polyacrylamide gels, and proteins were stained by the silver technique.

Acid hydrolysis of the extracellular polysaccharide recovered from liquid cultures of P. eryngii yielded 87% glucose, 11% mannose, and 2% galactose (the composition was not significantly affected by straw addition to the culture medium). Moreover, the FTIR spectra showed a band pattern that is typical of a -(13)-glucan, including 890, 1,000, 1,040, 1,110, and 1,150 cm¹ bands (29). Therefore, a complex of commercial -(13)-glucanase and colloidal gold (5-nm diameter) was prepared for glucan localization in TEM.

Semithin sections of mycelium from liquid medium, stained with FITC-coupled secondary antibody, revealed the presence of AAO as a thin green layer around hyphae (Fig. 3A). This green fluorescence was absent from controls without primary antibody, which showed reddish cell walls (Fig. 3B). In the same way, immunogold TEM showed that AAO was scarcely present inside hyphae; it mainly localized on the surface of the fungal cell wall (Fig. 4A). This was confirmed by double localization, which showed that glucan and AAO were present on both the cell wall and hyphal surface. The highest labeling was observed on the cell wall and hyphal surface, respectively (Fig. 4B), as evidenced by quantitation of the two sizes of gold particles used (Fig. 5).

View larger version (97K): [in this window] [in a new window]   FIG. 3.   AAO immunolocalization in mycelium from liquid culture of P. eryngii. (A) Fluorescence localization of AAO on the surfaces of hyphae (arrow) from 15-day cultures. (B) Control. Bar (both panels) = 10 µm.

View larger version (145K): [in this window] [in a new window]   FIG. 4.   AAO and polysaccharide localization in P. eryngii by immunogold and glucanase-gold TEM, respectively. (A) AAO immunolocalization in the fungal wall and proximity of the hyphal surface. (B) Double labeling, showing the localization of glucan (5-nm-diameter particles; arrows) and AAO (10-nm-diameter particles; arrowheads) in a hypha. (C) AAO immunolocalization in a hypha and different layers of the straw cell wall. (D) AAO immunolocalization control. Thirty-day cultures were used. Bar (all panels) = 1 µm.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Quantitative results from AAO and glucan double labeling in P. eryngii. Shown is the distribution of different-sized gold particles used for AAO (white bars) and glucan (dashed bars) localization in the cytoplasm, cell wall, sheath, and extracellular medium. Data are percentages of total particles per unit of area in TEM images.

In samples from SSF, it was found that the laminarinase-gold complex also reacted with wheat-straw glucan present in different cell wall layers (Fig. 6), with the most intense labeling observed in the primary wall (Fig. 6B). In agreement with the results obtained in liquid culture, fungal glucan was mainly localized on the outer surface of the mycelium, forming a thin hyphal sheath. This polysaccharide was also present in zones of contact between hyphae and the straw cell wall, as well as in hyphae penetrating the cell wall (Fig. 6A). Fluorescence microscopy of SSF samples showed that after 30 days of degradation, AAO was located in the highly degraded cell walls of phloem and inner parenchyma of straw (Fig. 7A and D). In the case of parenchyma, the separation of fibers from intercellular space throughout the middle lamella was observed (Fig. 7D). In less degraded straw tissues, such as sclerenchyma or outer parenchyma, AAO was attached to the secondary wall from the cell lumen (Fig. 7C). At this stage of degradation, contacts between hyphae and straw cell walls, as well as hyphae perforating cell walls, were frequently found (Fig. 6A). In some cases, old hypha aggregates (probably due to extracellular slime) with some AAO labeling were attached to the surfaces of straw cell walls (Fig. 7C). Semithin sections without primary antibody were used as controls in immunofluorescence studies (Fig. 7B). No FITC green fluorescence was observed in these controls, but the straw cell wall exhibited a yellow color due to lignin autofluorescence. AAO penetration into the wheat cell wall was better shown by TEM immunolocalization, revealing gold labeling at different cell wall layers (Fig. 4C). The presence of AAO was also observed in association with the fungal mycelium, mainly concentrated on the outer surfaces of hyphae. Immunogold labeling was absent from controls without primary antibody (Fig. 4D).

View larger version (128K): [in this window] [in a new window]   FIG. 6.   Polysaccharide localization with glucanase-gold complex by TEM. (A) Localization of fungal glucan on the surface of a hypha, causing a bore hole throughout the wheat-straw cell wall, which also showed strong glucan labeling (30-day SSF culture). (B) Labeling of wheat-straw glucan in a sound cell wall, revealing a higher concentration in the primary wall (PW). Abbreviations: ML, middle lamella; SW, secondary wall. Bar (both panels) = 1 µm.

View larger version (111K): [in this window] [in a new window]   FIG. 7.   Fluorescence microscopy of AAO immunolocalization during wheat-straw degradation by P. eryngii. (A) Enzyme localization during phloematic tissue degradation in a vascular bundle. (B) Control. (C) Enzyme localization in sclerenchymatic cell walls (arrowheads) and fungal hyphae (arrow). (D) Enzyme localization during degradation of parenchymatic tissue (arrows). Thirty-day SSF cultures were used. Bar (all panels) = 10 µm.

	DISCUSSION

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The hyphal sheath, an extracellular structure observed on the surfaces of hyphae and mainly consisting of polysaccharide, has been reported to play different roles in fungal physiology, including adhesion to plant cell walls and immobilization of ligninolytic enzymes (28). This could provide a favorable microenvironment for fungal enzymes that are involved in attacking the lignin macromolecule. Several authors have previously reported extracellular polysaccharide production by Pleurotus species (7, 9). In the present study, it was observed that the presence of straw stimulated the production of extracellular polysaccharide by P. eryngii, without any significant modification of its monosaccharide composition. Moreover, complete structural characterizations of the exopolysaccharides produced by six Pleurotus species were carried out in a parallel study (29). Methylation analysis, acetolysis, and ¹³C nuclear magnetic resonance spectroscopy of the major exopolysaccharide produced by P. eryngii revealed that 96% of it consisted of a -(13)-D-glucan with branches of one -(16)-linked glucose unit every two to three residues of the main chain. This structure is only slightly different from that of the extracellular glucan of Phanerochaete chrysosporium (43). Straw stimulation of polysaccharide production in P. eryngii may be related to the presence of a promoter in the soluble fraction of straw (37), but it may also be due to the involvement of this glucan in lignin degradation, as suggested by the detection of lignin-glucan complexes in lignin or lignocellulose-containing cultures of Pleurotus species (26). The existence of a correlation between the presence of a hyphal sheath and ligninolytic activity has previously been reported for the well-known ligninolytic fungus Phanerochaete chrysosporium (5).

H₂O₂-producing oxidases, including AAO, glyoxal oxidase, and glucose oxidases, are key enzymes in lignin degradation, and they are found in many ligninolytic fungi (30, 41, 47). It was early shown (16) that H₂O₂ is strictly required for the breakdown of this polymer, acting as an electron acceptor for ligninolytic peroxidases (33) or as a reactant for the formation of oxygen radicals involved in fungal attack of plant cell walls (2, 15, 25). Most previous studies of the immunolocalization of ligninolytic enzymes have focused on lignin peroxidase (LiP) and Mn-peroxidase (MnP) produced during wood degradation by Phanerochaete chrysosporium (6, 10-12, 18) and other fungi (12, 17). Recently, the extracellular presence of pyranose oxidase during wood degradation by three basidiomycetes has been described and considered as a source of H₂O₂ for MnP (13). AAO, an enzyme that is characteristic of ligninolytic fungi of the genera Pleurotus (36) and Bjerkandera (38) but has also previously been found intracellularly in Phanerochaete chrysosporium (1), has been fully characterized in P. eryngii (22, 23). Moreover, there is evidence that this enzyme is involved in extracellular H₂O₂ production (21, 24) from aromatic metabolites synthesized de novo by this fungus (27). The present study shows that the presence of wheat straw stimulated AAO production and provides the first evidence of AAO localization during lignocellulose degradation. This study also reveals the relationships between the hyphal sheath and the enzyme AAO in mycelia from liquid culture and wheat-straw SSF. Since the laminarinase used to prepare the enzyme-gold complex and localize fungal glucan by TEM shows endo-(13[4])--glucanase activity (i.e., hydrolysis of 13 or 14 linkages in -glucans when the residue whose reducing group is involved in the linkage to be hydrolyzed is itself replaced at C-3), it also reacts with (13;14)--glucans in the wheat cell wall together with cellulose (4).

In contrast with a widespread hyphal sheath produced by Phanerochaete chrysosporium (43), P. eryngii showed a thin glucan sheath closely attached to the fungal cell wall. The presence of AAO around hyphae in liquid cultures was revealed by fluorescence microscopy (Fig. 3A) and confirmed by TEM (Fig. 4A). Double labeling of AAO and glucan in TEM and subsequent particle quantitation (Fig. 5) showed that AAO was localized mainly in the extracellular sheath (smaller amounts were found in the cytoplasm and cell wall). In contrast, preferential localization of enzymes in the hyphal wall and cytoplasm has previously been found in Trametes versicolor and Rigidoporus lignosus, respectively, with some localization found in the hyphal sheath (17, 40). Pyranose oxidase of Phanerochaete chrysosporium grown in liquid culture (14) and on wood (13) was detected not only in the extracellular sheath but also in membrane-bound vesicles and the periplasmic space. Furthermore, MnP and LiP of this fungus have also previously been found in vesicle-like structures (12). Several wood-degrading enzymes, including LiP, laccases, and xylanases, have also previously been localized in the hyphal sheath, probably bound to glucan filaments (17, 19, 43).

Ultrastructural aspects of wheat-straw degradation by Phanerochaete chrysosporium and T. versicolor were studied by Barrasa et al. (3). Similar degradation aspects were observed in the straw degraded by P. eryngii, including early attack of the less lignified phloem and parenchyma (Fig. 7A and D), tissue defibriation and swelling of the secondary wall (Fig. 4C), and development of cell wall erosion and formation of bore holes (Fig. 6A). Fluorescence immunolocalization studies under SSF conditions showed that after 30 days of degradation, AAO was localized on the hyphal surface and on the remains of highly degraded cell walls of phloem and parenchyma (Fig. 7A and D); it was also localized in more lignified tissues such as sclerenchyma (Fig. 7C). The fungal colonization of straw tissues and the proximity of hyphae to the plant cell wall (Fig. 4C and 7B through D) suggest that the enzymatic attack of straw cell walls involves contact between hyphae and straw cell walls. When degradation progresses, some fungal hyphae can also progress throughout the straw cell wall, causing perforations, which implies the presence of cell wall-degrading enzymes in the thin slime layer (Fig. 6A). Furthermore, the penetration of AAO into straw cell wall layers was confirmed by TEM after 30 days of degradation (Fig. 4C and 7C and D). This is in agreement with the distribution of ligninolytic enzymes associated with the selective degradation pattern (in contrast with limited enzyme penetration during simultaneous degradation) described by Blanchette et al. (6) for fungal degradation of wood (although the M_r of AAO is larger than those of ligninolytic peroxidases and laccases). Whether a looser molecular architecture of wheat-straw polymeric components (i.e., polysaccharides and lignin) in different cell wall layers or tissues (e.g., in phloem or parenchyma) can contribute to easier penetration of lignin-degrading enzymes remains to be investigated.

Our TEM and fluorescence studies with P. eryngii showed preferential localization of AAO in the region corresponding to the hyphal sheath and its penetration into the wheat-straw cell wall during degradation under SSF conditions. Since the production of H₂O₂ is an important event in lignin degradation, information about AAO localization is important to our understanding of the mechanisms of cell wall attack by ligninolytic fungi. In particular, H₂O₂ generation at the plant cell wall can be envisaged, reducing toxicity risks for the fungus and limiting the possibility of premature chemical or enzymatic decomposition.