Laccase Localized in Hulle Cells and Cleistothecial Primordia of Aspergillus nidulans

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1983, p /83/ $02.00/0 Copyright C 1983, American for Microbiology Vol. 154, No. 2 Laccase Localized in Hulle Cells and Cleistothecial Primordia of Aspergillus nidulans THERON E. HERMANN, MYRA BERMAN KURTZ, AND SEWELL P. CHAMPE Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey Received 22 November 1982/Accepted 10 February 1983 Several species of the genus Aspergillus form sexual spores within minute (-0.2 mm) spherical shells (cleisthothecia) which are woven from specialized hyphae. Aspergillus nidulans cleistothecia are uniquely characterized by their dark red coloration and an envelope of thick-walled globose cells (hulle cells). By use of a new chromogenic substrate, we have shown that the constitutent hyphae of young cleistothecia and the hulle cells which surround the cleistothecia of A. nidulans exhibit a strong phenoloxidase activity which has the substrate specificity of a laccase. This enzyme (laccase II) is distinct from the previously described phenoloxidase (laccase I) that participates in the synthesis of the conidial pigment of A. nidulans: the two enzymes differ electrophoretically, do not cross-react immunologically, appear at different times during colonial development, and are under different genetic control. Examination of seven additional species of Aspergillus showed that the hulle cells of three acleistothecial species were also laccase positive, whereas the pale or unpigmented cleistothecia of four species (which lack hulle cells) were laccase negative. The relevance of these findings to the role of hulle cells in cleistothecial development is discussed. The presence of histologically detectable laccase in cleistothecial primordia provides a valuable tool, previously unavailable, for quantitating the early stages of sexual development in A. nidulans. Spore formation by the filamentous fungi is a useful model for the study of tissue differentiation. The spores themselves are specialized cells and are often associated with highly organized spore-bearing structures which ultimately arise from hyphal filaments. The homothallic ascomycete Aspergillus nidulans produces two kinds of spores, each during a specific stage of development. In a confluent mycelial lawn growing at 37 C, specialized aerial hyphae (conidiophores) appear at about 18 h after spore inoculation. These aerial hyphae rapidly develop terminal bulbs (vesicles) which become covered with knobby protuberances (sterigmata). Asexual spores (conidia) are generated in the sterigmata and are extruded as long conidial chains. By contrast, sexual spores (ascospores) develop several days later within closed spherical bodies (cleistothecia) which can number several thousand per square centimeter in the mycelial lawn. Young cleistothecia are soft, pale pink bodies which mature into firm, dark red shells with diameters up to 0.3 mm. From their earliest stage of development, cleistothecial primordia are surrounded by numerous globose glassyappearing cells of unknown function called hulle cells. During the early stages of sexual development, some hulle cell clusters are visible on the surface of the mycelial lawn, but most are buried Ċonidiation in wild-type A. nidulans is accompanied by the formation of a laccase (p-diphenol:oxygen oxidoreductase; EC ) which functions in the production of the dark green conidial pigment (6). Mutants with yellow conidia are deficient in this activity and define a single genetic locus (ya) which is almost certainly the structural gene for the conidial laccase as judged from the fact that a thermosensitive ya mutant produces a thermosensitive enzyme (6). Mutants which produce white conidia also exist but are not deficient in laccase (6); presumably, white mutants are defective in the synthesis of the yellow precursor of the green pigment, i.e., the laccase substrate. In the present paper, we demonstrate the presence of a second laccase of A. nidulans which appears later than the conidial laccase and 955 is localized in the hulle cells and cleistothecial primordia. The possibility that this enzyme plays an essential role in cleistothecial morphogenesis is discussed.

2 956 HERMANN, KURTZ, AND CHAMPE MATERLALS AND METHODS Organisms. A. nidulans R-21 (ya2 pabaal) and R-153 (wa3;pyroa4) were obtained from C. F. Roberts, University of Leicester, United Kingdom (2). Strains 0O-45 (ya2;wa2 bia3;sc12), GO-64 (ya2;wa2;clbj ;sc12), GO-68 (ya2 cla4;wa2;sc12), and GO-255 (meda15 biai) were obtained from John Clutterbuck, University of Glasgow, United Kingdom, and were used for the measurements shown in Table 2. Properties of the bl (blue cleistothecia) and cl (colorless cleistothecia) alleles are described by Apirion (1). Gene symbols for A. nidulans have been summarized by Clutterbuck (7). Other Aspergillus species were obtained from Kenneth Raper, University of Wisconsin, Madison, and are identified by their isolation numbers according to Raper and Fennell (23) as follows: A. fisheri (WB181), A. ornatus (WB2256), A. puniceus (WB5077), A. raperi (WB4778), A. ruber (WB52), A. silvaticus (WB2398), and A. tonophilus (WB5124). Media and culture conditions. For all experiments with A. nidulans, the growth medium was composed of 0.5% yeast extract (Difco Laboratories, Detroit, Mich.), 2% glucose, and trace elements at concentrations described previously (12). The medium was solidified with 1.5% agar (Difco). To obtain optimal and reproducible sexual development, deep petri plates (2 by 9 cm) containing 50 ml of medium were used and were incubated unstacked in the dark. Preparation of cell-free extracts. The mycelia from confluently inoculated plates (105 conidia per plate) incubated at 37 C were harvested by vigorously sweeping the center half of each plate (30 cm2) with a cotton-tipped applicator and suspending the dislodged material in 2.0 ml of extraction buffer (10 mm Trishydrochloride, 1.0 mm EDTA, 10 mm p-tosyl-l-arginine methyl ester [Sigma Chemical Co., St.Louis, Mo.; T-4626], and 1.0 mm phenylmethylsulfonyl fluoride [Sigma P-7626], ph 7.4). The slurry was homogenized in a glass Ten Broeck tissue homogenizer and centrifuged for 20 min at 30,000. x g to remove particulate material. The mycelial felt that adheres to and penetrates the agar surface contains a low level of DMP (N,Ndimethyl-p-phenylenediamine sulfate)-oxidizing activity which may correspond to the phenoloxidases of undifferentiated mycelium previously described by Martinelli and Bainbridge (20). Our method of harvesting was used to avoid interference by these activities in assays of laccase II. Laccase II purification. Laccase II was partially purified from a homogenate of a ya mutant as previously described for laccase I (13), except for the following changes in the chromatographic conditions. After centrifugation of a crude homogenate at 100,000 x g for 1 h, the supernatant was applied to a DEAEcellulose column equilibrated with 0.01 M Tris-hydrochloride, ph 7.4 (the standard buffer used in all operations). The column was washed, first with 4 column volumes of this buffer and then with 2 column volumes of the same buffer containing 0.04 M NaCI. Laccase activity were eluted with buffer containing 0.1 M NaCl. No further activity was eluted with higher salt concentrations. Fractions containing laccase activity were pooled, brought to 0.5 M NaCl, and applied to a phenyl-sepharose column. After washing with 3 column volumes of buffered 0.25 M NaCl, the activity J. BACTERIOL. was eluted with buffered 10% ethylene glycol. At this stage, the specific activity was about 90-fold greater than that of a crude homogenate, and on the basis of electrophoretic analysis, about 20% of the protein was estimated to be laccase. This material was used for determinations of substrate specificity. Further purification to obtain a sample suited to raising antibody was accomplished by electrophoresis in a nondenaturing polyacrylamide gel (14). Laccase assays. Laccase activity of extracts was determined by the method of Clutterbuck (6) with the chromogenic substrate DMP (Sigma D-4142). One unit of laccase activity is defined as the amount of enzyme that catalyzes the oxidation of 1 pumol of DMP per min at 25C. Initial rates were corrected for spontaneous oxidation of substrate. Specific activity was expressed as units per milligram of protein by the Bio-Rad protein assay. Oxygen uptake during laccase-catalyzed oxidation of various substrates was measured with a Polarigraph (Yellow Springs Instrument Co., Yellow Springs, Ohio) fitted with a Clark-type electrode. The reaction vessel contained 10 ml of 1.0 mm substrate in laccase assay buffer (37 mm citric acid, 126 mm Na2HPO4, ph 6.0) to which a suitable amount of partially purified enzyme was added. The uptake rate was monitored for 10 min at 25 C and corrected for the spontaneous rate measured before the addition of enzyme. Values are expressed as nmol of 02 per min. Histological stain for laccase. An improved, relatively stable histological reagent for staining laccasecontaining tissue was devised based on the oxidation of 4-amino-2,6-dibromophenol (ADBP) and the coupling of the reaction product to 3,5-dimethylaniline (DMA). The reagent was freshly prepared by mixing 10 volumes of ADBP (4 mg/ml; Eastman 2299) in 95% ethanol with 0.1 volume of DMA (Eastman 758) and diluting the mixture with 2 volumes of laccase assay buffer. Laccase-positive foci on a mycelial lawn were visualized in situ by flooding the petri plate wth the ADBP-DMA reagent, decanting the liquid after 5 min, and incubating the plate at 37 C for 30 min. The bright green laccase-positive foci were counted with a dissecting microscope by using transillumination and an eyepiece fitted with a 100-square reticle. Staining of hydrophobic nonwettable lawns was facilitated by flooding the lawn with chloroform which was allowed to evaporate before application of the substrate. Scanning electron microscopy. Cleistothecia scraped from a 100-h confluent surface culture were suspended in 0.01% Tween-80, gently fragmented with a loosefitting Teflon tissue homogenizer, and washed free of spores and hyphal debris by repeated sedimentation at 1 x g. After incubation with the ADBP-DMA reagent for 1 h at 37 C, the fragments were transferred to a small filter disk and washed thoroughly with water. The filter paper with adhering cleistothecial fragments was freeze-dried, sputter-coated with gold, and examined in a JOEL (JSM-35C) scanning electron microscope. RESULTS Tissue localization of laccase with an improved histological substrate. We have developed a new histological assay for laccase based on the oxidation of ADBP. Although the reaction product

3 VOL. 154, 1983 itself is colorless, an intense blue to blue-green pigment is formed by secondary reaction with DMA. Other characteristics of the reaction and the structure of the colored reaction product will be described elsewhere. In contrast with some other substrates such as N,N-dimethyl-p-phenylenediamine, which is used for quantitative assays, the ADBP reagent is stable to spontaneous oxidation at acid and neutral ph. In addition, the ADBP-DMA reaction product remains localized in laccase-containing tissue. When the substrate is applied to conidiating colonies of a white-spore strain (wa;ya+), the conidial heads become rapidly pigmented, with the pigment localized in conidia and in the conidiogenic sterigmata which protrude from the conidiophore vesicle. However, neither the conidiophore stalk nor the conidiophore vesicle is perceptibly stained. During screening of a variety of developmental mutants for the ability to form the conidial laccase, it was noted that surface cultures of strains expected to be laccase negative (e.g., strains carrying a ya mutation) contained numerous discrete intensely staining foci if the culture was older than about 60 h (Fig. 1). Microscopic examination showed that each of these foci was composed of an intensely stained spherical body surrounded by a large number of hulle cells, thus identifying the foci as cleistothecial primordia (Fig. 2). When the foci were first observable by means of the chromogenic reaction, most of the reaction product was localized in the spherical primordia, which at this stage had a diameter about twice that of a hulle cell. Later, as the primordia enlarged to many times this size, the hulle cells also became distinctly stainable. The pigment in these thick-walled cells was usually distributed uniformly throughout the cytoplasm, but under certain conditions it was deposited as microcrystals in the cell lumen (Fig. 3). The reaction of the cleistothecial wall and of hulle cells with the substrate became progressively weaker as the cleistothecia matured, to form hard spherical shells. However, if the mature, or almost mature, cleistothecia were disrupted and incubated with the ADBP-DMA substrate, minute asterisk-shaped crystals of reaction product could be observed by light microscopy in association with wall fragments. Examination of these fragments by scanning electron microscopy (Fig. 4) showed that these crystals were located on the inner spongy surface of the cleistothecial wall as opposed to the smooth outer surface. The ascospores, which developed within the cleistothecia, showed no evidence of reaction with the substrate at any stage of development. Properties of the cleistothecial laccase. As shown below, the conidial and cleistothecial A. NIDULANS LACCASE 957 phenoloxidases are different enzymes which will hereafter be called laccase I and laccase II, respectively. By using a strain that fails to make laccase I, the much less abundant laccase II could be quantitatively assayed. The two laccase activities appeared at distinctly different times for confluent plate cultures (Fig. Sa). Both activities declined at late times whether expressed as specific activity or as total activity per plate. Laccase II can thus serve as a convenient marker by which to quantitate early sexual development. Such quantitation can also be performed by merely counting the stainable foci as the confluent mycelial lawn ages, as shown in Fig. 5b, in which the appearance of foci is seen to parallel the appearance of soluble laccase II. Electrophoretic comparison of the two laccases on a nondenaturing gel stained for activity showed that the two activities had distinctly different mobilities (Fig. 6). Both activities migrated as broad multicomponent or diffuse bands. In the case of laccase I, as we have noted previously (13), the several electrophoretic components are apparently different forms of the same gene product because all are eliminated by the ya2 mutation present in strain R-21 (the source of laccase II). Although distinctly different electrophoretically, the two laccases do not differ significantly in molecular weight as determined by gel filtration through Sephacryl S-200. We previously reported that laccase I has a molecular weight of 110,000 by this method (13). We have also shown that laccase I is a glycoprotein by direct determination of carbohydrate content of purified enzyme and by its altered electrophoretic mobility upon incubation with endo H, which removes asparagine-linked oligosaccharide chains (13). Similarly, laccase II was also found to be alttred in electrophoretic mobility by incubation with endo H. Antibody prepared against laccase I did not cross-react with laccase II, nor did antibody prepared against laccase II cross-react with laccase I (Fig. 7). Although it is possible that the major antigenic determinant is the carbohydrate moiety, this is unlikely since laccase I treated with endo H still reacted with antibody. The specificity of laccase II for a variety of substrates was determined by oxygen uptake (Table 1). In general, the specificity conformed to those of other known laccases (3, 6, 10, 11, 16, 25). In particular, of the three isomers of amino phenol, only p-amino phenol is oxidized. On the other hand, p-dihydroxybenzene (quinol) is not oxidized by laccase II, but neither is it oxidized by laccase I (6). Other compounds, such as DOPA (3,4-dihydroxyphenylalanine), which are oxidized by some laccases (11, 16,

4 958 HERMANN, KURTZ, AND CHAMPE.i. A,.1 in.is _,.., J. BACTERIOL. *#,. *I, pt lb llxp S0 O."i.7. -, 7:: 'i., W..f. S.. C } >aaiv; o 4.4 FIG. 1. Mycelial lawn of strain R-21 treated with the ADBP-DMA chromogenic substrate. The dark foci, which become visible a few minutes after application of the reagent, are cleistothecial primordia as shown by higher magnification in Fig. 2. These foci are bright green on a field of yellow conidial heads. Under optimal conditions, the primordia are closely packed. For this culture, the density of primordia was intentionally limited by using thin (0.5 cm) agar. Bar, 1,000 pum. k A. : ' ''..l,.", f ",;i 24), are not oxidized by either laccase I or laccase II ofa. nidulans. Table 1 also shows that of the two components of our new histological stain (ADBP and DMA), only ADBP was oxidized. In the presence of H202, ADBP was oxidized by horseradish peroxidase. Laccase II, however, is not a peroxidase because preincubation of extracts with catalase to destroy peroxide did not diminish the reaction. Fungal laccases are usually extracellular, as judged from the fact that the enzyme is found largely in the culture medium or is extractable from tissue without cell disruption (11, 16). Indeed, the conidial laccase of A. nidulans is almost completely extracted without cell disruption (15). This is not true, however, for laccase II. Of the laccase II activity released by grinding mycelia in a tissue homogenizer, only about 30% is released by merely vortexing. In fact, although thoroughly washed cleistothecial fragments react well with the substrate (Fig. 4), we were not able to obtain soluble activity from wall fragments. This suggests that the soluble activity of extracts may be derived solely from hulle cells, and indeed if this is the case, we cannot be certain that the activity of primordia is the same enzyme as that of the hulle cells. Presence of laccase II in mutants of A. nidulans and in other Aspergillus species. Laccase II is a potentially useful marker by which to determine the stage at which sexually defective mutants are blocked. Clutterbuck (5) described an aconidial mutant called medusa which is also acleistothecial but which produces masses of hulle cells. We found that hulle cells of medusa are strongly laccase positive. We previously described (12) another class of acleistothecial mutant that produces hulle cells (yb mutants). In

5 VOL. 154, 1983 A. NIDULANS LACCASE 959 FIG. 2. Identification of the foci of Fig. 1 as cleistothecial primordia (dark spheres). The micrograph shows the contents of one focus lifted from the lawn with a needle. Surrounding the two primordia is a cloud of hulle cells and a few adhering conidial heads. Most foci contain only one primordium. Bar, 100 p.m. contrast to medusa, however, the hulle cells of yb mutants are laccase negative. If laccase II participates in the formation of the cleistothecial pigment, by analogy with the role of laccase I in the formation of the conidial pigment, mutants altered in cleistothecial pigment formation might be deficient in laccase II. Apirion (1) isolated two classes of cleistothecial color mutants. One allelic class makes blue rather than red cleistothecia, whereas the other class, which defines two loci, produces colorless (actually pink) cleistothecia. We tested representatives of each of these three loci and found that all produced cleistothecia which stained strongly with the laccase substrate. Quantitative assays of laccase II for the various mutants are given in Table 2. In the genus Aspergillus, most of the species catalogued by Raper and Fennel (23) do not exhibit a sexual cycle. Of those that do form cleistothecia, the structures are soft and lightcolored, except for A. nidulans. Other acleistothecial species develop masses of globose cells resembling the hulle cells of A. nidulans. The only species which exhibits both cleistothecia and hulle cells is A. nidulans, which is also the only species whose cleistothecia are dark pigmented. We tested seven species producing cleistothecia or hulle cells for reactivity with the ADBP-DMA laccase substrate (Table 3). None of the pale cleistothecia (of four species) reacted with the substrate, whereas the hulle cells (of three species) all showed positive reactions. The strongest reacting species was A. raperi, whose hulle cells formed compact spherical masses. The interiors of some of these masses contained a core of thin laccase-positive filaments resembling the cleistothecial primordia of A. nidulans.

6 960 HERMANN, KURTZ, AND CHAMPE J. BACTERIOL..Z. 01. Aoki. FIG. 3. Hulle cells stained with the ADBP-DMA reagent. Immediately after staining, the cells acquired a green tint. Upon chilling, opaque, deep blue microcrystals formed in the lumen of the cells as shown in the micrograph. The crystals formed most abundantly when the density of primordia was limited, as in Fig. 1. Bar, 10,um. These cores, however, showed no further development. DISCUSSION Cells and tissues having laccase activity can easily be identified by the use of a new reagent, ADBP-DMA, the reaction product of which is intensely pigmented. In the present study with A. nidulans, we have detected a laccase, laccase II, that is localized in the hyphae of cleistothecial primordia and the surrounding hulle cells. Even very young minute primordia react strongly with the substrate and are highly conspicuous if the mycelial lawn is viewed by transillumination. For mature cleistothecia, the outer surface of the wall does not visibly react with the substrate, but the inner surface of wall fragments become covered with microcrystals of the reaction product. Although extractable activity of laccase II per unit of mycelial mass is an order of magnitude less than that of laccase I (Fig. 5), the concentration of laccase II in tissues that have the activity is high: upon incubation with substrate, hulle cells and primordia stain as rapidly with the substrate as do conidial heads having laccase I. Laccases have been detected for many different fungi, both ascomycetes and basidiomycetes (8, 11, 16, 19, 21), and are often correlated temporally with the formation and pigmentation of sexual sporocarps. However, the precise role TABLE 1. Substrate specificity of laccase II 02 uptakea Substrate nmol/10 min % p-phenylenediamine m-phenylenediamine 0 0 DMPb p-aminophenol 8 27 m-aminophenol 0 0 o-aminophenol 0 0 p-dihydroxybenzene (quinol) 0 0 DOPA 0 0 Tyrosine 0 0 ADBP DMA 0 0 a Oxygen uptake was measured with a Polarigraph as described in the text. All substrates were present at a concentration of 1 mm. A value of 0 indicates an uptake of less than 1 nmol/10 min. b With DMP as the substrate, the activity determined by 02 uptake agreed with the activity determined spectrophotometrically to within 20%.

7 VOL. 154, 1983 A. NIDULANS LACCASE 961 FIG. 4. Scanning electron micrograph of cleistothecial wall fragments incubated with the ADBP-DMA reagent. The crystals, which appear as blue asterisk-shaped bodies in light microscopy, formed on the spongy inner surface of the wall but not on the smooth outer surface. Bar, 10 pum. of this enzyme in sexual development is unknown. Bu'lock (4) proposed that phenoloxidases may catalyze the formation of phenolic polymers that cross-link adjacent hyphae and thus produce organ-like structures from aggregates of filametous cells. The presence of laccase in the hyphae of the cleistothecial primordia of A. nidulans is consistent with this hypothesis. When first observable by the ADBP-DMA staining reaction, the primordia are already highly stable to disruption. What, then, is the role of hulle cells in this developmental process? They could serve to protect the immature ascocarps. However, the fact that hulle cells, like the primordia, are laccase positive suggests that these cells may play a more direct role in cleistothecial morphogenesis. The laccase of hulle cells and primordia could be synthesized in both of these tissues, or it could be synthesized in one and transported to the other. TABLE 2. Laccase II activity in various strains of A. nidulans Genotypea Relevant phenotype Laccase II sp actb Wild-type Normal 200 medal Acleistothecial; hulle cells 200 ybi Acleistothecial; hulle cells <40 bla3 Blue cleistothecia 470 cla4 Colorless cleistothecia 390 cibi Colorless cleistothecia 420 a All strains except ybi carry the yellow-conidial mutation ya2, which eliminates laccase I. The yb mutation itself also eliminates laccase I. The wild-type strain used in this comparison was R-21. b Expressed as nanomoles of DMP oxidized per milligram of protein per minute. Assays were performed on homogenates of 110-h confluent surface cultures as described in the text. For the ybi mutant, the assay time was 135 h because this mutant has a germination delay of about 24 h (12).

8 962 HERMANN, KURTZ, AND CHAMPE TABLE 3. Presence of laccase in sexual structures of various Aspergillus species Cleistothecia Hulle cells Species" Presence Color Laccaseb Presence Laccaseb A. nidulans + Red A. raperi A. puniceus A. silvaticus A. ornatus + Yellow 0 0 A. ruber + Yellow 0 0 A. tonophilus + White 0 0 A. fisheri + White 0 0 J. BACTERIOL. a Species were grown as surface colonies on the optimal media recommended by Raper and Fennell (23). b Laccase was detected histologically by the ADBP-DMA reaction. We know, however, that hulle cells do not obtain their laccase from primordia because the hulle cells of certain mutant strains that lack cleistothecia are laccase positive. This leaves open the possibility that enzyme transport is from the hulle cells to the primordia. If laccase functions to cross-link the hyphae of the cleistothecial walls, the need for oxidative enzymes may be more than can be supplied by the constituent hyphae of the primordia, especially if these hyphae become metabolically inert during the process. The hulle cells may thus serve as nurse cells for the primordia. Such a role is consistent with our finding that, unlike the conidial laccase, HOURS (37*) FIG. 5. (a) Time course of laccase I and laccase II development by confluent surface cultures of A. nidulans. The strains R-153 (wa3;pyroa4) and R-21 (ya2 pabaal) were the sources of laccase I and laccase II, respectively. (b) Time course of development of laccase-positive foci in a confluent lawn of strain R-21. the laccase of hulle cells is not extracellular but is located in the cytoplasm. To determine the topological relationship beween cleistothecia and the surrounding hulle cells, we have examined partially stripped mature cleistothecia by scanning electron microscopy. The cleistothecial surface is smooth and featureless, having the appearance of a continuous wall. Superimposed on this surface, however, is a network of thin hyphal filaments which closely adhere to the surface, sometimes appearing to merge with the surface, and which are connected directly to the hulle cells by short radial stalks. A microscopic study of the early stages of cleistothecial development is in progress and will be reported elsewhere. The pattern of occurrence of cleistothecial laccase in mutants of A. nidulans and other Aspergillus species does not resolve the function of this enzyme but does suggest certain possibilities. On the one hand, the fact that species which produce pale cleistothecia and lack hulle cells have no cleistothecium-associated laccase suggests that in A. nidulans laccase may participate in the synthesis of the cleistothecial pigment which has been identified as a polyhydroxylanthroquinone called asperthecin (22). The presence of laccase II in mutants with colorless cleistothecia does not argue against this hypothesis; pigment synthesis is undoubtedly a multistep process which could be aborted by a deficiency in some other enzyme. The existence of such mutants, however, shows that the pigment is not essential for cleistothecial development and is thus not the cross-linking agent of the cleistothecial wall. On the other hand, the characteristics of the dominant yellow-conidial mutants (yb) suggests that laccase II is essential for cleistothecial development. These mutants produce neither laccase I nor laccase II and are acleistothecial, although hulle cells are formed in abundance

9 VOL. 154, 1983 A. NIDULANS LACCASE 963 CUILTURE AGE (lotutrs) L ccasc 11 X * *, P- + elocaase I ya ya+ FIG. 6. Electrophoretic comparison of laccase I and laccase II. Equivalent extracts of strains R-21 and R-153 were prepared by the same method used for the assays shown in Fig. 5a (see text) and were analyzed on a 7.5% nondenaturing polyacrylamide gel. The gels were stained with the chromogenic laccase substrate DMP. To obtain visible staining for both samples without overloading, the R-21 extract was concentrated threefold by lyophilization, and the R-153 extract was diluted threefold. (12). Furthermore, the sexual defect of yb mutants is remedied by copper, which is an essential cofactor of all known laccases and tyrosinases. If cleistothecial pigmentation and hyphal cross-linking are in fact independent processes, it is possible that laccase participates in both processes but that only the cross-linking function is essential for morphogenesis. In Aspergillus species whose cleistothecia are laccase negative, some analogous enzyme presumably serves the cross-linking function. An essential role for phenoloxidases in sexual morphogenesis is also 6r. fi g..if :'p.'...i..1.-o 11 p-it P. 1. FIG. 7. Ouchterlony immunodiffusion assay of the cross-reaction of laccase I and laccase II. Anti-laccase II was raised in two rabbits, the sera of which were assayed separately together with their preimmune sera (p.i.). Immunological methods were as described previously (12, 13). I indicated from studies with other fungi. In Podospora anserina, many mutants defective in the formation of perithecia have an altered phenoloxidase spectrum (8, 9). In Schizophyllum commune, inhibitors of phenoloxidase block basidiocarp development at early stages without affecting vegetative growth (17-19). Quantitative studies of cleistothecial development in A. nidulans have heretofore been difficult or impossible because the primordia are mostly embedded in the mycelial mat. Thus, as shown by the experiment of Fig. 5b, the presence of histologically detectable laccase in primordia provides a valuable technique for quantifying the density of primordia in a mycelial lawn at very early stages of development. ACKNOWLEDGMENTS This research was supported in part by Public Health Service grant GM from the National Institutes of Health and by the Charles and Johanna Busch Memorial Fund. We thank Lee Simon for assistance with scanning electron microscopy and Amy Chang for superb technical assistance. LITERATURE CITED 1. Apirion, D Formal and physiological genetics of ascospore colour in Aspergillus nidiulans. Genet. Res. 4: Armitt, S., W. McCullough, and C. F. Roberts Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J. Gen. Microbiol. 92: Blaich, R., and K. Esser Function of enzymes in wood destroying fungi. II. Multiple forms of laccase in white rot fungi. Arch. Microbiol. 103: Bu'lock, J. D Fungal metabolites with structural function, p In Essays in biosynthesis and microbial development, E. R. Squibb lectures on chemistry of microbial products. John Wiley & Sons, New York. 5. Clutterbuck, A. J A mutational analysis of conidial development in Aspergillus nidtulans. Genetics 63: Clutterbuck, A. J Absence of laccase from yellow-

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