Mycologia
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DOI: 10.3852/mycologia.98.5.682
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Mycologia, 98(5), 2006, pp. 682-689.
© 2006 by The Mycological Society of America

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies


Carmen Sánchez 1

     Laboratory of Biotechnology, Research Center for Biological Sciences, Universidad Autónoma de Tlaxcala, Apartado postal 129, Tlaxcala, Tlax., CP 90000, México

David Moore

     Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom

Gerardo Díaz-Godínez

     Laboratory of Biotechnology, Research Center for Biological Sciences, Universidad Autónoma de Tlaxcala, Apartado postal 129, Tlaxcala, Tlax., CP 90000, México

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.

Key words: basidiomycetes, electron microscopy techniques, extracellular matrix, hyphal ultrastructure


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal cells do not proliferate in the way that animal and plant cells do. In microscopic sections fungal tissue might appear to be composed of tightly packed cells resembling plant tissue but the tubular (hyphal) nature of the components always can be demonstrated by reconstruction from serial sections or by scanning electron microscopy. Plants, animals and fungi are distinct eukaryotic kingdoms and there are fundamental differences among the three kingdoms in the way that the morphology of multicellular structures is determined. A characteristic of animal embryology is the movement of cells and cell populations. In contrast plant morphogenesis depends on control of the orientation and position of the daughter cell wall, which forms at the equator of the mitotic division spindle. Fungi also have walls, like plants, but their basic structural unit, the hypha, exhibits two features that cause fungal morphogenesis to be totally different from plant morphogenesis; these are that (i) hyphae extend only at their apex and (ii) cross walls form only at right angles to the long axis of the hypha. One consequence of these ‘‘rules’’ is that fungal morphogenesis depends on the placement of hyphal branches. Increasing the number of growing tips by hyphal branching is the equivalent of cell proliferation in animals and plants. To proliferate the hypha must branch and form an organized tissue; the position of branch emergence and its direction of growth must be controlled (Moore 1998aGo).

Another way in which fungal morphogenesis differs from that in other organisms is that no lateral contacts have been found between fungal hyphae analogous to the plasmodesmata, gap junctions and cell processes, which interconnect neighboring cells in plant and animal tissues. Their absence suggests that morphogens used to regulate development in fungi will be communicated through the extracellular environment (Moore 1998Go) and implies that the environment immediately surrounding the hypha could be of prime importance in the regulation of fungal morphogenesis.

Over the years several studies have been published on a range of aspects of the developmental biology of different members of the Agaricales (using the classification scheme of Kirk et al [2001]Go). These include Agaricus in the Agaricaceae (Flegg et al 1985Go, Umar and van Griensven 1997aGo, bGo, cGo), Coprinopsis (now placed in the Psathyrellaceae) but as Coprinus in the Coprinaceae (Chiu and Moore 1993Go, Greening and Moore 1996Go, Greening et al 1993Go, 1997Go, Kher et al 1992Go, Moore 1984Go, Moore et al 1979Go), Volvariella in the Pluteaceae (Chiu 1993Go, Chiu and Moore 1990Go, 1993Go, 1999Go, Chiu et al 1989Go, 1995Go), Schizophyllum in the Schizophyllaceae (Schuren et al 1993Go, Wessels 1992Go, 1993Go) and Flammulina and Lentinula in the Marasmiaceae (Chiu et al 1996Go, 1999Go, Tan and Moore 1994Go, 1995Go, Williams et al 1985Go).

Unfortunately, with the expansion of interest in molecular analyses, studies of structure and physiology have become unfashionable. However data mining of fungal genomes (which is fashionable) has demonstrated that genomes of filamentous fungi lack sequences showing homology to gene sequences that are considered to be crucial to regulation of development of animals and plants (Moore et al 2005Go, Moore and Meskauskas 2006Go). This observation carries the implication that the unique cell biology of filamentous fungi has caused control of multicellular development in fungi to evolve in a radically different fashion from that in animals and plants. If what is known about animals and plants sheds little or no light on development in fungi there remains therefore a dire need for basic studies of structure and physiology of fungal fruit bodies, if only to broaden the foundation of information on which theories of fungal morphogenesis can be based.

What we do know already hints at some of the complexity of the relationships that exist. Expansion of the fruit body cap in Lentinula edodes and Pleurotus pulmonarius occurs by hyphal multiplication for example whereas the same process in Agaricus bisporus and Coprinopsis cinerea (published as Coprinus cinereus) occurs by hyphal inflation (Moore 1996Go, 1998aGo, Wessels 1993Go, 1994Go; this report). Both strategies require water uptake and therefore a metabolic osmoregulator, but Agaricus and Lentinula accumulate mannitol as osmoregulator (Hammond and Wood 1985Go, Tan and Moore 1994Go). In Coprinopsis mannitol is undetectable but urea accumulates to drive water into the cap (Moore 1984Go, Moore et al 1979Go); while in at least one species of Pleurotus, both mannitol and urea accumulate in the cap (Chiu and To 1993Go).

Pleurotus (in the Pleurotaceae) has several cultivated species that are becoming increasingly economically important as a food source (Moore and Chiu 2001Go), but the genus also has promise in bioremediation as the fungus and even its spent compost is able to degrade many organic pollutants effectively (Chiu et al 1998Go).

In the research reported here different stages of the development of fruit bodies of Pleurotus pulmonarius formed on potato-extract agar were studied with light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Strain.— – Pleurotus pulmonarius (Fr.) Quél. strain PPL27 was provided from the culture collection of the Chinese University of Hong Kong (Shatin, Hong Kong). Stock cultures were grown on malt-extract agar (Oxoid, England) without illumination at 25 C for 10 d in Petri dishes and then stored at 4 C. All experimental fruiting cultures were grown on potato-extract agar (PEA) (Sánchez and Moore 1999Go) contained in standard 9 cm Petri dishes. Fruit body primordia were collected after 10 d growth of the colony. Fruit body primordia approximately 0.5, 1, 2 and 4 mm long were studied.

Transmission electron microscopy.— – Samples were fixed, dehydrated, embedded, sectioned and stained as previously reported (Sánchez 2000Go, Sánchez and Moore 1999Go). Sections were observed with either a Philips 201 or a Hitachi 600 transmission electron microscope. Micrographs were recorded by conventional photography on Kodak ESTAR 4489 film.

Light microscopy.— – Embedding and sectioning of the samples were carried out with the same procedure as for transmission electron microscopy (Sánchez 2000Go, Sánchez and Moore 1999Go). Sections 1.5 µm thick were placed on glass slides and dried on a hot plate for 10 min before staining. The sections were stained with 1% toluidine blue (w/v) in 1% boric acid (w/v), which stains most of the cytoplasm, the cell walls and nuclei of the cells. The slides were left in the stain on a hotplate for 15 min and rinsed with distilled water and dried (Sánchez and Moore 1999Go). The measurements of the cross-sectional area of the pileus and stipe zones represented in sections of the fruit body were done with a Quantimet Q570 image analysis system and a video camera (Panasonic model WV-CD20), which sent the fruit body image to the computer monitor. An image analysis program written by G.C. Paul (Birmingham University) was used. Light micrographs were recorded with either a wild MPS 51S SPOT or Nikon M-35S camera attached respectively to the Leitz or the Nikon microscope. Either TMAX100 Kodak or Technical pan Kodak B&W film was used, according to the contrast in the stained specimens.

Environmental-scanning electron microscopy.— – Fresh specimens were observed with a Philips Electroscan E3 environmental-scanning electron microscope. Micrographs were taken with an attached 35 mm camera and Ilford B&W Delta film.

Cryoscanning electron microscopy.— – Fresh fruit bodies were frozen by immersing them in liquid nitrogen slush (at –210 C). They were transferred under vacuum to a cooled microscope stage where ice was sublimed from the tissue (at –70 C), after which the samples were again cooled to –180 C and coated with gold. Samples were examined in the hydrated frozen state with a Cambridge Instruments S200 scanning electron microscope fitted with an Oxford Instruments CT1000 low temperature stage. Micrographs were made with an attached 35 mm camera and Ilford B&W Delta film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
In 500 µm long fruit body initials, the pileus and stipe were not yet differentiated (FIGS. 1Go, 5Go). Differentiation of the pileus was observed in fruit body primordia approximately 1 mm long (FIGS. 2, 6). The cross-sectional area occupied by the pileus in these primordia represented about 20% of the total area of the section. The Pleurotus fruit body is symmetrical in these early stages of development, so the volumetric proportions will be the same as the cross-sectional area proportions in these median longitudinal sections (FIGS. 1–4Go).


Figure 1
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  		FIGS. 1–4. Light micrographs of longitudinal sections of fruit bodies of P. pulmonarius. 1. 500 µm long. 2. 1 mm long. 3. 2 mm long. 4. 4 mm long.

 

Figure 2
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  		FIGS. 5, 6. Cryoscanning electron micrographs of fruit body initials of P. pulmonarius. Top bar = 500 µm; bottom bar = 1 mm.

 
As the fruit body develops, the pileus of the fruit body became further separated from the stipe base by elongation of the middle zones of the stipe (FIGS. 1–4Go). As a result of this increase in magnitude of the stipe’s girth and length in the 2.0 mm fruit body, the area of the pileus represented about 6% of the total cross-sectional area (FIG. 3Go). By this stage the stipe had become clearly differentiated, but in sharp contrast to the situation in Coprinopsis cinerea (Moore et al 1979Go) the stipe tissue of the primordia fruit body of P. pulmonarius showed little evidence of cell inflation (FIGS. 7a, bGo; 8a, bGo; 9a–cGo).


Figure 3
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  		FIG. 7. Transmission electron micrographs of longitudinal sections of the bases of a fruit body initial (a) and of a 1 mm long fruit body (b) of P. pulmonarius. Bar = 5 µm.

 

Figure 4
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  		FIG. 8. Transmission electron micrographs of the stem (a) and the pileus (b) of a 1 mm long fruit body of Pleurotus pulmonarius. Bar = 5 µm.

 

Figure 5
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  		FIG. 9. Cryoscanning electron micrographs from the base (a), stem (b) and pileus (c) of a 1 mm long fruit body, and from the pileus (d) of a 4 mm long fruit body. Direction of growth from stipe to pileus in a horizontal longitudinal section of the fruit body. Bar = 20 µm.

 
In fruit body primordia approximately 4 mm long, the pileus clearly had begun to expand and now represented about 13% of the cross-sectional area of the section. In general hyphae in the base of the fruit body primordium of P. pulmonarius were interwoven in all directions and in the pileus also were twisted together (FIG. 9a, c, dGo). On the other hand, in the central parts of the stipe, they were disposed mainly in a longitudinal or parallel manner (FIG. 9bGo).

Extensive sheaths of ECM were observed in all stages of P. pulmonarius fruit body development. They were evident in transmission electron micrographs as slightly electron dense regions between the hyphal profiles (FIG. 10aGo) and were clearly revealed surrounding live hyphae by environmental-scanning EM (FIG. 10bGo). Sometimes this surface material formed a sheath over the whole surface of the young fruit body.


Figure 6
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  		FIG. 10. Transmission electron (a) and environmental-scanning electron (b) micrographs of hyphae from the stem of a 1 mm long fruit body surrounded by sheaths of ECM. Bar (a) = 2 µm. Arrows show ECM. Diamond-headed arrows show hyphae.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Sánchez et al (2004)Go showed that the same staining technique as that used in this study was able to stain specifically the peripheral growth zone of vegetative colonies. These authors found that glycogen (identified as a histologically stainable accumulation in fruit body initials and primordia of Coprinopsis cinerea [Moore 1998bGo, Moore et al 1979Go, Niederpruem 1978Go]) was not the only compound responsible for the dense staining of the hyphae in the peripheral growth zone; other components that also might be accumulated include high levels of protein, phospholipids and wall polysaccharides.

A much lesser role for glycogen in fruit body development of Pleurotus sajorcaju also has been demonstrated by direct measurements (Chiu and To 1993Go). Whatever the exact identity of the accumulation products, the fact that the histological staining technique identifies the peripheral growth zone suggests that the zones of the P. pulmonarius fruit body primordia that were most densely stained were the most active growth zones of the primordium. They also were populated by hyphae that contained few vacuoles.

The base of 1 mm long P. pulmonarius fruit bodies had a similar hyphal arrangement to that seen in the base of younger fruit body initials, but the hyphae were more highly vacuolated even at this early stage (FIG. 7a, bGo). As development proceeded and distinct stipe tissue differentiated, the extent of vacuolation increased (FIG. 8aGo). Cells in the pileus also were more intensely stained (FIG. 8bGo) and generally smaller than those in the stipe (Sánchez 2004Go) (reflected in FIG. 8a, bGo).

Vacuolation might be important in the creation of the turgor pressures required during stipe extension. Localized production of large vacuoles certainly has been associated with specific stipe bending caused by the gravitropic response (Greening and Moore 1996Go, Greening et al 1993Go, 1997Go).

Williams et al (1985)Go reported that, in 2 mm long fruit bodies of Flammulina velutipes, the tissue of the stipe consisted mainly of elongated, longitudinal hyphae arranged parallel to each other, whereas in the base the hyphal arrangement was irregular and the hyphae appeared to be highly interwoven. Similar hyphal arrangement was observed in a 1 mm fruit body long (FIG. 9a, dGo). The longitudinal hypha arranged in a parallel manner (FIG. 9bGo) presumably was due to the stretching tensions resulting from the increasing vacuolation. Even the most extensive sheaths of ECM seems to have little mass because material prepared for conventional cryoscanning EM preserves only a slight sprinkling of material over hyphal surfaces that we assume to be dehydrated ECM (FIG. 10aGo).

The expansion of the young fruit body involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix, the components of which must be under the control of the hyphae the ECM surrounds. The ECM clearly offers an environment through which intercellular signals could be communicated and even might be composed of such signaling molecules. However the ECM in these fruit bodies is dilute. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae.


   ACKNOWLEDGMENTS
 
We thank Dr A.L. Demain for critical reading of the manuscript and Dr S.W. Chiu for kindly providing P. pulmonarius PPL27. This work was supported by the Mexican Council of Science and Technology (CONACYT) and the British Council.


   FOOTNOTES
 
Accepted for publication August 21, 2006.

1 Corresponding author. E-mail: sanher6{at}hotmail.com


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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