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Stipe cell wall architecture varies with the stipe elongation of the mushroom Coprinopsis cinerea
f u n g a l b i o l o g y 1 1 9 ( 2 0 1 5 ) 9 4 6 e9 5 6
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Stipe cell wall architecture varies with the stipe elongation of the mushroom Coprinopsis cinerea Xin NIU1, Zhonghua LIU1, Yajun ZHOU, Jun WANG, Wenming ZHANG, Sheng YUAN* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China
article info
abstract
Article history:
A large amount of granular protrusions overlie the outer cell wall surfaces in both elongat-
Received 29 May 2015
ing and non-elongating stipe regions but overlie the inner cell wall surfaces only in non-
Received in revised form
elongating stipe regions. Removal of granular protrusions using alkali, amorphous mate-
10 July 2015
rials overlying on both the inner and outer cell wall surfaces were explored in the non-
Accepted 24 July 2015
elongating stipe regions. b-1,3-Glucanase treatment not only removed above those granu-
Available online 5 August 2015
lar protrusions and underlying amorphous materials on the wall surfaces but also removed
Corresponding Editor:
wall matrices embedding chitin microfibrils on the cell walls of most stipe regions, except
Matt Walmsley
for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained. The chitin microfibrils were closely and transversely arranged on both
Keywords:
the inner and outer cell wall surfaces in the elongating apical stipe region, whereas they
Chitin microfibril
were loosely and transversely arranged on the inner cell wall surfaces and further became
b-1,3-glucan
sparser and even randomly arranged on the outer cell wall surface in the non-elongating
Granular protrusion
stipe regions. We propose that the surface deposition of granular protrusions and amor-
Overlying amorphous material
phous materials and the change of microfibril architecture and wall matrices may cause
Wall matrix
loss of wall plasticity and cessation of stipe elongation. ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction The development of fruiting bodies of basidiomycete involves a characteristic elongation growth of the stipe, which is dependent on the elongation of stipe cell walls (Mol et al. 1990; Kamada et al. 1991; Voisey 2010; Fang et al. 2014; Zhang et al. 2014). Stipe cells are surrounded by a layer of wall that acts like a strait-jacket to constrain and shape the cell. For the elongation of stipe cells, cell walls must provide the cell
with sufficient strength to withstand the large mechanical forces that result from cell turgor pressure while concurrently retaining adequate plasticity to extend under the strain of turgor pressure so that the newly synthesized wall components can be inserted at the existing wall to increase the cell surface areas (Bowman & Free 2006). Kamada et al. (1991) reported that chitin microfibrils are arranged transversely in stipe cell walls supported by the cross-linking between chitins and glucans in Coprinus cinereus,
* Corresponding author. College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing 210023, PR China. Tel./fax: þ86 25 85891067. E-mail address: [email protected] (S. Yuan). 1 Co-first authors. http://dx.doi.org/10.1016/j.funbio.2015.07.008 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Architecture of Stipe Wall
although they were observed as randomly orientated microfibrils on the outer surface of stipe wall preparations. Disruption of these transversely oriented microfibrils requires hydrolysis with concomitant insertion of new chitin chains and reformation of their transverse arrangement; these are mediated by both glucanases and chitinases, resulting in the required cell wall plasticity for stipe elongation (Kamada et al. 1982; 1985; 1991). However, Mol et al. (1990) also reported that chitin microfibrils on the inner surface of the elongating stipe cell walls of Agaricus bisporus were transversely arranged, neglecting the randomly oriented microfibrils on the outer surface of the stipe wall preparation. They suggested that a higher percentage of b-1,6-linked branches existed in the b-1,3-glucan backbone of elongating stipe cell walls compared to mature stipe cell walls. This makes hydrogen bonds between the glucan chains unstable (Mol & Wessels 1990) and subject to be directly disrupted by the strain of turgor pressure rather than by hydrolysis of lytic enzymes (Mol et al. 1990). Recently, we purified an expansin-like protein from snail stomach juice that could reconstitute heat-inactivated stipe wall extensions of Flammulina velutipes (Fang et al. 2014) and Coprinopsis cinerea (Zhang et al. 2014) stipes without hydrolytic activity. Our results do not support the previous hypotheses that hydrolysis of wall polymers by enzymes (Kamada et al. 1991) or the disruption of hydrogen bonding in wall polymers by the stress of turgor pressure (Mol et al. 1990) directly correspond to stipe wall extension. Instead, we propose that similar to higher plant wall extension (Cosgrove 2000, 2005; Wang et al. 2013), stipe wall extension may be mediated by some endogenous fungal expansin-like proteins that facilitate the separation of wall microfibrils without the passive reorientation of microfibrils by hydrogen bond disruption between parallel microfibrils. Furthermore, different regions of F. velutipes and C. cinerea stipes have different susceptibilities to the snail expansin-like protein, which may reflect differences in wall structure and chemical composition in the different stipe regions, as shown in yeasts (Cabib & Arroyo 2013) and plants (Zhao et al. 2008). Therefore, it is necessary to further clarify the architecture of stipe cell walls to understand how wall structure relates to stipe elongation. In this study, we developed methods to examine stipe cell wall architecture using field emission scanning electron microscopy (FESEM) on essentially intact stipe tissues, and compared the architectural changes of cell walls along the length of the elongating stipes from the apex to the base.
Materials and methods Strain and culture Coprinus cinereus ATCC 56838 was purchased from ATCC. Mycelium grown on the PDYA medium agar (300 g of diced potatoes, 20 g of glucose, 5 g of yeast extract, 15 g of agar and 1 L of distilled water) were inoculated on a fresh PDYA medium agar in 9-cm Petri dishes and incubated at 28 C under the conditions described by Kamada et al. (1982) until appropriate lengths of fruiting bodies were formed.
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Measurement of elongation growth of the developing stipe Approximately one-third of a cap was vertically removed from an approximately 60-mm length fruiting body of Coprinopsis cinerea grown on the PDYA medium agar in a Petri dish to expose the apical part of the stipe enclosed by the cap. The entire stipe was marked into six divisions from the apex to the basal swollen plectenchyma at 10-mm intervals with a marker pen. The marked fruiting bodies were grown continuously on the agar medium, and the change in length of each stipe division was measured at 1- and 2-h intervals (Cox & Niederpruem 1975).
Measurement of stipe cell wall extension Approximately 1e10 mm apical, 21e30 mm median, 41e50 basal (i.e., above the swollen plectenchyma), and 51e60 mm swollen plectenchyma region segments were excised with a razor blade from the 60-mm elongating fruiting body stipes. The fresh Coprinopsis cinerea stipe segments were abraded with carborundum, frozen at 20 C for approximately 24 h or more, and used within one week for the measurement of wall extension activity with an extensometer, as described by Zhang et al. (2014).
FESEM Excised segments identical to those described above were further bisected longitudinally into two approximately equal halves. The specimens were prepared for FESEM by modification using the method of Marga et al. (2005). Briefly, after slicing off the thin inner surface longitudinally from each half segment, the segments were washed three times with distilled water, fixed in 4 % paraformaldehyde in 0.2 M Na2HPO4eNaH2PO4 buffer (PBS, pH 7.4) for 4 h at 4 C, and rinsed in PBS. The specimens were then dehydrated in a graded ethanol series (30 %, 50 %, 70 %, and 95 % for approximately 45 min per step) followed by 100 % ethanol overnight, and critical point dried using CO2. For alkali treatment, stipe segments were incubated in 1 ml of 1 M KOH for 60 min, rotated at 600 rpm at 60 C (changing for fresh 1 M KOH at 15 min intervals), and washed five times with distilled water and once with PBS. The segments were then fixed in 4 % paraformaldehyde, dehydrated and dried as described as above. For hydrolase treatments, native stipe segments or alkali-treated stipe segments were incubated in 7.8 102 U/ml partially purified chitinase from Streptomyces griseus (Sigma) in 50 mM NaAc (pH 6.0) for 24 h at 37 C, 23.3 U/ml partially purified combined exo- and endo-b-1,3glucanases from Coprinopsis cinerea (purified in our lab) in 50 mM NaAc (pH 6.0) for 24 h at 37 C, or 3.2 U/ml partially purified pronase (Sigma) in 50 mM TriseHCl (pH 7.5) for 24 h at 37 C at 600 rpm in a rotator. The incubated segments were then washed three times with distilled water and further fixed, dehydrated, and dried as described above. Chitinase was partially purified from commercial chitinase from S. griseus (Sigma) by TSKgel G2000SW (13 m, 21.5 600 mm, Tosoh) size exclusion HPLC, with a mobile phase of 0.1 M NaCl-50 mM NaAc (pH 6.0) (Sietsma & Wessels 1979) containing 278 103 units mg1 protein chitinase activity but no b-1,3-glucanase activity (on laminarin) or proteinase activity (on casein). Pronase was partially
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purified from commercial pronase from S. griseus (Sigma) by CM300 (7 m, 250 4.6 mm, Eprogen) cation exchange HPLC, with a 0e0.3 M NaCl linear gradient in 50 mM NaAc (pH5.0) mobile phase (Jurasek et al. 1971) containing 20.1 units mg1 protein proteinase activity (on casein) but no hydrolytic activity on laminarin or chitin. Combined exo- and endo-b-1,3-glucanases were partially purified from C. cinerea pilei (published elsewhere) containing 93.9 units mg1 protein b-1,3-glucanase activity (on laminarin) but no hydrolytic activity on chitin or casein. One enzyme unit is defined as the release of 1 mmol of glucose or equivalent, or tyrosine per min at the indicated measurement conditions from laminarin, chitin, or casein. All specimens were mounted on stubs with double-sided sticky carbon tape with the cut surface facing upward and coated with gold on an SCD 500 sputter coater (BAL-TEC) at 37 mA for 45 s. The cell wall architecture was examined in a Hitachi S4800 cold-cathode field emission scanning electron microscope (Hitachi, Japan) fitted with a solid state backscatter upper electron detector at 5 kV with a working distance between 6 and 8 mm.
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plectenchyma regions were 1 %.h1, 0.5 %.h1, 0.1 %.h1, and 0.1 %.h1, respectively (Fig 1B). The remaining minute acidinduced wall extension activity of basal segments was due to the non-protein mediated short-term wall extension, which is not involved in stipe elongation growth (Zhang et al. 2014).
Architecture of native cell walls in different stipe regions
The stipe segments of Coprinopsis cinerea were trimmed to 1e2 mm2 and placed in 0.2 ml 2.5 % glutaraldehyde in 0.1 M phosphate buffer, with a pH of 7.2. After 4 h at 4 C, the specimens were washed thoroughly with 0.1 M PBS and post-fixed for 2 h in 1 % osmium tetroxide in 0.1 M phosphate buffer, with a pH of 7.2, at 4 C. Then, the specimens were washed and dehydrated with a graded acetone-water series and embedded in Spurr epoxy resin. Sections with approximately 70 nm thickness were cut with a diamond knife on a Reichert Ultracut E. ultramicrotome (Laica) and double staining with Uranyl acetate and lead citrate. The specimens were mounted on copper grids and observed with a Hitachi 7650 TEM at 80 kV.
For observation of architecture of cell walls in different stipe regions, the 1e10 mm apical segment, the 21e30 mm median segment, the 41e50 mm basal segment above the swollen plectenchyma, and the 51e60 mm swollen plectenchyma segment from 60-mm length elongating fruiting bodies were bisected longitudinally into two halves, so that some of cells at inside of stipe segments were cut to be opened to expose their inner surface of the cell walls, as indicated by the low magnification images in FESEM (Supplement Fig S1). Because buffer washing and the series of ethanol dehydration completely released the cytoplasm contents and plasma membrane from the opened cells during specimen preparation (Marga et al. 2005), the inner wall surface of cells in the fast elongating apical segments showed a transversely arranged microfibril architecture embedded in the matrix under high FESEM magnification (Fig 2A1). In the non-elongating basal and swollen plectenchyma segments, however, the inner wall surfaces of the opened cells were overlain by massive amounts of granular protrusions, so the microfibril architecture was not observed there (Fig 2C1 and D1). The inner wall surface of cells in the slow elongating median segments exhibited a dim transversely arranged microfibril architecture embedded in the matrix, which seemed to be overlain by some faint amorphous materials with a few granular protrusions (Fig 2B1). In contrast, all of the outer wall surfaces of cells in the various stipe regions of Coprinopsis cinerea fruiting bodies were overlain by a mass of granular protrusions (Fig 2A2eD2).
Results
Architecture of alkali-treated cell walls in different stipe regions
Transmission electron microscopy (TEM)
Elongation growth and cell wall extension activity in different stipe regions In a previous experiment, we measured the profile of elongation growth of an approximately 27-mm length young Coprinopsis cinerea fruiting body and the relative profile of cell wall extension activity in different stipe regions (Zhang et al. 2014). To obtain a more distinct contrast of the differences in wall architecture between different stipe regions, appropriately 60-mm lengths C. cinerea fruiting bodies were selected for FESEM observations (Fig 1A). So it is necessary to determine the elongation growth features and cell wall extension activity of different stipe regions of the 60-mm lengths C. cinerea fruiting bodies. As shown in Fig 1A, after 2 h of marking, the 1e10 mm apical region and the 21e30 mm median region far from the apex elongated by approximately 67.3 % and 25.2 %, respectively, whereas the 41e50 mm basal region above the swollen plectenchyma and the 51e60 mm swollen plectenchyma region (Kues 2000) did not significantly elongate (t-test, P 0.05). The acid-induced cell wall extension activities of the apical, median, basal, and swollen
To further study the architecture of stipe walls, stipe segments were treated with 1 M KOH. As shown in Fig 3A2, B2, C1e2, D1e2, the KOH treatment removed granular protrusions from all of the existing wall surfaces of the stipes to explore different features of cell walls in different stipe regions. The KOH-treated outer wall surface of cells in the elongating apical and median stipe regions showed clear transversely arranged microfibril architecture embedded in the matrix (Fig 3A2 and B2), whereas the KOH-treated inner and outer wall surfaces of cells in the non-elongating basal and swollen plectenchyma stipe regions exhibited an amorphous material architecture rather than microfibril architecture (Fig 3C1e2 and D1e2). Furthermore, the KOH treatment made the dim microfibril architecture clear on the inner wall surfaces of the cells in the slowly elongating median segments (Fig 3B1).
Architecture of cell walls treated with hydrolytic enzymes in different stipe regions To further elucidate the architecture of the stipe walls, stipes were treated with some hydrolytic enzymes to observe their
Architecture of Stipe Wall
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Fig 1 e The distribution of elongation growth (A) and wall extension activity (B) along the 60-mm length developing C. cinerea stipe. To determine the elongation growth of the developing stipe, approximately one-third of the cap was vertically removed from the 60-mm fruiting body grown on the agar medium in a Petri dish to expose the part of the stipe enclosed by the cap. The entire stipe was marked into six different divisions: 1e10, 11e20, 21e30, 31e40, 41e50, and 51e60 mm from the apex to the base at 10-mm intervals with a marker pen. The length change of each division was measured at 1-h intervals. The elongation photography and elongation growth summary of the stipes (n [ 9) are shown in A1 and A2, respectively. Data are means ± SD of nine replicates. ‘a’ indicates a significant difference between the length change of the fruiting body at 0 h and 2 h (t-test, P £ 0.001). ‘b’ indicates no significant difference between the length change of the fruiting body at 0 h and 2 h (t-test, P > 0.05). For analysis of the wall extension activity, the 1e10 mm apical, 21e30 mm median, 41e50 mm basal, or 51e60 mm basal swollen plectenchyma segments from the 60-mm long C. cinerea stipes were treated and subjected to extensometry analysis, as described in the materials and methods section. The length and extension rate changes of the stipe walls during measurements are shown in B1 and B2, respectively. The curves are the means of more than three independent experiments (n [ 9).
effects on the wall architecture. We used a partially purified b1,3-glucanase preparation (containing exo-b-1,3-glucanase and endo-b-1,3-glucanase activities but no chitinase or proteinase activity) isolated from Coprinopsis cinerea pilei to treat native stipe segments. The results show that the b-1,3glucanase treatment not only removed all granular protrusions on all existing wall surfaces of the stipes but also removed the alkali-resistant amorphous material on both of the inner and outer wall surfaces of the non-elongating stipe regions, exposing the underlying wall architecture. In addition to the transversely arranged microfibril architecture observed on the outer wall surfaces of cells in the elongating apical and median regions (Fig 4A2 and B2), microfibril architecture
under amorphous materials on both of the inner and outer wall surfaces of the cells in the non-elongating basal and swollen plectenchyma regions of the stipes were exposed (Fig 4C1e2 and D1e2). Furthermore, compared to corresponding KOH-treated stipe segments (Fig 3), b-1,3-glucanase treatment apparently removed most amorphous matrices embedding the microfibrils on both the inner and outer wall surfaces of the elongating apical stipe segments (Fig 4A and B). As a result these microfibrils appeared to have little matrix left between them, exposing a superimposed microfibril network that was clear and distinguishable on these walls surfaces. However, the b-1,3-glucanase treatment only removed partial amorphous matrices embedding the microfibrils on
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Fig 2 e FESEM images of the architecture of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60-mm long elongating stipes of C. cinerea. Stipe segments were bisected longitudinally into two halves to expose the inner stipe cells, followed by buffer washing, fixing, critical point drying, and coating with gold, allowing the wall architecture of cells to be examined. Images are oriented so that the transverse cell axis is parallel to the short axis of the page. Scale lines indicate 500 nm.
both the inner and the outer wall surfaces of the nonelongating basal and swollen plectenchyma segments, and the microfibril network was still filled with many amorphous matrices between these microfibrils (Fig 4C and D). Notably,
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Fig 3 e FESEM images of the architectures of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segment (D) of 60-mm long elongating stipes of C. cinerea that were incubated in 1 M KOH for 60 min at 60 C before fixing, critical-point drying, and coating with gold as described in Fig 2. Scale lines indicate 500 nm.
there are some differences in the wall microfibril architecture between the elongating and non-elongating stipe regions. The microfibrils were closely and transversely arranged on both the inner and outer wall surfaces of the fast elongating apical region (Fig 4A1 and A2), whereas they were often loosely and
Architecture of Stipe Wall
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transversely arranged on both the inner and outer wall surfaces of the non-elongating stipe regions (Fig 4C1e2 and E1e2) and were sometimes even sparsely and randomly arranged on the outer wall surfaces of the non-elongating stipe regions (Fig 4D2 and F2). Compared to the fast elongating 1e10 mm apical stipe segments (Fig 4A2), the microfibril network of the outer wall surface of the slowly elongating 21e30 mm median stipe segments (Fig 4 B2) contained some matrix between the microfibrils and exhibited some loosely and randomly arranged architecture sometimes, whereas the microfibril network of the inner wall surface was similar to that of the fast elongating apical stipe segment, which represented a transition type of cell wall architecture from elongating to non-elongating stipes. Fig 5 shows that the proteinase (pronase) treatment also could remove all granular protrusions on all of the existing wall surfaces of the stipes. With the removal of granular protrusions by pronase, the outer wall surface of cells in elongating apical and median stipe regions exhibited microfibril architecture (Fig 5A2 and B2), whereas both the inner and outer wall surface of cells in the non-elongating basal and swollen plectenchyma stipe regions appeared amorphous materials architecture (Fig 5C1e2 and D1e2). When the KOH-treated stipe walls were treated further with chitinase, the microfibril structures were completely eliminated at both the inner and outer wall surfaces of the elongating apical and median stipe regions, only leaving amorphous matrix architecture (Fig 6), some of which showed apparent microfibril-like indentations or cavities (Fig 6A1, A2, B1, B2) left from the digestion of microfibrils. This indicates that the microfibrils consist of chitin chains, as described previously by others (Gow & Gooday 1983; Kamada et al. 1991).
Cell wall thickness in different stipe regions The cell-wall thickness of different regions of stipes were determined from electron micrographs of ultrathin sections (Fig 7). As shown in Fig 7E, the cell wall thickness of the fast elongating apical stipe region was 134.6 15.1 nm, whereas compared to the apical stipe region, the cell-wall thickness of the slow elongating median stipe region increased by 24.6 % and reached 170.1 27.8 nm. The cell-wall thickness of the non-elongating basal and swollen plectenchyma stipe regions further increased by above 66 % and reached 224.4 33.2 nm and 237.4 31.8 nm, respectively, both which were similar to each other, showing no statistical difference.
Fig 4 e FESEM images of the architecture of the inner wall surfaces (A1eF1) and the outer wall surfaces (A2eF2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C, D), and the 51e60 mm basal swollen plectenchyma segment (E, F) of 60-mm long elongating stipes of C. cinerea that were incubated in combined exo-, endo-b-1,3-glucanases from C. cinerea in 50 mM NaAc (pH 6.0) for 24 h at 37 C before fixing, critical-point drying, and coating with gold, as described in Fig 2. Scale lines indicate 500 nm.
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Fig 5 e FESEM images of the architectures of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segment (D) of 60-mm long elongating stipes of C. cinerea that were incubated in pronase from Streptomyces griseus (Sigma) in 50 mM TriseHCl (pH 7.5) for 24 h at 37 C before fixing, critical point drying, and coating with gold as described in Fig 2. Scale lines indicate 500 nm.
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Fig 6 e FESEM images of the architecture of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60 mm long elongating stipes of C. cinerea, which were first incubated in 1 M KOH for 60 min at 60 C, and then incubated in chitinase from Streptomyces griseus (Sigma) in 50 mM NaAc (pH 6.0) for 24 h at 37 C after washing with water, followed by fixing, critical point drying, and coating with gold, as described in Fig 2. Scale lines indicate 500 nm.
Discussion It is known that the classic ultrathin section method of TEM is not well suited for the observation of cell walls because polysaccharides cannot be efficiently stained by heavy metals
(Marga et al. 2005). Using the Pt/Pd shadow-cast technique, the architecture of stipe cell walls can be imaged by TEM (Mol et al. 1990; Kamada et al. 1991). However, this shadowcast approach depends on mechanical disruption and
Architecture of Stipe Wall
chemical extraction to prepare samples of wall pieces free of cytoplasm (Mol et al. 1990; Kamada et al. 1991). This macerated cell wall preparation may reflect the architecture of rearranged microfibrils to some extent (Itoh 1975; Mol et al. 1990; Kamada et al. 1991; Marga et al. 2005). Our approach uses a stipe fragment that is bisected longitudinally but kept intact, sparing a disruptive homogenization. This simple cutting and solution rinsing preparation offers a gentle and convenient way to explore the architecture of stipe cell walls. Recently, some reports show that high-resolution FESEM images the cell wall cellulose microfibrils directly in essentially intact plant tissues that were coated thickly enough to make the sample conduct electrons. Marga et al. (2005) demonstrated that in plant cell walls, the FESEM images closely resemble images of partially hydrated cell walls obtained by atomic force microscopy (AFM); the AFM images did not support the notion that fixation, dehydration, or critical-point drying during the preparation of FESEM specimens influences the images to any appreciable extent. Thus, we presume that the FESEM images in this study represent the real architecture of stipe cell walls. This study explored that the outer wall surfaces of cells in all stipe regions were overlain completely by massive amounts of granular protrusions, consistent with the outer wall surface architecture of filament fungi Aspergillus nidulans hyphae reported in Ma et al. (2005). Because these granular protrusions could be removed by KOH, pronase, and b-1,3glucanase, they may be composed of glycoproteins as in A. nidulans hyphae (Ma et al. 2005). In fact, Michalenko et al. (1976) even reported that there is a KOH-soluble outer layer on the outside of the chitin microfibril layer in basidiomycetes hyphal walls and that this alkali-soluble layer is composed primarily of b-1,3-glucan with b-1,6-linked branches and proteins in Coprinus cinereus (Schaefer 1977; Marchant 1978). These alkali-soluble b-1,3-glucans and proteins are usually
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non-covalently linked to chitin microfibrils and are removed by alkali treatment or b-1,3-glucanase (Wessels 1986; Latge 2007; Ruiz-Herrera 2012). Previous reports in which the granular protrusions were not observed on the outer wall surface (Mol et al. 1990; Kamada et al. 1991) might be due to mechanical disruption and alkali extraction for their preparation of stipe cell walls. Interestingly, we found that the inner wall surfaces of cells in the elongating apical and median segments showed little or a little granular protrusions, whereas in the nonelongating basal and swollen plectenchyma segments, the inner wall surfaces were also overlain completely by massive amounts of granular protrusions like the outer wall surfaces. Because the regions where the inner wall surface were overlain by the protrusions lost their capacities for elongated growth and wall extension, these protrusions appeared on the inner wall surface of cells in the non-elongating regions may be related to wall maturation. A significant discovery in this study is that, except for surface protrusions, amorphous materials overlying both the inner and outer wall surfaces of cells appear different distributions and distinctive features in different stipe regions. Removal of surface protrusions with alkali treatment, the layer of amorphous material overlying both the inner and outer wall surfaces of cells was explored in the nonelongating basal and basal swollen plectenchyma segments of stipes, whereas little amorphous material was overlain on both the inner and outer wall surfaces of the elongating apical and median regions, i.e., the faint amorphous material overlying on the inner wall surface of cells in the median stipe region could be removed by alkali treatment. The alkali-resistant overlying amorphous materials on both of the inner and outer wall surfaces of the non-elongating basal and basal swollen plectenchyma regions could be removed by b-1,3-glucanase treatment. Therefore, these amorphous materials must consist of b-1,3-glucan covalently linked to chitin chains
Fig 7 e Electron micrographs of cross sections of the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60 mm long elongating stipes of C. cinerea (scale lines indicate 1 mm). Comparison of the thickness of cell walls of different stipe segments in AeD (E). Same letters on the top of the bars indicate no significant difference between the thickness of cell walls of relative stipe segments, and different letters on the top of the bars indicate significant differences between the thicknesses of cell walls of relative stipe segments. Each thickness value is the mean wall thickness of 30 cells from the indicated stipe region.
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2007; Ruiz-Herrera 2012). Due to the lim(Wessels 1986; Latge itation of the previous macerated cell wall preparation, earlier reports did not dissolve the distribution difference of overlying amorphous materials on the wall surfaces in different stipe regions. We propose that with wall maturation, the overlying amorphous materials are gradually deposited on the wall surfaces and further modified, such as covalent linking r et al. 1997; of b-1,3-glucans to chitin microfibrils (Kolla Kopecek & Raclavsky 1999; Bowman & Free 2006), becoming alkali-insoluble. The remodeling of these overlying amorphous materials on the wall surfaces during the wall maturation may alter the plasticity of the stipe cell wall because the regions overlain by the amorphous material lose their capacities for elongation growth and wall extension. In fungal cell walls, there are masses of amorphous wall matrices that filled the space between chitin microfibrils acting as a cementing substance. These amorphous wall matrices are considered to be composed of polysaccharides and glycoproteins (Farkas 1979). This study shows that the wall matrix embedding microfibrils on both the inner and outer wall surfaces of cells in the elongating regions can be removed by b-1,3-glucanase treatment, rather than alkali treatment or pronase, to expose a distinctive microfibril network with little matrix between microfibrils, implying that these matrix components mainly consist of b-1,3-glucan covalently linked to chitin microfibrils. However, the b-1,3-glucanase-treated non-elongating regions of stipes showed some matrices remained between the microfibrils on both of the inner and outer wall surfaces, especially the outer wall surface. These results imply that the decrease of alkali-insoluble b-1,3glucans with wall maturation may be due to cross-linking between matrix components and microfibrils that are resistant to the b-1,3-glucanase treatment, or, alternatively, some other new components instead of b-1,3-glucans are added into the matrix between the microfibrils. Along with the removal of overlying protrusions and amorphous materials, as well as partial wall matrix, this study explores the features of chitin microfibril architecture on cell walls in the different regions of developing C. cinerea stipes: (1) In the fast elongating apical stipe region, the microfibrils were closely and transversely arranged on both the inner and outer wall surfaces; (2) In the non-elongating basal and basal swollen plectenchyma stipe regions, the microfibrils were loosely and transversely arranged on the inner wall surface, and became sparser and more loosely transversely arranged or sometimes even randomly arranged on the outer wall surface; (3) In the slow elongating median stipe region, the outer wall surface showed some loosely arranged microfibril architecture, though the inner wall surface still showed a closely transversely oriented microfibril architecture, exhibiting a transition type of cell wall architecture from elongating to non-elongating regions. Kamada et al. (1991) even reported that shadow-cast electron micrographs of alkali-extracted cell wall preparations of Coprinus cinereus stipes showed transversely arranged microfibrils on the inner surface, whereas the microfibrils on the outer surface were often randomly arranged. They attributed the observed randomly orientated microfibrils to disordering during wall preparation. Our experiments suggest that the randomly oriented microfibril structure previously reported by their lab for the
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Fig 8 e Schematic representation of a compositional, architectural and thick gradient of cell walls from the elongation apical region to the non-elongating basal swollen plectenchyma regions of a developing Coprinopsis cinerea fruiting body stipe (see text for explanation). , Granular protrusions, which are removed by KOH, pronase and b-1,3glucanase. , Chitin microfibrils, which are closely and transversely arranged at the wall of the elongating region, whereas loosely and transversely, sometimes even sparsely and randomly arranged at the wall of the non-elongating regions. , Wall matrix which is removed by b-1,3glucanase. , Wall matrix which is difficult to be removed by b-1,3-glucanase. , A thin layer of amorphous materials on the wall surface which is removed by KOH. , A thick layer of amorphous materials on the wall surface which is removed by b-1,3-glucanase but not by KOH. , Plasma membrane.
outer surfaces of stipe walls may reflect wall architecture of the outer wall surface in the full elongated (ceasing-elongating) stipes because their chemically extracting wall pieces were prepared from stipes at the end of elongation (21 h stage) (Kamada et al. 1991). We propose that at the fast
Architecture of Stipe Wall
elongating stipe region, newly synthesized chitin microfibrils are continually deposited on the inner wall surface and inserted between the existing microfibrils in a transverse orientation, and moved to the outer wall surface to increase the cell surface area so that there is no apparent difference of microfibril architecture between the inner and outer wall surfaces. With stipe elongation and wall maturation, though the old wall microfibrils are still progressively stretched, and the synthesis of new chitin microfibrils is reduced and finally terminated, b-1,3-glucan are largely synthesized and filled into the matrix spaces between chitin microfibrils by covalently linking to chitin chains at the inner wall surface of the non-elongating stipe segments. Thus, chitin microfibrils at the older outer wall layers far from the inner wall surface are further separated from each other into a more loose or random orientation. Simultaneously, the addition and further cross-linking of more matrix components occurs on the outer wall surface, so the wall matrix becomes resistant to the b-1,3-glucanase and alkali treatments, and chitin microfibrils become relatively sparse. This remodeling of matrix components protects the wall from weakening caused by fewer microfibrils while preventing the slippage between chitin microfibrils for wall elongation. In conclusion, the cell walls exhibit a compositional, architectural, and thick gradient from the elongation apical regions to the non-elongating basal swollen plectenchyma regions of the fruiting-body stipes (Fig 8). In the fast elongating apical region, chitin microfibrils are aligned transversely, and b-1,3-glucans with b-1,6 branches are linked covalently to chitin chains as molecular binders to connect two adjacent chitin microfibrils. This architecture facilitates the separation of wall microfibrils without passive reorientation so that newly synthesized chitin microfibrils are inserted in between separated microfibrils to longitudinally increase the wall surface area. Although the turgor pressure is the driving force for the fungal wall extension, the rate of elongation growth of stipes depends on the extensibility of the wall rather than the turgor pressure (Money & Ravishankar 2005; Lew 2011). With wall maturation, b-1,3-glucans with b-1,6 branches are largely synthesized instead of chitin chains to fill into the matrix spaces between chitin microfibrils and deposit as amorphous materials on both the inner and outer wall surfaces. Meanwhile, glycoprotein granular protrusions are further deposited on the inside adjacent to the plasma membrane of the amorphous materials on the inner wall surface to prohibit secretion of chitin microfibrils. Therefore, the wall thickness increases and the wall plasticity gradually reduces along the stipe from the elongating apical region to the non-elongating basal region. We propose that the surface deposition of protrusions and amorphous materials, and the change of microfibril architectures and wall matrices lead to the rigidification of cell walls and cessation of stipe cell elongation.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31170028) and the Priority Academic
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Development Institutions.
Program
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Jiangsu
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Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2015.07.008.
references
Bowman SM, Free SJ, 2006. The structure and synthesis of the fungal cell wall. Bioessays 28: 799e808. Cabib E, Arroyo J, 2013. How carbohydrates sculpt cells: chemical control of morphogenesis in the yeast cell wall. Nature Reviews Microbiology 11: 648e655. Cosgrove DJ, 2000. Loosening of plant cell walls by expansins. Nature 407: 321e326. Cosgrove DJ, 2005. Growth of the plant cell wall. Nature Reviews Molecular Cell Biology 6: 850e861. Cox R, Niederpruem D, 1975. Differentiation in Coprinus lagopus. Archives of Microbiology 105: 257e260. Fang H, Zhang W, Niu X, Liu Z, Lu C, Wei H, Yuan S, 2014. Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa. Fungal Biology 118: 1e11. Farkas V, 1979. Biosynthesis of cell walls of fungi. Microbiological Reviews 43: 117. Gow N, Gooday G, 1983. Ultrastructure of chitin in hyphae of Candida albicans and other dimorphic and mycelial fungi. Protoplasma 115: 52e58. Itoh T, 1975. Application of freeze etching technique for investigating cell wall organization of parenchyma cells in higher plants. Wood Research: bulletin of the Wood Research Institute Kyoto University 58: 20e32. Jurasek J, Johnson P, Olafson RW, Smillie LB, 1971. An improved fractionation system for pronase on CM-sephadex. Canadian Journal of Biochemistry 49: 1195e1201. Kamada T, Fujii T, Nakagawa T, Takemaru T, 1985. Changes in (1/3)-b-glucanase activities during stipe elongation in Coprinus cinereus. Current Microbiology 12: 257e259. Kamada T, Hamada Y, Takemaru T, 1982. Autolysis in vitro of the stipe cell wall in Coprinus macrorhizus. Journal of General Microbiology 128: 1041e1046. Kamada T, Takemaru T, Prosser J, Gooday G, 1991. Right and left handed helicity of chitin microfibrils in stipe cells in Coprinus cinereus. Protoplasma 165: 64e70. r R, Reinhold BB, Petra kova E, Yeh HJ, Ashwell G, Drgonova J, Kolla Kapteyn JC, Klis FM, Cabib E, 1997. Architecture of the yeast cell wall b(1/6)-glucan interconnects mannoprotein, b(1/3)glucan, and chitin. Journal of Biological Chemistry 272: 17762e17775. Kopecek P, Raclavsky V, 1999. Comparison of chitin content in the apical and distal parts of fungal hyphae in Basidiobolus ranarum, Neurospora crassa and Coprinus sterquilinus. Folia Microbiologica (Praha) 44: 397e400. Kues U, 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiology and Molecular Biology Reviews 64: 316e353. Latge JP, 2007. The cell wall: a carbohydrate armour for the fungal cell. Molecular Microbiology 66: 279e290. Lew RR, 2011. How does a hypha grow? The biophysics of pressurized growth in fungi. Nature Reviews Microbiology 9: 509e518. Ma H, Snook LA, Kaminskyj SG, Dahms TE, 2005. Surface ultrastructure and elasticity in growing tips and mature regions of
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journal homepage: www.elsevier.com/locate/funbio
Stipe cell wall architecture varies with the stipe elongation of the mushroom Coprinopsis cinerea Xin NIU1, Zhonghua LIU1, Yajun ZHOU, Jun WANG, Wenming ZHANG, Sheng YUAN* Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, PR China
article info
abstract
Article history:
A large amount of granular protrusions overlie the outer cell wall surfaces in both elongat-
Received 29 May 2015
ing and non-elongating stipe regions but overlie the inner cell wall surfaces only in non-
Received in revised form
elongating stipe regions. Removal of granular protrusions using alkali, amorphous mate-
10 July 2015
rials overlying on both the inner and outer cell wall surfaces were explored in the non-
Accepted 24 July 2015
elongating stipe regions. b-1,3-Glucanase treatment not only removed above those granu-
Available online 5 August 2015
lar protrusions and underlying amorphous materials on the wall surfaces but also removed
Corresponding Editor:
wall matrices embedding chitin microfibrils on the cell walls of most stipe regions, except
Matt Walmsley
for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained. The chitin microfibrils were closely and transversely arranged on both
Keywords:
the inner and outer cell wall surfaces in the elongating apical stipe region, whereas they
Chitin microfibril
were loosely and transversely arranged on the inner cell wall surfaces and further became
b-1,3-glucan
sparser and even randomly arranged on the outer cell wall surface in the non-elongating
Granular protrusion
stipe regions. We propose that the surface deposition of granular protrusions and amor-
Overlying amorphous material
phous materials and the change of microfibril architecture and wall matrices may cause
Wall matrix
loss of wall plasticity and cessation of stipe elongation. ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction The development of fruiting bodies of basidiomycete involves a characteristic elongation growth of the stipe, which is dependent on the elongation of stipe cell walls (Mol et al. 1990; Kamada et al. 1991; Voisey 2010; Fang et al. 2014; Zhang et al. 2014). Stipe cells are surrounded by a layer of wall that acts like a strait-jacket to constrain and shape the cell. For the elongation of stipe cells, cell walls must provide the cell
with sufficient strength to withstand the large mechanical forces that result from cell turgor pressure while concurrently retaining adequate plasticity to extend under the strain of turgor pressure so that the newly synthesized wall components can be inserted at the existing wall to increase the cell surface areas (Bowman & Free 2006). Kamada et al. (1991) reported that chitin microfibrils are arranged transversely in stipe cell walls supported by the cross-linking between chitins and glucans in Coprinus cinereus,
* Corresponding author. College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing 210023, PR China. Tel./fax: þ86 25 85891067. E-mail address: [email protected] (S. Yuan). 1 Co-first authors. http://dx.doi.org/10.1016/j.funbio.2015.07.008 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Architecture of Stipe Wall
although they were observed as randomly orientated microfibrils on the outer surface of stipe wall preparations. Disruption of these transversely oriented microfibrils requires hydrolysis with concomitant insertion of new chitin chains and reformation of their transverse arrangement; these are mediated by both glucanases and chitinases, resulting in the required cell wall plasticity for stipe elongation (Kamada et al. 1982; 1985; 1991). However, Mol et al. (1990) also reported that chitin microfibrils on the inner surface of the elongating stipe cell walls of Agaricus bisporus were transversely arranged, neglecting the randomly oriented microfibrils on the outer surface of the stipe wall preparation. They suggested that a higher percentage of b-1,6-linked branches existed in the b-1,3-glucan backbone of elongating stipe cell walls compared to mature stipe cell walls. This makes hydrogen bonds between the glucan chains unstable (Mol & Wessels 1990) and subject to be directly disrupted by the strain of turgor pressure rather than by hydrolysis of lytic enzymes (Mol et al. 1990). Recently, we purified an expansin-like protein from snail stomach juice that could reconstitute heat-inactivated stipe wall extensions of Flammulina velutipes (Fang et al. 2014) and Coprinopsis cinerea (Zhang et al. 2014) stipes without hydrolytic activity. Our results do not support the previous hypotheses that hydrolysis of wall polymers by enzymes (Kamada et al. 1991) or the disruption of hydrogen bonding in wall polymers by the stress of turgor pressure (Mol et al. 1990) directly correspond to stipe wall extension. Instead, we propose that similar to higher plant wall extension (Cosgrove 2000, 2005; Wang et al. 2013), stipe wall extension may be mediated by some endogenous fungal expansin-like proteins that facilitate the separation of wall microfibrils without the passive reorientation of microfibrils by hydrogen bond disruption between parallel microfibrils. Furthermore, different regions of F. velutipes and C. cinerea stipes have different susceptibilities to the snail expansin-like protein, which may reflect differences in wall structure and chemical composition in the different stipe regions, as shown in yeasts (Cabib & Arroyo 2013) and plants (Zhao et al. 2008). Therefore, it is necessary to further clarify the architecture of stipe cell walls to understand how wall structure relates to stipe elongation. In this study, we developed methods to examine stipe cell wall architecture using field emission scanning electron microscopy (FESEM) on essentially intact stipe tissues, and compared the architectural changes of cell walls along the length of the elongating stipes from the apex to the base.
Materials and methods Strain and culture Coprinus cinereus ATCC 56838 was purchased from ATCC. Mycelium grown on the PDYA medium agar (300 g of diced potatoes, 20 g of glucose, 5 g of yeast extract, 15 g of agar and 1 L of distilled water) were inoculated on a fresh PDYA medium agar in 9-cm Petri dishes and incubated at 28 C under the conditions described by Kamada et al. (1982) until appropriate lengths of fruiting bodies were formed.
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Measurement of elongation growth of the developing stipe Approximately one-third of a cap was vertically removed from an approximately 60-mm length fruiting body of Coprinopsis cinerea grown on the PDYA medium agar in a Petri dish to expose the apical part of the stipe enclosed by the cap. The entire stipe was marked into six divisions from the apex to the basal swollen plectenchyma at 10-mm intervals with a marker pen. The marked fruiting bodies were grown continuously on the agar medium, and the change in length of each stipe division was measured at 1- and 2-h intervals (Cox & Niederpruem 1975).
Measurement of stipe cell wall extension Approximately 1e10 mm apical, 21e30 mm median, 41e50 basal (i.e., above the swollen plectenchyma), and 51e60 mm swollen plectenchyma region segments were excised with a razor blade from the 60-mm elongating fruiting body stipes. The fresh Coprinopsis cinerea stipe segments were abraded with carborundum, frozen at 20 C for approximately 24 h or more, and used within one week for the measurement of wall extension activity with an extensometer, as described by Zhang et al. (2014).
FESEM Excised segments identical to those described above were further bisected longitudinally into two approximately equal halves. The specimens were prepared for FESEM by modification using the method of Marga et al. (2005). Briefly, after slicing off the thin inner surface longitudinally from each half segment, the segments were washed three times with distilled water, fixed in 4 % paraformaldehyde in 0.2 M Na2HPO4eNaH2PO4 buffer (PBS, pH 7.4) for 4 h at 4 C, and rinsed in PBS. The specimens were then dehydrated in a graded ethanol series (30 %, 50 %, 70 %, and 95 % for approximately 45 min per step) followed by 100 % ethanol overnight, and critical point dried using CO2. For alkali treatment, stipe segments were incubated in 1 ml of 1 M KOH for 60 min, rotated at 600 rpm at 60 C (changing for fresh 1 M KOH at 15 min intervals), and washed five times with distilled water and once with PBS. The segments were then fixed in 4 % paraformaldehyde, dehydrated and dried as described as above. For hydrolase treatments, native stipe segments or alkali-treated stipe segments were incubated in 7.8 102 U/ml partially purified chitinase from Streptomyces griseus (Sigma) in 50 mM NaAc (pH 6.0) for 24 h at 37 C, 23.3 U/ml partially purified combined exo- and endo-b-1,3glucanases from Coprinopsis cinerea (purified in our lab) in 50 mM NaAc (pH 6.0) for 24 h at 37 C, or 3.2 U/ml partially purified pronase (Sigma) in 50 mM TriseHCl (pH 7.5) for 24 h at 37 C at 600 rpm in a rotator. The incubated segments were then washed three times with distilled water and further fixed, dehydrated, and dried as described above. Chitinase was partially purified from commercial chitinase from S. griseus (Sigma) by TSKgel G2000SW (13 m, 21.5 600 mm, Tosoh) size exclusion HPLC, with a mobile phase of 0.1 M NaCl-50 mM NaAc (pH 6.0) (Sietsma & Wessels 1979) containing 278 103 units mg1 protein chitinase activity but no b-1,3-glucanase activity (on laminarin) or proteinase activity (on casein). Pronase was partially
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purified from commercial pronase from S. griseus (Sigma) by CM300 (7 m, 250 4.6 mm, Eprogen) cation exchange HPLC, with a 0e0.3 M NaCl linear gradient in 50 mM NaAc (pH5.0) mobile phase (Jurasek et al. 1971) containing 20.1 units mg1 protein proteinase activity (on casein) but no hydrolytic activity on laminarin or chitin. Combined exo- and endo-b-1,3-glucanases were partially purified from C. cinerea pilei (published elsewhere) containing 93.9 units mg1 protein b-1,3-glucanase activity (on laminarin) but no hydrolytic activity on chitin or casein. One enzyme unit is defined as the release of 1 mmol of glucose or equivalent, or tyrosine per min at the indicated measurement conditions from laminarin, chitin, or casein. All specimens were mounted on stubs with double-sided sticky carbon tape with the cut surface facing upward and coated with gold on an SCD 500 sputter coater (BAL-TEC) at 37 mA for 45 s. The cell wall architecture was examined in a Hitachi S4800 cold-cathode field emission scanning electron microscope (Hitachi, Japan) fitted with a solid state backscatter upper electron detector at 5 kV with a working distance between 6 and 8 mm.
X. Niu et al.
plectenchyma regions were 1 %.h1, 0.5 %.h1, 0.1 %.h1, and 0.1 %.h1, respectively (Fig 1B). The remaining minute acidinduced wall extension activity of basal segments was due to the non-protein mediated short-term wall extension, which is not involved in stipe elongation growth (Zhang et al. 2014).
Architecture of native cell walls in different stipe regions
The stipe segments of Coprinopsis cinerea were trimmed to 1e2 mm2 and placed in 0.2 ml 2.5 % glutaraldehyde in 0.1 M phosphate buffer, with a pH of 7.2. After 4 h at 4 C, the specimens were washed thoroughly with 0.1 M PBS and post-fixed for 2 h in 1 % osmium tetroxide in 0.1 M phosphate buffer, with a pH of 7.2, at 4 C. Then, the specimens were washed and dehydrated with a graded acetone-water series and embedded in Spurr epoxy resin. Sections with approximately 70 nm thickness were cut with a diamond knife on a Reichert Ultracut E. ultramicrotome (Laica) and double staining with Uranyl acetate and lead citrate. The specimens were mounted on copper grids and observed with a Hitachi 7650 TEM at 80 kV.
For observation of architecture of cell walls in different stipe regions, the 1e10 mm apical segment, the 21e30 mm median segment, the 41e50 mm basal segment above the swollen plectenchyma, and the 51e60 mm swollen plectenchyma segment from 60-mm length elongating fruiting bodies were bisected longitudinally into two halves, so that some of cells at inside of stipe segments were cut to be opened to expose their inner surface of the cell walls, as indicated by the low magnification images in FESEM (Supplement Fig S1). Because buffer washing and the series of ethanol dehydration completely released the cytoplasm contents and plasma membrane from the opened cells during specimen preparation (Marga et al. 2005), the inner wall surface of cells in the fast elongating apical segments showed a transversely arranged microfibril architecture embedded in the matrix under high FESEM magnification (Fig 2A1). In the non-elongating basal and swollen plectenchyma segments, however, the inner wall surfaces of the opened cells were overlain by massive amounts of granular protrusions, so the microfibril architecture was not observed there (Fig 2C1 and D1). The inner wall surface of cells in the slow elongating median segments exhibited a dim transversely arranged microfibril architecture embedded in the matrix, which seemed to be overlain by some faint amorphous materials with a few granular protrusions (Fig 2B1). In contrast, all of the outer wall surfaces of cells in the various stipe regions of Coprinopsis cinerea fruiting bodies were overlain by a mass of granular protrusions (Fig 2A2eD2).
Results
Architecture of alkali-treated cell walls in different stipe regions
Transmission electron microscopy (TEM)
Elongation growth and cell wall extension activity in different stipe regions In a previous experiment, we measured the profile of elongation growth of an approximately 27-mm length young Coprinopsis cinerea fruiting body and the relative profile of cell wall extension activity in different stipe regions (Zhang et al. 2014). To obtain a more distinct contrast of the differences in wall architecture between different stipe regions, appropriately 60-mm lengths C. cinerea fruiting bodies were selected for FESEM observations (Fig 1A). So it is necessary to determine the elongation growth features and cell wall extension activity of different stipe regions of the 60-mm lengths C. cinerea fruiting bodies. As shown in Fig 1A, after 2 h of marking, the 1e10 mm apical region and the 21e30 mm median region far from the apex elongated by approximately 67.3 % and 25.2 %, respectively, whereas the 41e50 mm basal region above the swollen plectenchyma and the 51e60 mm swollen plectenchyma region (Kues 2000) did not significantly elongate (t-test, P 0.05). The acid-induced cell wall extension activities of the apical, median, basal, and swollen
To further study the architecture of stipe walls, stipe segments were treated with 1 M KOH. As shown in Fig 3A2, B2, C1e2, D1e2, the KOH treatment removed granular protrusions from all of the existing wall surfaces of the stipes to explore different features of cell walls in different stipe regions. The KOH-treated outer wall surface of cells in the elongating apical and median stipe regions showed clear transversely arranged microfibril architecture embedded in the matrix (Fig 3A2 and B2), whereas the KOH-treated inner and outer wall surfaces of cells in the non-elongating basal and swollen plectenchyma stipe regions exhibited an amorphous material architecture rather than microfibril architecture (Fig 3C1e2 and D1e2). Furthermore, the KOH treatment made the dim microfibril architecture clear on the inner wall surfaces of the cells in the slowly elongating median segments (Fig 3B1).
Architecture of cell walls treated with hydrolytic enzymes in different stipe regions To further elucidate the architecture of the stipe walls, stipes were treated with some hydrolytic enzymes to observe their
Architecture of Stipe Wall
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Fig 1 e The distribution of elongation growth (A) and wall extension activity (B) along the 60-mm length developing C. cinerea stipe. To determine the elongation growth of the developing stipe, approximately one-third of the cap was vertically removed from the 60-mm fruiting body grown on the agar medium in a Petri dish to expose the part of the stipe enclosed by the cap. The entire stipe was marked into six different divisions: 1e10, 11e20, 21e30, 31e40, 41e50, and 51e60 mm from the apex to the base at 10-mm intervals with a marker pen. The length change of each division was measured at 1-h intervals. The elongation photography and elongation growth summary of the stipes (n [ 9) are shown in A1 and A2, respectively. Data are means ± SD of nine replicates. ‘a’ indicates a significant difference between the length change of the fruiting body at 0 h and 2 h (t-test, P £ 0.001). ‘b’ indicates no significant difference between the length change of the fruiting body at 0 h and 2 h (t-test, P > 0.05). For analysis of the wall extension activity, the 1e10 mm apical, 21e30 mm median, 41e50 mm basal, or 51e60 mm basal swollen plectenchyma segments from the 60-mm long C. cinerea stipes were treated and subjected to extensometry analysis, as described in the materials and methods section. The length and extension rate changes of the stipe walls during measurements are shown in B1 and B2, respectively. The curves are the means of more than three independent experiments (n [ 9).
effects on the wall architecture. We used a partially purified b1,3-glucanase preparation (containing exo-b-1,3-glucanase and endo-b-1,3-glucanase activities but no chitinase or proteinase activity) isolated from Coprinopsis cinerea pilei to treat native stipe segments. The results show that the b-1,3glucanase treatment not only removed all granular protrusions on all existing wall surfaces of the stipes but also removed the alkali-resistant amorphous material on both of the inner and outer wall surfaces of the non-elongating stipe regions, exposing the underlying wall architecture. In addition to the transversely arranged microfibril architecture observed on the outer wall surfaces of cells in the elongating apical and median regions (Fig 4A2 and B2), microfibril architecture
under amorphous materials on both of the inner and outer wall surfaces of the cells in the non-elongating basal and swollen plectenchyma regions of the stipes were exposed (Fig 4C1e2 and D1e2). Furthermore, compared to corresponding KOH-treated stipe segments (Fig 3), b-1,3-glucanase treatment apparently removed most amorphous matrices embedding the microfibrils on both the inner and outer wall surfaces of the elongating apical stipe segments (Fig 4A and B). As a result these microfibrils appeared to have little matrix left between them, exposing a superimposed microfibril network that was clear and distinguishable on these walls surfaces. However, the b-1,3-glucanase treatment only removed partial amorphous matrices embedding the microfibrils on
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Fig 2 e FESEM images of the architecture of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60-mm long elongating stipes of C. cinerea. Stipe segments were bisected longitudinally into two halves to expose the inner stipe cells, followed by buffer washing, fixing, critical point drying, and coating with gold, allowing the wall architecture of cells to be examined. Images are oriented so that the transverse cell axis is parallel to the short axis of the page. Scale lines indicate 500 nm.
both the inner and the outer wall surfaces of the nonelongating basal and swollen plectenchyma segments, and the microfibril network was still filled with many amorphous matrices between these microfibrils (Fig 4C and D). Notably,
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Fig 3 e FESEM images of the architectures of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segment (D) of 60-mm long elongating stipes of C. cinerea that were incubated in 1 M KOH for 60 min at 60 C before fixing, critical-point drying, and coating with gold as described in Fig 2. Scale lines indicate 500 nm.
there are some differences in the wall microfibril architecture between the elongating and non-elongating stipe regions. The microfibrils were closely and transversely arranged on both the inner and outer wall surfaces of the fast elongating apical region (Fig 4A1 and A2), whereas they were often loosely and
Architecture of Stipe Wall
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transversely arranged on both the inner and outer wall surfaces of the non-elongating stipe regions (Fig 4C1e2 and E1e2) and were sometimes even sparsely and randomly arranged on the outer wall surfaces of the non-elongating stipe regions (Fig 4D2 and F2). Compared to the fast elongating 1e10 mm apical stipe segments (Fig 4A2), the microfibril network of the outer wall surface of the slowly elongating 21e30 mm median stipe segments (Fig 4 B2) contained some matrix between the microfibrils and exhibited some loosely and randomly arranged architecture sometimes, whereas the microfibril network of the inner wall surface was similar to that of the fast elongating apical stipe segment, which represented a transition type of cell wall architecture from elongating to non-elongating stipes. Fig 5 shows that the proteinase (pronase) treatment also could remove all granular protrusions on all of the existing wall surfaces of the stipes. With the removal of granular protrusions by pronase, the outer wall surface of cells in elongating apical and median stipe regions exhibited microfibril architecture (Fig 5A2 and B2), whereas both the inner and outer wall surface of cells in the non-elongating basal and swollen plectenchyma stipe regions appeared amorphous materials architecture (Fig 5C1e2 and D1e2). When the KOH-treated stipe walls were treated further with chitinase, the microfibril structures were completely eliminated at both the inner and outer wall surfaces of the elongating apical and median stipe regions, only leaving amorphous matrix architecture (Fig 6), some of which showed apparent microfibril-like indentations or cavities (Fig 6A1, A2, B1, B2) left from the digestion of microfibrils. This indicates that the microfibrils consist of chitin chains, as described previously by others (Gow & Gooday 1983; Kamada et al. 1991).
Cell wall thickness in different stipe regions The cell-wall thickness of different regions of stipes were determined from electron micrographs of ultrathin sections (Fig 7). As shown in Fig 7E, the cell wall thickness of the fast elongating apical stipe region was 134.6 15.1 nm, whereas compared to the apical stipe region, the cell-wall thickness of the slow elongating median stipe region increased by 24.6 % and reached 170.1 27.8 nm. The cell-wall thickness of the non-elongating basal and swollen plectenchyma stipe regions further increased by above 66 % and reached 224.4 33.2 nm and 237.4 31.8 nm, respectively, both which were similar to each other, showing no statistical difference.
Fig 4 e FESEM images of the architecture of the inner wall surfaces (A1eF1) and the outer wall surfaces (A2eF2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C, D), and the 51e60 mm basal swollen plectenchyma segment (E, F) of 60-mm long elongating stipes of C. cinerea that were incubated in combined exo-, endo-b-1,3-glucanases from C. cinerea in 50 mM NaAc (pH 6.0) for 24 h at 37 C before fixing, critical-point drying, and coating with gold, as described in Fig 2. Scale lines indicate 500 nm.
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Fig 5 e FESEM images of the architectures of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segment (A), the 21e30 mm median segment (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segment (D) of 60-mm long elongating stipes of C. cinerea that were incubated in pronase from Streptomyces griseus (Sigma) in 50 mM TriseHCl (pH 7.5) for 24 h at 37 C before fixing, critical point drying, and coating with gold as described in Fig 2. Scale lines indicate 500 nm.
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Fig 6 e FESEM images of the architecture of the inner wall surfaces (A1eD1) and the outer wall surfaces (A2eD2) taken from the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60 mm long elongating stipes of C. cinerea, which were first incubated in 1 M KOH for 60 min at 60 C, and then incubated in chitinase from Streptomyces griseus (Sigma) in 50 mM NaAc (pH 6.0) for 24 h at 37 C after washing with water, followed by fixing, critical point drying, and coating with gold, as described in Fig 2. Scale lines indicate 500 nm.
Discussion It is known that the classic ultrathin section method of TEM is not well suited for the observation of cell walls because polysaccharides cannot be efficiently stained by heavy metals
(Marga et al. 2005). Using the Pt/Pd shadow-cast technique, the architecture of stipe cell walls can be imaged by TEM (Mol et al. 1990; Kamada et al. 1991). However, this shadowcast approach depends on mechanical disruption and
Architecture of Stipe Wall
chemical extraction to prepare samples of wall pieces free of cytoplasm (Mol et al. 1990; Kamada et al. 1991). This macerated cell wall preparation may reflect the architecture of rearranged microfibrils to some extent (Itoh 1975; Mol et al. 1990; Kamada et al. 1991; Marga et al. 2005). Our approach uses a stipe fragment that is bisected longitudinally but kept intact, sparing a disruptive homogenization. This simple cutting and solution rinsing preparation offers a gentle and convenient way to explore the architecture of stipe cell walls. Recently, some reports show that high-resolution FESEM images the cell wall cellulose microfibrils directly in essentially intact plant tissues that were coated thickly enough to make the sample conduct electrons. Marga et al. (2005) demonstrated that in plant cell walls, the FESEM images closely resemble images of partially hydrated cell walls obtained by atomic force microscopy (AFM); the AFM images did not support the notion that fixation, dehydration, or critical-point drying during the preparation of FESEM specimens influences the images to any appreciable extent. Thus, we presume that the FESEM images in this study represent the real architecture of stipe cell walls. This study explored that the outer wall surfaces of cells in all stipe regions were overlain completely by massive amounts of granular protrusions, consistent with the outer wall surface architecture of filament fungi Aspergillus nidulans hyphae reported in Ma et al. (2005). Because these granular protrusions could be removed by KOH, pronase, and b-1,3glucanase, they may be composed of glycoproteins as in A. nidulans hyphae (Ma et al. 2005). In fact, Michalenko et al. (1976) even reported that there is a KOH-soluble outer layer on the outside of the chitin microfibril layer in basidiomycetes hyphal walls and that this alkali-soluble layer is composed primarily of b-1,3-glucan with b-1,6-linked branches and proteins in Coprinus cinereus (Schaefer 1977; Marchant 1978). These alkali-soluble b-1,3-glucans and proteins are usually
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non-covalently linked to chitin microfibrils and are removed by alkali treatment or b-1,3-glucanase (Wessels 1986; Latge 2007; Ruiz-Herrera 2012). Previous reports in which the granular protrusions were not observed on the outer wall surface (Mol et al. 1990; Kamada et al. 1991) might be due to mechanical disruption and alkali extraction for their preparation of stipe cell walls. Interestingly, we found that the inner wall surfaces of cells in the elongating apical and median segments showed little or a little granular protrusions, whereas in the nonelongating basal and swollen plectenchyma segments, the inner wall surfaces were also overlain completely by massive amounts of granular protrusions like the outer wall surfaces. Because the regions where the inner wall surface were overlain by the protrusions lost their capacities for elongated growth and wall extension, these protrusions appeared on the inner wall surface of cells in the non-elongating regions may be related to wall maturation. A significant discovery in this study is that, except for surface protrusions, amorphous materials overlying both the inner and outer wall surfaces of cells appear different distributions and distinctive features in different stipe regions. Removal of surface protrusions with alkali treatment, the layer of amorphous material overlying both the inner and outer wall surfaces of cells was explored in the nonelongating basal and basal swollen plectenchyma segments of stipes, whereas little amorphous material was overlain on both the inner and outer wall surfaces of the elongating apical and median regions, i.e., the faint amorphous material overlying on the inner wall surface of cells in the median stipe region could be removed by alkali treatment. The alkali-resistant overlying amorphous materials on both of the inner and outer wall surfaces of the non-elongating basal and basal swollen plectenchyma regions could be removed by b-1,3-glucanase treatment. Therefore, these amorphous materials must consist of b-1,3-glucan covalently linked to chitin chains
Fig 7 e Electron micrographs of cross sections of the 1e10 mm apical segments (A), the 21e30 mm median segments (B), the 41e50 mm basal segment above the basal swollen plectenchyma (C), and the 51e60 mm basal swollen plectenchyma segments (D) of 60 mm long elongating stipes of C. cinerea (scale lines indicate 1 mm). Comparison of the thickness of cell walls of different stipe segments in AeD (E). Same letters on the top of the bars indicate no significant difference between the thickness of cell walls of relative stipe segments, and different letters on the top of the bars indicate significant differences between the thicknesses of cell walls of relative stipe segments. Each thickness value is the mean wall thickness of 30 cells from the indicated stipe region.
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2007; Ruiz-Herrera 2012). Due to the lim(Wessels 1986; Latge itation of the previous macerated cell wall preparation, earlier reports did not dissolve the distribution difference of overlying amorphous materials on the wall surfaces in different stipe regions. We propose that with wall maturation, the overlying amorphous materials are gradually deposited on the wall surfaces and further modified, such as covalent linking r et al. 1997; of b-1,3-glucans to chitin microfibrils (Kolla Kopecek & Raclavsky 1999; Bowman & Free 2006), becoming alkali-insoluble. The remodeling of these overlying amorphous materials on the wall surfaces during the wall maturation may alter the plasticity of the stipe cell wall because the regions overlain by the amorphous material lose their capacities for elongation growth and wall extension. In fungal cell walls, there are masses of amorphous wall matrices that filled the space between chitin microfibrils acting as a cementing substance. These amorphous wall matrices are considered to be composed of polysaccharides and glycoproteins (Farkas 1979). This study shows that the wall matrix embedding microfibrils on both the inner and outer wall surfaces of cells in the elongating regions can be removed by b-1,3-glucanase treatment, rather than alkali treatment or pronase, to expose a distinctive microfibril network with little matrix between microfibrils, implying that these matrix components mainly consist of b-1,3-glucan covalently linked to chitin microfibrils. However, the b-1,3-glucanase-treated non-elongating regions of stipes showed some matrices remained between the microfibrils on both of the inner and outer wall surfaces, especially the outer wall surface. These results imply that the decrease of alkali-insoluble b-1,3glucans with wall maturation may be due to cross-linking between matrix components and microfibrils that are resistant to the b-1,3-glucanase treatment, or, alternatively, some other new components instead of b-1,3-glucans are added into the matrix between the microfibrils. Along with the removal of overlying protrusions and amorphous materials, as well as partial wall matrix, this study explores the features of chitin microfibril architecture on cell walls in the different regions of developing C. cinerea stipes: (1) In the fast elongating apical stipe region, the microfibrils were closely and transversely arranged on both the inner and outer wall surfaces; (2) In the non-elongating basal and basal swollen plectenchyma stipe regions, the microfibrils were loosely and transversely arranged on the inner wall surface, and became sparser and more loosely transversely arranged or sometimes even randomly arranged on the outer wall surface; (3) In the slow elongating median stipe region, the outer wall surface showed some loosely arranged microfibril architecture, though the inner wall surface still showed a closely transversely oriented microfibril architecture, exhibiting a transition type of cell wall architecture from elongating to non-elongating regions. Kamada et al. (1991) even reported that shadow-cast electron micrographs of alkali-extracted cell wall preparations of Coprinus cinereus stipes showed transversely arranged microfibrils on the inner surface, whereas the microfibrils on the outer surface were often randomly arranged. They attributed the observed randomly orientated microfibrils to disordering during wall preparation. Our experiments suggest that the randomly oriented microfibril structure previously reported by their lab for the
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Fig 8 e Schematic representation of a compositional, architectural and thick gradient of cell walls from the elongation apical region to the non-elongating basal swollen plectenchyma regions of a developing Coprinopsis cinerea fruiting body stipe (see text for explanation). , Granular protrusions, which are removed by KOH, pronase and b-1,3glucanase. , Chitin microfibrils, which are closely and transversely arranged at the wall of the elongating region, whereas loosely and transversely, sometimes even sparsely and randomly arranged at the wall of the non-elongating regions. , Wall matrix which is removed by b-1,3glucanase. , Wall matrix which is difficult to be removed by b-1,3-glucanase. , A thin layer of amorphous materials on the wall surface which is removed by KOH. , A thick layer of amorphous materials on the wall surface which is removed by b-1,3-glucanase but not by KOH. , Plasma membrane.
outer surfaces of stipe walls may reflect wall architecture of the outer wall surface in the full elongated (ceasing-elongating) stipes because their chemically extracting wall pieces were prepared from stipes at the end of elongation (21 h stage) (Kamada et al. 1991). We propose that at the fast
Architecture of Stipe Wall
elongating stipe region, newly synthesized chitin microfibrils are continually deposited on the inner wall surface and inserted between the existing microfibrils in a transverse orientation, and moved to the outer wall surface to increase the cell surface area so that there is no apparent difference of microfibril architecture between the inner and outer wall surfaces. With stipe elongation and wall maturation, though the old wall microfibrils are still progressively stretched, and the synthesis of new chitin microfibrils is reduced and finally terminated, b-1,3-glucan are largely synthesized and filled into the matrix spaces between chitin microfibrils by covalently linking to chitin chains at the inner wall surface of the non-elongating stipe segments. Thus, chitin microfibrils at the older outer wall layers far from the inner wall surface are further separated from each other into a more loose or random orientation. Simultaneously, the addition and further cross-linking of more matrix components occurs on the outer wall surface, so the wall matrix becomes resistant to the b-1,3-glucanase and alkali treatments, and chitin microfibrils become relatively sparse. This remodeling of matrix components protects the wall from weakening caused by fewer microfibrils while preventing the slippage between chitin microfibrils for wall elongation. In conclusion, the cell walls exhibit a compositional, architectural, and thick gradient from the elongation apical regions to the non-elongating basal swollen plectenchyma regions of the fruiting-body stipes (Fig 8). In the fast elongating apical region, chitin microfibrils are aligned transversely, and b-1,3-glucans with b-1,6 branches are linked covalently to chitin chains as molecular binders to connect two adjacent chitin microfibrils. This architecture facilitates the separation of wall microfibrils without passive reorientation so that newly synthesized chitin microfibrils are inserted in between separated microfibrils to longitudinally increase the wall surface area. Although the turgor pressure is the driving force for the fungal wall extension, the rate of elongation growth of stipes depends on the extensibility of the wall rather than the turgor pressure (Money & Ravishankar 2005; Lew 2011). With wall maturation, b-1,3-glucans with b-1,6 branches are largely synthesized instead of chitin chains to fill into the matrix spaces between chitin microfibrils and deposit as amorphous materials on both the inner and outer wall surfaces. Meanwhile, glycoprotein granular protrusions are further deposited on the inside adjacent to the plasma membrane of the amorphous materials on the inner wall surface to prohibit secretion of chitin microfibrils. Therefore, the wall thickness increases and the wall plasticity gradually reduces along the stipe from the elongating apical region to the non-elongating basal region. We propose that the surface deposition of protrusions and amorphous materials, and the change of microfibril architectures and wall matrices lead to the rigidification of cell walls and cessation of stipe cell elongation.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31170028) and the Priority Academic
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Development Institutions.
Program
of
Jiangsu
Higher
Education
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2015.07.008.
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