f u n g a l b i o l o g y 1 1 8 ( 2 0 1 4 ) 1 e1 1

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Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa Hejian FANG1, Wenming ZHANG1, Xin NIU, Zhonghua LIU, Changmei LU, Hua WEI, 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

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Article history:

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen

Received 10 April 2013

bonding between cell wall polysaccharides. However, it is unknown if this mechanism is

Received in revised form

operative in mushrooms. Herein we report that the native wall extension activity was located

6 October 2013

exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation

Accepted 7 October 2013

growth was restricted also to the 9 mm apical region of the stipes where the elongation growth

Available online 31 October 2013

of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall

Corresponding Editor:

extension activity represents elongation growth of the stipe. The low concentration of expan-

Teun Boekhout

sin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansinlike protein from snail stomach juice which reconstituted heat-inactivated stipe wall exten-


sion without hydrolytic activity. So the previous hypotheses that stipe wall extension was re-

Cell walls

sulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall

Fruit body

polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension


may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slip-

Stipe elongation

page by disrupting noncovalent bonding between glucan chains or chitin chains.

Wall extension activity

ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The development of mushroom fruit bodies involves a phase of cell multiplication and differentiation, followed by a phase of cell elongation (Gruen 1963). In the initial phase a small mycelial aggregate is formed, which subsequently develops into the early primordium that finally differentiates into the button with cap, gills, and stipe regions. In the elongation phase, the cap and gills are expanded and the stipe is elongated

rapidly with the development of the fruit body (Gruen 1963; Craig et al. 1977). Stipe elongation is mainly the result of manifold cell elongation rather than cell division (Kamada & Takemaru 1977a; Gooday 1985), and cell division occurs only in the apex meristematic region of the stipe (Kues 2000). In Agaricus bisporus elongation of the stipe occurs mainly in the apical portion and there is a gradient of decreasing elongation from the top to the base of the stipe (Craig et al. 1977). Similarly, in Flammulina velutipes, elongation growth is restricted

* Corresponding author. College of Life Science, Nanjing Normal University, 1 Wenyuan Rd, Xianlin University Park, Nanjing 210023, PR China. Tel./fax: þ86 25 85891067 (O). E-mail address: [email protected] (S. Yuan). 1 Co-first author. 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.10.003


to the first 2e3 mm apical zone of the stipe (Kern et al. 1997). Additionally, in Coprinus lagopus the most active zone of elongation is the upper-mid region of the stipe, while little or no elongation occurs at either the free apex or the base (Cox & Niederpruem 1975). In Coprinus radiates, the upper 2/3 of the stipe is responsible for 80e90 % of the total elongation of the stipe (Eilers 1974). Like all of fungal cells, the stipe cell is enwrapped by a thin wall that consists primarily of a chitin network embedded in a b-glucan matrix with a small amount of dispersive proteins and lipids (Michalenko et al. 1976; Gooday 1979; Mol et al. 1990; Kamada et al. 1991; Kamada & Tsuru 1993). The stipe wall must keep an essential strength and plasticity to withstand the large mechanical forces that arise from cell turgor pressure for maintaining of its stable shape and at the same time expand for creation of space for holding of the enlarging protoplast (Kamada et al. 1991; Bartnicki-Garcia 1999). The cell elongation in the stipe was proposed as the result of enzymatic hydrolysis of matrix polysaccharides (Kamada et al. 1985, 1991) according to the modifications of the component polysaccharides (Gooday 1977; Kamada & Takemaru 1983), the changes of the mechanical properties (Kamada & Takemaru 1977b), and the existence of wall lytic enzyme activities (glucanase and chitinase) in the stipe cell wall (Kamada et al. 1980, 1982; Sakamoto et al. 2005; Fukuda et al. 2008) during the stipe elongation period. However, Mol et al. (1990) suggested an altered model for the stipe elongation of fruit bodies which does not involve lytic enzymes. They proposed an elongation growth of the wall via creep of the polymers due to continuous breakage and reformation of hydrogen bonds among the glucan chains by turgor pressure-mediated stress, and passive orientation of the chitin chains. Indeed, several studies support the proposition that lytic enzymes are not central to the mechanism of stipe elongation. Kamada et al. (1985) have reported that a glucanase activity remained almost constant during stipe elongation and still remained high near the end of stipe elongation. Moreover, Gooday et al. (1992) have demonstrated that filamentous fungi produce chitinases at all stages of active growth. Relatedly, the chitinase inhibitor allosamidin inhibited spore germination of Macrophthalmus rouxii and daughter cell separation in budding yeast cells of Candida albicans, but it did not affect apical extension or branching of hyphae. Indeed, Lim & Choi (2010) have reported that expression of a chitinase from mushroom Coprinus congregatus actually inhibited yeast cell growth. Similar to fungal cell, plant cells possess cell wall, while plant cell walls mainly consist of cellulose microfibrils embedded in a matrix of hemicelluloses (e.g. b-glucan, xyloglucan, arabinoxylan, etc.) and pectins, which form a cohesive network through noncovalent and covalent linking (Cosgrove 2005). Plant cell walls need to be expanded for cell growth. Until the early 1990s, this expansion was thought to be primarily facilitated by hydrolysis of matrix polysaccharides (Masuda 1978), but the discovery of expansins has uncovered a nonhydrolytic mechanism of plant cell wall expansion (McQueen-Mason et al. 1992; Cosgrove 2000). McQueen-Mason et al. (1992), by a reconstituted wall extension assay in an extensometer, isolated and purified two proteins, from growing cucumber hypocotyl walls extract, that induced the cell wall extension of heat-inactivated cucumber hypocotyls; thus the proteins were termed expansins.

H. Fang et al.

McQueen-Mason et al. subsequently demonstrated that expansins did not have polysaccharides lytic activity (McQueenMason et al. 1992, 1993), and that they induced plant cell wall extension by disrupting noncovalent bonding of wall polysaccharides (McQueen-Mason & Cosgrove 1994, 1995). The fruit body of the mushroom F. velutipes has a thin and long stipe. The diameter of the stipe in young fruit bodies is approximately 2 mm which is similar to that of cucumber hypocotyls and of wheat coleoptiles, both of which have been used to measure cell wall extension via extensometers. Therefore, extensometer may be applicable to measure cell wall extension of isolated stipes, thereby facilitating the study of the mechanism of stipe elongation. In this study, we detected an acid-induced wall extension of F. velutipes stipe by extensometer. Further, we purified and functionally characterized a protein, from the stomach juice of the snail Helix aspersa, which induced hydrolysis-free cell wall extension of heatinactivated F. velutipes stipes under the acidic conditions. Our results suggest that similar to plant cell wall extension, cell wall extension of mushroom fruit body stipes is mediated by an expansin-like protein that functions via the disruption of hydrogen bonds between glucan and/or chitin chains.

Materials and methods Strain and culture Culture of Flammulina velutipes (Curtis) Karst was purchased from Jiangsu Academy of Agricultural Sciences, China. The culture compost, consisting of 78 % cotton seed hull, 20 % wheat bran, 1 % sugar, and 1 % CaCO3 with approximately 65 % moisture content, was put in polypropylene bags (16 cm  33 cm) and autoclaved. The mycelia were inoculated on the culture compost in the bags and cultured for 4 weeks at 23e25  C in the dark. Fruit body buttons were induced at 13  C under scattered light. Then the compost bag was opened on the top and the open top was covered with another bag for appropriate air. Fruit bodies were grown for indicated length at 13  C. Cucumber seeds (Cucumis sativus cv. Burpee Pickler) and wheat seeds (Triticum aestivum L. Yangmai 87158), purchased from Jiangsu Academy of Agricultural Sciences, China, were sown on ten layers of gauze soaked with water in flat containers, covered with lids, and grown for 3e4 d at 30  C under darkness. Snail Helix aspersa was purchased from Shijiazhuang Yakun Brown Garden Snail Development Ltd., Co., China.

Fruit body stipe elongation measurement A marker pen was used to mark 1 mm interval regions along 30 mm length of stipes of fruit bodies grown on culture compost in polypropylene bags. The change in length of each region was measured daily during cultivation (Cox & Niederpruem 1975).

Wall extension measurement by extensometer Approximately 10 mm fresh segments of different regions of Flammulina velutipes stipes, apical cucumber hypocotyls, or

Stipe wall extension of F. velutipes

apical wheat coleoptiles, were quickly excised and frozen at 80  C for about 24 h or more, and were used within 1 week. Frozen segments were then thawed at room temperature, abraded with carborundum, and pressed between two glass slides. Where indicated, the segments were submerged in boiling water and heated for 120 s in a microwave oven to inactivate native enzyme activity. Treated samples were clamped under an applied force of 20g for plant cell walls or 12g for fruit body stipe walls. Specimen length between clamps was 5 mm. Tissues between clamps were incubated in 0.1 mL bathing solution and extension was recorded using a linear voltage displacement transducer in the extensometer (McQueen-Mason et al. 1992). For measurement of native wall extension, native segments of tissues were first incubated in 50 mM Hepes, pH 6.8, for 30 min, after which the bathing solution was replaced by 50 mM sodium acetate, pH 4.5, for another 120 min of incubation. For measurement of reconstituted, heat-inactivated wall extension, heated segments of tissues were suspended first in 50 mM sodium acetate, pH 4.5, for 30 min, after which the bathing solution was refreshed by the same bathing buffer with or without expansin or expansin-like protein for another 120 min of incubation. For native wall extension, the maximum extension rate is defined as the change in length of tissue per minute (McQueen-Mason et al. 1992), which is calculated by subtracting the wall extension rate in neutral buffer from the wall extension rate after 1 min of shifting to acidic buffer. For reconstituted, heat-inactivated wall extension, the maximum extension rate is calculated from the arithmetic difference in stable wall extension rate before and after addition of expansins or snail expansin-like protein (Zhao et al. 2008). All assays reported in this study were done in triplicate in each of three independent experiments.

Isolation, purification, and characterization of an expansinlike protein from snail stomach juice After snail Helix aspersa were starved for 40 h, stomach juice was collected (Wang et al. 2003) and diluted five-fold with buffer A (50 mM sodium acetate, pH 4.5) containing 5 mM Ethylene Diamine Tetraacetic Acid (EDTA) at 4  C. The mixture was centrifuged at 10 000g for 10 min at 4  C. The supernatant was precipitated with ammonium sulphate and the wall extension activity precipitated between 40 and 50 % saturation. The precipitate was recovered by centrifugation at 10 000g for 10 min and subsequently dissolved in buffer A. After centrifugation, the supernatant was desalted on a column of Bio-Gel P-6 (Bio-Rad) preequilibrated with buffer A. The desalted solution was loaded onto a CM-Sepharose Fast Flow column (Amersham, 1  18 cm, 4  C) that was preequilibrated with buffer A. Protein fractions were eluted from the column at a flow rate of 0.5 mL per minute with a linear gradient of 0e0.5 M NaCl in buffer A. The active fractions were pooled, freeze-dried, and loaded onto a Bio-Gel P-60 column (Bio-Rad, 1.5  120 cm) that was preequilibrated with buffer A. Proteins were eluted from the column at a flow rate of 0.1 mL per minute with buffer A. Fractions were tested for wall extension activity and the fractions with the activity were pooled. The protein purity was analyzed by Sodium Dodecyl Sulfate-Polyacrylamid Gel Electrophoresis (SDS-PAGE) in


11 % acrylamide gels (Laemmli 1970). Protein concentration was determined by Coomassie Brilliant Blue staining (Bradford 1976) using bovine serum albumin as a standard. For sequencing of purified protein we used Edman degradation or mass spectrometry. For the former, the protein in the SDS-PAGE gel was electroblotted onto a polyvinylidene difluoride membrane, and sequenced at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China. For mass spectrometry, the protein was digested with trypsin and subjected to HPLC for peptide separation, followed by sequencing via liquid chromatography-electron spectroscopic imagingetandem mass spectroscopy at National Center of Biomedical Analysis, Academy of Military Medical Sciences, Beijing, China.

Extraction of plant expansin Crude a-expansin was extracted from cucumber hypocotyls as described by McQueen-Mason et al. (1992), and crude a-expansin was extracted from maize (Zea mays) pollen as described by Cosgrove et al. (1997).

Analysis of hydrolytic activity of snail expansin-like protein on various wall components b-Glucan from yeast and pachyman from Poria cocos were purchased from Megazyme (USA). Glucan and mannan from baker’s yeast (Saccharomyces cerevisiae), and chitin and chitosan from crab shells were purchased from Sigma (USA). Stipe wall preparation was made from Flammulina velutipes stipes. Briefly, 10 g of the upper 3 mm segments of stipes were homogenized with 200 mL of 20 mM sodium acetate, pH 4.5, in a Waring blender. The wall fragments were collected by filtration through Micracloth, washed twice with diH2O, treated with 20 mM Hepes buffer, pH 6.8, containing 1 M NaCl and 2 mM EDTA, washed again with diH2O, and then freeze-dried. To assay the hydrolytic activity of snail expansin-like protein on wall polysaccharides, 0.1 mL of the reaction mixture containing 50 mM sodium acetate buffer, pH 4.5, 1 % one of aforementioned polysaccharides, and 15 mg mL1 purified protein fraction was incubated at 37  C for 2 h. Subsequently, the reaction mixture was mixed with 0.5 mL of 100 mM borate buffer (pH 9.0) and 0.1 mL 1 % 2-cyanoacetamide, heated at 100  C for 10 min, cooled to room temperature, and finally colourimetrically assayed at 276 nm to measure concentration of reduced sugars released from polysaccharides (Honda et al. 1982; Zhao et al. 2008). One enzyme unit is defined as the amount of protein required to release 1 mmol of reduced sugar per minute. All assays reported in this study were done in duplicate in each of four independent experiments.

Results The characteristics of Flammulina velutipes stipe elongation When 10 mm apical segments of isolated 30 mm F. velutipes stipes were preincubated in neutral buffer (50 mM Hepes, pH


H. Fang et al.

6.8) in the cuvette of extensometer for 30 min, and then changed to acidic buffer (50 mM sodium acetate, pH 4.5), rapid wall extension was induced immediately (Fig 1). However, this native wall extension decayed to a basal level after approximately 15 min of exposure to acid pH buffer. For a comparison, native wall extension of cucumber hypocotyls also was measured which showed an extension profile similar to that of F. velutipes, although both the peak extension rate and the final basal rate of the former were greater than that of the latter. When apical stipe segments were preheated with boiling water for 120 s, wall extension was not induced by the acid buffer, implying that native wall extension activity was inactivated. Moreover, the maximum wall extension rate of apical, median, and basal regions of stipe is 3.0 mm min1, 0.05 mm min1, and 0.015 mm min1, respectively. Therefore the median and basal parts essentially don’t extend. As to a smaller sharp extension peak in wall extension profile of the median and basal parts, it only transiently peaked in about 30 s and then dropped down to zero, therefore it is not stable, which attributed to a transient vibration caused by replacing bath solution rather than stable wall extension, apparently different from extension pattern of apical part. Fig 2 shows the pH dependence of native wall extension of F. velutipes stipes. pH 3.5e5.5 was the optimal range for native wall extension of apical stipe segments. Native wall extension activity gradually decreased as pH increased and this activity was completely lost at neutral or basic pH conditions. The distribution of elongation growth along the length of the developing stipes was determined by measurement of stipe length changes during fruit body development. The region of the stipe from below the cap to the base of 30 mm fruit bodies, grown on culture compost in polypropylene bags, was demarcated into 1 mm intervals by marker pen. The change in length of each millimetre region was measured daily for 7 d. This demarcation process did not affect stipe growth. As shown in Fig 3, the apical-most 1 mm region of the stipes elongated throughout the duration of the experiment and peaked at 98.6 mm after 7 d of measurement; the 2nd and 3rd millimetre regions elongated until the 4th day peaking at 11.7 mm and 7.8 mm, respectively. The 4the8th millimetre regions elongated to a peak length of 4.8 mm, 3.9 mm, 2.6 mm, 2.0 mm, and 1.7 mm, respectively, after 3 d of stipe growth; there was nominal growth of the 9th millimetre region until the 2nd day, after which no further elongation of this segment was observed. We observed no change in elongation of any demarcated segments below the apical 10 mm region throughout the 7 d of measurement.

inactivated cucumber hypocotyls, and that expansin-like proteins were detected in these extracts when subjected to Western blot analysis. Further, previous studies have indicated that expansin synergistically enhances the activity of polysaccharide hydrolytic enzymes (Cosgrove et al. 1998; Zhao et al. 2008; Wei et al. 2010), and that such enzymes and expansin are often coexpressed in plant, fungal, and animal tissues (Cosgrove & Durachko 1994; Saloheimo et al. 2002; Qin et al. 2004; Balestrini et al. 2005). Considering that gastric juice is usually used as an enzyme preparation to hydrolyze fungal cell walls for the preparation of protoplasts (Anderson & Milibank 1966), we hypothesized that proteins that function to loosen stipe cell walls may be present in snail gastric juice. Thus, we tested snail H. aspersa stomach juice for its potential to modulate stipe wall extension and found that appropriately diluted stomach juice (0.5 mg mL1 protein) induced cell wall extension in heatinactivated stipes at an initial rate of 4.3 mm min1. This stomach juice reconstituted heat-inactivated cell wall extension lasted about 100 min and then the stipe broken which was due to degradation of wall network by hydrolytic enzymes in the stomach juice (Fig 4). To identify the active protein in snail stomach juice that is responsible for stipe wall extension, stomach juice was precipitated with 40e50 % saturation of ammonium sulphate. The precipitate was dissolved in buffer, desalted, and fractionated on a CM-Sepharose Fast Flow column. Among the five protein fractions eluted from the column, fraction II showed stipe wall extension activity (Fig 5A). Fraction II was further purified on a Bio-Gel P-60 column to yield five fractions among which fraction 5 showed stipe wall extension activity (Fig. 5B) (Table 1). Subjecting this fraction to SDS-PAGE analysis revealed a single protein band (Fig 5C). This protein functioned in F. velutipes stipe walls in a manner similar to plant expansin, and therefore we termed the purified protein as expansin-like protein. Sequence analysis of the amino terminus of expansin-like protein by Edman degradation showed the following 18 amino acids sequence: ACGLDGRNDLINCPHSRA. By sequencing trypsin-digested fragments of expansin-like protein via liquid chromatography-electron spectroscopic imagingetandem mass spectroscopy (LCESIeMS/MS), we obtained the following peptide fragments’ sequences: CVTNVSTYGWVASCCRG, GSSYGTCRC, and RGSTTWCCR. We then conducted Basic Local Alignment Search Tool (BLAST) searches for these peptide fragment sequences but did not find any homologues in GeneBank.

An expansin-like protein from stomach juice of snail Helix aspersa induces cell wall extension of Flammulina velutipes stipes

Characteristics of induced stipe wall extension by snail expansin-like protein

Since plant cell wall extension is mediated by expansin proteins, we reasoned that a similar protein may exist in F. velutipes. However we failed to isolate and purify such a protein from F. velutipes, possibly because of much low content of this putative protein in the much limited region of stipes (see Discussion). Interestingly, Cosgrove & Durachko (1994) have reported that crude protein preparation from the digestive tracts of snails induced cell wall extension of heat-

We next determined the effect of purified snail expansin-like protein on stipe wall extension. As shown in Fig 6B1, 20 mg mL1 of snail expansin-like protein significantly and immediately induced wall extension of heat-inactivated Flammulina velutipes stipe to a maximum rate of 6.6 mm min1, after which there was gradual rate decay down to basal levels over a period of 2 h, showing a sharp extension profile. This expansin-like protein-mediated wall extension was speciesspecific as it did not induce plant cell wall expansion of dicot

Stipe wall extension of F. velutipes


Fig 1 e Length change (A) or extension rate (B) of isolated stipe wall specimens under constant load during measurement. Ten-millimetre fragments of F. velutipes stipes were frozen, thawed, abraded, pressed, and then clamped to an extensometer with a loading force of 12g. The 5 mm region of the stipe fragments between the clamps was suspended in 50 mM Hepes, pH 6.8, in a cuvette in the extensometer for 30 min, after which the bathing solution was replaced by 50 mM sodium acetate, pH 4.5 (arrow). Stipe fragment extension was recorded using a linear voltage displacement transducer (Cosgrove 1989). The 10 mm apical fragments of F. velutipes stipes were heated in boiling water for 120 s to serve as a negative control. Tenmillimetre apical fragments of cucumber hypocotyls were used as a positive control (using a loading force of 20g). From top to bottom: 10 mm apical fragment, 10 mm apical fragment inactivated by heating for 120 s in boiling water, 10 mm median fragment and 10 mm basal fragment of 30 mm isolated F. velutipes stipes, and 10 mm apical fragment of isolated cucumber hypocotyls.

cucumber hypocotyls or of monocot wheat coleoptiles (Fig 6A1 and B1). Conversely, neither cucumber a-expansin (Fig 6A2 and B2) nor maize b-expansin (Fig 6A3 and B3) induced F. velutipes stipe wall extension, yet they induced wall extension of cucumber hypocotyls and of wheat coleoptiles, respectively. To determine if the snail expansin-like protein-mediated wall extension activity was concentration-dependent, we exposed F. velutipes stipes to various concentrations of the snail expansin-like protein. Fig 7A shows that the increase in the rate of reconstituted stipe wall extension correlated with snail expansin-like protein concentration. Although wall extension rate slowed beyond 20 mg mL1 snail expansin-like protein, the rate continued to increase until the maximum (saturation) protein concentration of 80 mg mL1. Furthermore, the pH dependence of this activity was examined in a range of pH values. Fig 7B shows that 10 mg mL1 snail expansin-like protein exhibited a slim induction of wall extension at pH  4 or pH  7, while the optimal pH for reconstituted wall extension activity was between 4.5 and 6.5. Given that native wall extension is concentrated in the apical region of F. velutipes stipes (Figs 1 and 3), it was of interest to profile the effect of snail expansin-like protein on wall extension of different stipe regions. When the protein was applied to different regions of F. velutipes stipes, it induced different extension rates (Fig 7C). Exposure of heatinactivated stipes to 10 mg mL1 snail expansin-like protein resulted in a maximum wall extension rate of 6.0 mm min1,

of 3.6 mm min1, and of 1.1 mm min1 in the apical, median, and basal regions, respectively. To investigate the lytic activity of snail expansin-like protein, the wall polysaccharides b-glucan (alkali soluble, from

Fig 2 e Maximum extension rate in isolated apical F. velutipes stipe walls as a function of pH. Stipe fragments were prepared and subsequently subjected to extensometry analysis as in Fig 1 except that the pH in the replacing bathing solution ranged from 3.5 to 7.5.


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Fig 3 e Location of elongation growth zones in F. velutipes stipes. The stipes of 30 mm F. velutipes fruit bodies grown in culture compost were marked from their apical region below the cap to the base at 1 mm intervals by marker pen. The fruit bodies were then incubated at 13  C for further growth.

yeast), pachyman (1,3-b-D-glucan, from basidiomycotina Poria cocos), glucan and mannan (from baker’s yeast Saccharomyces cerevisiae), laminarin (from brown alga Laminaria digitata), chitin and chitosan (from crab shells) were each incubated with 15 mg mL1 snail expansions-like protein for 2 h. No reduced sugar was released from these polysaccharides in the incubation solution (Table 2). Similarly, treatment by snail expansinlike protein did not release reduced sugar from F. velutipes stipe wall preparation. These results show that snail expansin-like protein does not hydrolyze stipe wall polysaccharides.

Discussion This study is the first to report native wall extension activity in fruit body stipes of Flammulina velutipes. Native wall extension activity was located in the apical 10 mm segment of 30 mm

stipes, while absent in the median and basal segments. This wall extension profile is consistent with distribution of elongation growth along the fruit body stipe. Elongation growth in F. velutipes stipes was restricted primarily to the 1e2 mm apical zone of the stipe below the cap, while elongation growth in the stipe between the 3th and 9th millimetre apical zones was very slow; there was no elongation growth in the 10 mm region. Therefore, the wall extension activity represents elongation growth activity of the stipe, which provides a new approach for the study of stipe elongation of basidiomycete fruit bodies. This study shows that the native wall extension activity of F. velutipes stipes is acid-dependent and can be heatinactivated, which are features consistent with those of plant cell wall extension. Furthermore, we purified a protein, from snail stomach juice, that had wall extension activity similar to that of the plant protein expansin. This snail expansinlike protein reconstituted the wall extension of heat-

Fig 4 e Induction of extension in heat-inactivated apical fragments of F. velutipes stipes by stomach juice preparations of snail, Helix aspersa. (A) Length traces; (B) Rate traces. Stipe fragments were heated by boiling as described in Fig 1, and incubated in 50 mM sodium acetate (pH 4.5) under 12g of loading force in an extensometer for 30 min. Subsequently, the bathing solution was replaced by snail stomach juice that was diluted 200 folds in 50 mM sodium acetate, pH 4.5.

Stipe wall extension of F. velutipes

Fig 5 e Purification of a protein with wall extension activity from stomach juice of snail, Helix aspersa. (A) Fractionation of ammonium sulphate precipitate by CM-Sepharose chromatography yielded a single peak of activity (peak II). (B) Peak II from A was fractionated by Bio-Gel P-60 chromatography, yielding a single peak of activity (peak 5). (C) Coomassie Brilliant Blue stained SDS-PAGE. SJ, stomach juice from snail; AS, ammonium sulphate precipitate; II, active peak II in (A); 5, active peak 5 in (B). Molecular size markers (in kilodaltons) are shown on left. Arrow indicates the active protein at approximately 21 kD.

inactivated F. velutipes stipes in a concentration-dependent and pH-dependent manner. We conclude that, similar to plant cell wall extension, wall extension of F. velutipes stipes may be mediated by endogenous expansin-like proteins. In comparison to plant cell wall extension, F. velutipes stipes exhibited a lower and sharper wall extension profile after transition from neutral buffer (pH 6.8) to acid buffer (pH 4.5). This stipe-specific extension profile may be resulted from


the measurement method in which the w2 mm apical region of the 10 mm length of stipe which is the fastest elongation region, along with its w2 mm basal terminal region, was clamped by the extensometer and therefore unavailable for measurement. Therefore, our extensometer measurements reflect the low wall extension activity of the subapical region between clamps, and do not accurately reflect the maximum extension activity from the apical region of F. velutipes (note: elongation of the 1st millimetre region of the stipe was almost 40-fold greater than that of the 5th millimetre region during 7 d of growth). In contrast to native wall extension activity, which was limited to the apical region, snail expansin-like protein not only induced greater wall extension in the apical region but also in the median and basal regions. This suggests that the exogenous expansin-like protein rescued the lost or inactivated endogenous expansin-like protein activity in the stipe. This also indicated that the content of endogenous expansin-like protein in the subapical region of stipe is really much low, and that loss or inactivation of endogenous expansin-like protein is one of the main causes of elongation cessation in the median and basal regions of stipe, besides biochemical modification of the cell wall (Mol & Wessels 1990; Kopeck & Raclavsky 1999) which make the wall less susceptible to expansin-like protein. This is different from wall extension features of plant cucumber hypocotyls or of wheat coleoptiles, in which nonelongating, basal fragments could not be induced to extend by exogenous expansin because their walls are rigidified by biochemical modification and not susceptible to expansin during maturation (McQueen-Mason et al. 1992; Zhao et al. 2008). It is notable that, similar to the native wall extension profile of stipe, snail expansin-like protein reconstituted stipe wall also exhibited an initially sharp extension and a rapid subsequent decay of extension during the course of measurement. However, plant expansin reconstituted plant cell wall extended almost at an approximate steady extension rate during the course of measurement though exposure of native plant walls from neutral buffer to acid buffer resulted in a sharp wall extension induction followed by decay in extension (compared Fig 1 with Fig 6). This difference may be due to endogenous hydrolytic enzyme synergism in native plant walls (Cosgrove et al. 1998; Zhao et al. 2008; Wei et al. 2010). That is, endogenous hydrolytic enzymes may synergize with endogenous expansin to quickly loosen native walls for maximum extension, after which the extension decays to basal levels. Conversely, exogenous expansin preparations lack hydrolytic enzymes (McQueen-Mason et al. 1992; Wei et al. 2010) and therefore only gradually loosen the heat-inactivated wall, resulting in a lower and steadier extension rate. In contrast to plant expansin preparation, the snail expansin-like protein preparation also lacked hydrolytic enzymes yet yielded a profile of reconstituted stipe wall extension similar to that of native wall extension. As such, we propose that the cell wall structure specific to stipes accounts for the profile of snail expansin-like protein reconstituted wall extension. The stipe cell wall is thin and its structure is simpler than that of plants, without build-up of layer upon layer of chitin fibrils and multimet growth (Kamada et al. 1991). Thus, the stipe wall may be loosened immediately and extended quickly to the maximum


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Table 1 e Summary of purification of an expansin-like protein from stomach juice of Helix aspersa. Fraction Stomach juice (NH4)2SO4 precipitation Fraction II from CM-sepharose Fraction 5 from BioGel-P60 a

Total Protein (mg)

Total active (Units)

Specific activity (units mg1 protein)a

Fold purification

Yield (%)

786 264 25.4 0.492

80052 20011 2800 1005

102 75.8 110 2051

1.03 0.742 1.13 20.1

100 25.0 3.51 1.33

One unit is defined as the amount of protein for inducing 1 mm change in cell wall length per minute.

extension rate by snail expansin-like protein, even in the absence of synergistic hydrolytic enzymes, resulting in a sharp extension profile. Our study shows that snail expansin-like protein acts specifically on F. velutipes stipe walls rather than on plant cucumber hypocotyls or on wheat coleoptiles; conversely, aexpansin from cucumber hypocotyles or b-expansin from wheat coleoptiles cannot induce stipe wall extension of F. velutipes. These results are consistent with chemical composition differences between plant cell walls and basidiomycete stipe cell walls. Plant cell walls consist of cellulose microfibrils and a matrix of hemicelluloses, pectins, and a small amount of dispersed proteins (Cosgrove 2005), whereas basidiomycete stipe walls consist of chitin microfibrils and a matrix of various b-glucans along with few dispersed proteins (Mol & Wessels 1990). Moreover, partial amino acid sequencing indicated that snail expansin-like protein and plant expansins are not homologous. This investigation showed that snail expansin-like protein did not hydrolyze chitins or b-glucans. Therefore, its function in inducing stipe wall extension is not dependent on the degradation of networks of b-glucans and/or chitins, which is antithetical to the hypothesis that hydrolytic enzymes are involved in the extension of stipe walls (Kamada et al. 1985, 1991; Sakamoto et al. 2005; Fukuda et al. 2008). Plant expansins also appear to lack polysaccharides hydrolytic activity (McQueen-Mason et al. 1992, 1993). Plant expansions result in polymer slippage under turgor pressure-mediated stress via disrupting hydrogen bonding between wall polysaccharides, such as b-glucans and celluloses, therefore their action does not lead to the degradation of polymers and subsequent overall weakening of wall structure during wall expansion (McQueen-Mason & Cosgrove 1995). It is known that in fungal cell walls, chitin microfibrils are formed from interchain hydrogen bonding (Ramakrishnan & Prasad 1972) and intermolecular hydrogen bond is present also in b-glucan chains (Mol et al. 1990), though b-glucan chains are covalently linked to chitin chains. These hydrogen bonds between glucan chains and chitin chains enable these polymers have an enormous tensile strength and significantly contribute to the overall integrity of the cell wall. We propose that, similar to plant expansins, snail expansin-like protein may induce wall extension by disrupting hydrogen bonds between the b-glucan chains or the chitin chains in stipe walls (Mol & Wessels 1990; Bowman & Free 2006). Indeed, Mol & Wessels (1990) have shown that elongating stipe walls of young fruit bodies show a higher proportion of (1-6)-b-glucan side-branches in (1-3)-b-glucan, which hampers hydrogen bond formation

among (1-3)-b-glucan chains. They have suggested that these loose hydrogen bonds between the glucan chains are not stable in growing stipe hyphae, and are subjected to disruption by turgor pressure stress. Accordingly, this instability in glucan hydrogen bonds enables the polymers in the wall of fruit body hyphae to reorientate, in a lytic enzyme-free manner, under turgor pressure, and permits expansion in the axial direction (Mol et al. 1990). However, Money & Ravishankar (2005) measured the osmotic pressure and turgor of Coprinopsis cinerea fruit bodies and have found no significant difference in the osmolality of the stipes of two strains; one strain produced normal fruit bodies with relatively short stipes, whereas a mutant produced abnormally elongated stipes. Our data further indicate that stipe wall extension is not induced exclusively by turgor pressure stress (mimicked by loading stress in extensometer), because the native wall extension activity of stipes under loading stress in the extensometer could be inactivated by boiling, and this inactivated extension activity could be rescued by snail expansin-like protein. It is known that filament fungi grows by means of hyphae that grow at their tips and that branch subapically to form a mycelium or a colony. The primary growth zone is located at the periphery of the colony, whereas the colony centre is an old zone. Secreted wall proteins at the growing periphery and the old centre of the colony of Aspergillus niger were different (Levin et al. 2007; Krijgsheld et al. 2012, 2013). Cycloheximide, a protein synthesis inhibitor, resulted in hyphal elongation cease and thicker cell walls at the periphery of colonies, however, a reduced thickness of the cell walls in the centre of the colony which is due to the net degradation of the cell wall in the colony centre (Krijgsheld et al. 2013). These results imply that the expansin-like protein may function in hyphal tip growth which is actively synthesized and turn over, whereas hydrolysis enzymes are retained mainly in nongrowing cell walls in the colony centre. In conclusion, this study offers insight into the mechanism of mushroom stipe wall extension. We suggest that some endogenous expansin-like proteins exist in cell walls of mushroom stipes. Like plant expansin, mushroom expansin-like protein may facilitate stress-mediated polymer slippage for wall extension, by disrupting noncovalent bonding between wall polymers, e.g. hydrogen bonds between glucan chains or chitins, rather than cleaving covalent bonds. Therefore, degradation of wall components, and subsequent weakening of the wall structure during stipe elongation is not involved. Although snail expansin-like protein significantly induced F. velutipes stipe cell wall extension, it is necessary to confirm the presence and activity of endogenous expansin-like

Stipe wall extension of F. velutipes


Fig 6 e Induction of extension in heat-inactivated walls from growing tissues of different species by expansin from plants or by expansin-like protein from snail. (A) Length traces; (B) Rate traces. Ten-millimetre apical fragments from F. velutipes stipes, cucumber hypocotyls, and wheat coleoptiles were prepared, heated, and clamped as described in Fig 1. The tissues were first suspended in 50 mM sodium acetate, pH 4.5, for 30 min, after which, the bathing solution was replaced with 0.1 mL of the same buffer containing 20 mg mLL1 snail expansin-like protein (A1 and B1), or 50 mg mLL1 a-expansin from cucumber hypocotyls (A2 and B2), or 1.9 mg mLL1 b-expansin from maize pollen (A3 and B3).


H. Fang et al.

Table 2 e Hydrolysis of polysaccharides by Helix aspersa expansin-like protein. Substrate Yeast b-glucan (Megazyme) Glucan from baker yeast (Sigma) Mannan from S. cerevisiae (Sigma) Chitin from crab shells (Sigma) Chitosan from crab shells (Sigma) Pachyman from Poria cocos (Megazyme) Laminarin (Sigma) Cell wall preparation from 10-mm apical stipe of F. velutipes (selfprepared)

Hydrolytic activity (units mg1 protein)a 0.11  0.09  0.10  0.06  0.04  0.44  0.25  0.16 

0.07 0.38 0.11 0.11 0.04 0.25 0.17 0.06


One unit is defined as the amount of protein for releasing 1 mmol of reduced sugar per minute.

protein. To that end, we are screening mushroom fruit bodies containing higher concentrations of endogenous expansinlike proteins so as to purify and further characterize their function and mechanism.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31170028) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.


Fig 7 e Concentration dependence (A), pH dependence (B), and region dependence of snail expansin-like protein-mediated induction of wall extension in heat-inactivated F. velutipes stipes (C). Heat-inactivated 10 mm apical stipe fragments were treated as in Fig 6 except that the bathing solution was replaced with 0.1 mL of the same buffer (pH 4.5) containing various concentrations of snail expansinlike protein for concentration dependence, or 10 mg of snail expansin-like protein with varying pH for pH dependence. Apical, median, or basal heat-inactivated stipe fragments were respectively determined for region dependence, as described in Fig 6, except with 0.1 mL of the same buffer containing 10 mg mLL1 snail expansin-like protein with pH 4.5.

Anderson FB, Milibank JW, 1966. Protoplast formation and yeast cell-wall structure-the action of the enzymes of the snail, Helix pomatia. Biochemical Journal 90: 682e687. Balestrini R, Cosgrove DJ, Bonfante P, 2005. Differential location of alpha-expansin proteins during the accommodation of root cells to an arbuscular mycorrhizal fungus. Planta 220: 889e899. Bartnicki-Garcia S, 1999. Glucans, walls, and morphogenesis: on the contributions of J. G. H. Wessels to the golden decades of fungal physiology and beyond. Fungal Genetics and Biology 27: 119e127. Bowman SM, Free SJ, 2006. The structure and synthesis of the fungal cell wall. BioEssays 28: 799e808. Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248e254. Craig GD, Gull K, Wood DA, 1977. Stipe elongation in Agaricus bisporus. Journal of General Microbiology 102: 337e347. Cosgrove DJ, 1989. Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls. Planta 177: 121e130. Cosgrove DJ, 2000. Loosening of plant cell walls by expansins. Nature 407: 321e326. Cosgrove DJ, 2005. Growth of the plant cell walls. Nature Reviews Molecular Cell Biology 6: 850e861. Cosgrove DJ, Bedinger P, Durachko DM, 1997. Group I allergens of grass pollen as cell wall-loosening agents. Proceedings of the National Academy of Sciences 94: 6559e6564. Cosgrove DJ, Durachko DM, 1994. Autolysis and extension of isolated walls from growing cucumber hypocotyls. Journal of Experimental Botany 45: 1711e1719. Cosgrove DJ, Durachko DM, Li LC, 1998. Expansins may have cryptic endoglucanase activity and can synergize the

Stipe wall extension of F. velutipes

breakdown of cellulose by fungal cellulases. In: Annual Meeting of American Society of Plant Physiologists (Abstract: 171). Cox RJ, Niederpruem DJ, 1975. Differentiation in Coprinus lagopus III. Expansion of excised fruitbodies. Archives of Microbiology 105: 257e260. Eilers FI, 1974. Growth regulation in Coprinus radiatus. Archives of Microbiology 96: 353e364. Fukuda K, Hiraga M, Asakuma S, Arai I, Sekikawa M, Urashima T, 2008. Purification and characterization of a novel exo-b-1, 3-1, 6-gluconase from the fruiting body of the edible mushroom Enoki (Flammulina velutipes). Bioscience, Biotechnology, and Biochemistry 72: 3107e3113. Gooday GW, 1977. Biosynthesis of the fungal cell wall. Mechanisms and implications, the first fleming lecture. Journal of General Microbiology 99: 1e11. Gooday GW, 1979. Chitin synthesis and differentiation in Coprinus cinereus. In: Burnett JH, Trinci APJ (eds), Fungal Walls and Hyphal Growth. Cambridge University Press, Cambridge, pp. 203e223. Gooday GW, 1985. Elongation of the stipe of Coprinus cinereus. In: Moore D, Casselton D (eds), Developmental Biology of Higher Fungi. Cambridge University Press, Cambridge, pp. 311e332. Gooday GW, Zhu W-Y, O’Donnell RW, 1992. What are the roles of chitinases in the growing fungus? FEMS Microbiology Letters 100: 387e392. Gruen HE, 1963. Endogenous growth regulation in carpophores of Agaricus bisporus. Plant Physiology 38: 652e666. Honda S, Nishimura Y, Takahashi M, Chiba H, Kakehi K, 1982. A manual method for the spectrophotometric determination of reducing carbohydrates with 2-cyanoacetamide. Analytical Biochemistry 119: 194e199. 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: 257e260. Kamada T, Takemaru T, 1977a. Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: mechanical properties of stipe cell wall. Plant and Cell Physiology 18: 831e840. Kamada T, Takemaru T, 1977b. Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: changes in polysaccharide composition of stipe cell wall during elongation. Plant and Cell Physiology 18: 1291e1300. Kamada T, Fujii T, Takemaru T, 1980. Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: changes in activity of cell wall lytic enzymes. Transactions of the Mycological Society of Japan 21: 359e367. 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, 1983. Modifications of cell-wall polysaccharides during stipe elongation in the basidiomycete Coprinus cinereus. Journal of General Microbiology 129: 703e709. Kamada T, Takemaru T, Prosser JI, Gooday GW, 1991. Right and left handed helicity of chitin microfibrils in stipe cells in Coprinus cinereus. Protoplasma 165: 64e70. Kamada T, Tsuru M, 1993. The onset of the helical arrangement of chitin microfibrils fruit-body development of Coprinus cinereus. Mycological Research 97: 884e888. Kern VD, Mendgen K, Hock B, 1997. Flammulina as a model system for fungal graviresponses. Planta 203: S23eS32. Kopeck 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 44: 397e400. € ller WH, Krijgsheld P, Altelaar AFM, Post H, Ringrose JH, Mu € sten HAB, 2012. Spatially resolving the secreHeck AJR, Wo tome within the mycelium of the cell factory Aspergillus niger. Journal of Proteome Research 11: 2807e2818.


€ ller WH, Krijgsheld P, Bleichrodt R, van Veluw GJ, Wang F, Mu € sten HAB, 2013. Development in Aspergillus. Dijksterhuis J, Wo Studies in Mycology 74: 1e29. Kues U, 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiology and Molecular Biology Reviews 64: 316e353. Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: S680eS685. Levin AM, de Vries RP, Conesa A, de Bekker C, Talon M, € sten HAB, 2007. Spatial differMenke HH, van Peij NNME, Wo entiation in the vegetative mycelium of Aspergillus niger. Eukaryotic Cell 6: 2311e2322. Lim H, Choi HT, 2010. Growth inhibition of the yeast transformant by the expression of a chitinase from Coprinellus congregatus. The Journal of Microbiology 48: 706e708. Masuda Y, 1978. Auxin-induced cell wall loosening. The Botanical Magazine Tokyo S1: 103e123. McQueen-Mason SJ, Cosgrove DJ, 1994. Disruption of hydrogenbonding between plant cell wall polymers by proteins that induce wall extension. Proceedings of the National Academy of Sciences 91: 6574e6578. McQueen-Mason SJ, Cosgrove DJ, 1995. Expansin mode of action on cell walls e analysis of wall hydrolysis, stress-relaxation, and binding. Plant Physiology 107: 87e100. McQueen-Mason SJ, Durachko DM, Cosgrove DJ, 1992. Two endogenous proteins that induce cell-wall extension in plants. Plant Cell 4: 1425e1433. McQueen-Mason SJ, Fry SC, Durachko DM, Cosgrove DJ, 1993. The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls. Planta 190: 327e331. Michalenko GO, Hohl HR, Rast D, 1976. Chemistry and architecture of the mycelial wall of Agaricus bisporus. Journal of General Microbiology 92: 251e262. Mol PC, Vermeulen CA, Wessels JGH, 1990. Diffuse extension of hyphae in stipes of Agaricus bisporus may be based on a unique wall structure. Mycological Research 94: 480e488. Mol PC, Wessels JGH, 1990. Differences in wall structure between substrate hyphae and hyphae of fruit-body stipes in Agaricus bisporus. Mycological Research 94: 472e479. Money NP, Ravishankar JP, 2005. Biomechanics of stipe elongation in the basidiomycete Coprinopsis cinerea. Mycological Research 109: 627e634. Qin L, Kudla U, Roze EH, Goverse A, Popeijus H, Nieuwland J, Overmars H, Jones JT, Schots A, Smant G, Bakker J, Helder J, 2004. Plant degradation: a nematode expansin acting on plants. Nature 427: 30. Ramakrishnan C, Prasad N, 1972. Rigid-body refinement and conformation of a-chitin. Biochimica et Biophysica Acta 261: 123e135. Sakamoto Y, Irie T, Sato T, 2005. Isolation and characterization of a fruiting body-specific exo-b-1, 3-glucanase-encoding gene, exg1, from Lentinula edodes. Current Genetics 47: 244e252. Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssonen E, Bhatia A, Ward M, Penttila M, 2002. Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. European Journal of Biochemistry 269: 4202e4211. Wang J, Ding M, Li YH, Chen QX, Xu GJ, Zhao FK, 2003. A monovalent anion affected multi-functional cellulase EGX from the mollusca, Ampullaria crossean. Protein Expression and Purification 31: 108e114. Wei W, Yang C, Luo J, Lu C, Wu Y, Yuan S, 2010. Synergism between cucumber-expansin, fungal endoglucanase and pectin lyase. Journal of Plant Physiology 167: 1204e1210. Zhao Q, Yuan S, Wang X, Zhang Y, Zhu H, Lu C, 2008. Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro. Plant Physiology 147: 1874e1885.

Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa.

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it i...
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