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Keywords:

  • arbuscular mycorrhizal (AM) fungus;
  • cryo-TEM;
  • energy-filtering transmission electron microscopy (EF-TEM);
  • enzyme-affinity immuno-gold labelling;
  • Gigaspora margarita;
  • polyphosphate (poly P);
  • tubular vacuole

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    In arbuscular mycorrhizas (AM), the supply of phosphorus from the fungi is one of the most important benefits to the host plant. Here we describe for the first time the ultrastructure and polyphosphate (poly P) distribution in rapidly frozen and freeze-substituted germ tubes of the AM fungus Gigaspora margarita.
  • • 
    At the ultrastructural level, phosphorus distribution was analysed using energy-filtering transmission electron microscopy, and poly P was detected using an enzyme-affinity method. Semithin sections and live cells were also stained with 4′,6-diamidino-2-phenylindole, which is not specific but fluoresces yellow when viewed under UV irradiation by binding with poly P.
  • • 
    The cryotechnique method showed that extensive elongate ellipsoid vacuoles containing a uniform electron-opaque material occupied most of the cell volume. Combining the results of multiple methods revealed that poly P was localized in a dispersed form in vacuoles and in the outer fungal cell wall.
  • • 
    These results show the significant potential of AM fungi for phosphorus storage based on its localization in the extensive complement of vacuoles in thick hyphae. The mechanism of translocation of poly P in tubular vacuoles, and the role of poly P in the cell wall, need to be elucidated.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Most terrestrial plant species have symbiotic relationships with arbuscular mycorrhizal (AM) fungi belonging to the phylum Glomeromycota. The AM association involves host roots colonized by intercellular and intracellular hyphae, as well as extraradical hyphae extending into the soil (Peterson et al., 2004). The AM fungi depend on live host plants for their carbon source. In return, they provide minerals absorbed from the soil to the host, possibly via arbuscules formed within cortical cells (Smith & Gianinazzi-Pearson, 1988, 1990; Harrison, 1999). Within hyphae, therefore, a mass movement of minerals occurs from extra- to intraradical compartments, and a mass movement of carbon occurs in the opposite direction. As a result of establishment of this mutualistic relationship, spores are formed on the extraradical hyphae and also within colonized roots in some species; these play a crucial role in reproduction of the AM fungi. The hyphae produced from germinating spores are called germ tubes; these extend into the soil and contact host roots, initiating the colonization process.

Phosphorus is one of the essential elements for plant growth. However, most forms of P in the soil have low availability to terrestrial plants because of immobilization or sorption in soil, which causes a depletion zone around the root surface. Extraradical hyphae of AM fungi extend the distance from the root surface from which P can be absorbed. Inorganic phosphate (Pi) absorbed by extraradical hyphae is rapidly condensed into inorganic polyphosphate (poly P) within the hyphae (Ezawa et al., 2004). Poly P is a linear polymer of Pi connected by high-energy bonds, and occurs in all organisms studied so far (Kornberg et al., 1999; Kulaev et al., 2004). Poly P can be detected cytologically by its metachromatic reaction with toluidine blue O (TBO) at low pH, producing a distinctive pink colour (Lapeyrie et al., 1984; Grellier et al., 1989) or by yellow fluorescence under UV irradiation when stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Allan & Miller, 1980; Kawaharasaki et al., 1999; Klauth et al., 2006; Saito et al., 2006). When fungal hyphae are fixed chemically and dehydrated in ethanol, the metachromatic reaction is confined to granules in vacuoles, and the presence of P can be detected by energy-dispersive X-ray spectroscopy (EDXS) in electron-opaque bodies within vacuoles (Ashford et al., 1986).

Approaches using fluorescent dyes that accumulate in vacuoles in live cells have shown fungal vacuoles to be organelles composed of both tubular and spherical forms that are motile (Shepherd et al., 1993; Allaway & Ashford, 2001). It has been hypothesized that the tubular vacuole system is involved in long-distance P translocation within hyphae (Orlovich & Ashford, 1993; Cole et al., 1998; Ashford et al., 1999; Ashford, 2002; Darrah et al., 2006). The existence of a tubular vacuolar system has been reported in a wide range of filamentous microorganisms, including Basidiomycetes, Ascomycetes, Zygomycetes, Oomycota and Glomeromycota (Shepherd et al., 1993; Rees et al., 1994; Allaway & Ashford, 2001; Ashford et al., 2001; Uetake et al., 2002; Saito et al., 2006; Shoji et al., 2006). These findings led to a reassessment of fungal ultrastructure by applying techniques such as rapid freezing and freeze-substitution combined with elemental analysis. These approaches revealed that in the ectomycorrhizal fungus Pisolithus tinctorius, poly P is distributed in a dispersed form in tubular vacuoles (Orlovich & Ashford, 1993; Cole et al., 1998; Ashford et al., 1999), and the poly P granules often seen in fungal vacuoles are artefacts precipitated by the addition of TBO or ethanol to either living or glutaraldehyde-fixed cells (Orlovich & Ashford, 1993). Later, dispersed distribution of poly P was reported in vacuoles of the budding yeast Saccharomyces cerevisiae (Saito et al., 2005) and hyphae of the Ascomycete fungus Phialocephala fortinii, a dark septate root endophyte (Saito et al., 2006). In both studies, materials were freeze-substituted and labelled with a novel technique specific for poly P detection, which involved the application of a poly P-binding protein. The use of this method by these authors proved, for the first time at the ultrastructural level, the presence of poly P in fungal vacuoles.

In AM fungi, an extensive tubular vacuole system occurs in living germ tubes, as well as intra- and extraradical hyphae (Uetake et al., 2002). Although many ultrastructural studies have been published on intraradical structures of AM fungi, these have focused mainly on the interface between host cells and fungal structures (interfacial matrix) (Bonfante & Perotto, 1995; Genre & Bonfante, 2005) and the hyphal cell wall (Grandmaison et al., 1988; Bonfante-Fasolo et al., 1990); less attention has been paid to cytological aspects of the intraradical hyphae. Also, applications of freezing techniques to AM fungi have so far involved only Gigaspora margarita spores (Bonfante et al., 1994; Cruz, 2004). To understand the mechanism of long-distance transport of P in AM fungi, it is important to re-evaluate the form of poly P in AM fungal hyphae, where a tubular vacuole system has to be retained and any techniques that cause an artefact of poly P deposition need to be avoided.

The objectives of this study were therefore to observe the ultrastructure of AM fungal hyphae free from host tissues prepared with rapid freezing and freeze-substitution methods, and to determine the location and form of poly P within the hyphae. Germ tubes of the AM fungus G. margarita were used; for detection of poly P in situ, a number of methods were applied to resin-embedded materials or live cells. At the ultrastructural level, P was detected by energy spectroscopic imaging (ESI) and parallel electron energy-loss spectroscopy (PEELS) using an energy-filter transmission electron microscope (EF-TEM). Advantages of EELS against EDS are better detection of light elements (e.g. P and N), and an allowance of osmium fixation for P detection that gives better ultrastructural information. So far, a few studies have been published using EELS on AM fungal intraradical hyphae (Dexheimer et al., 1996) and spores (Cruz, 2004). Also, the direct detection of poly P on ultrathin sections was conducted by a method using the affinity of poly P-binding protein to the substrate (Saito et al., 2005, 2006; Werner et al., 2007). Although the reaction is less specific compared with the above method, semithin sections and live cells of germ tubes were stained with DAPI, which, combined with poly P, emits yellow fluorescence using UV excitation combined with epifluorescence microscopy (Tijssen et al., 1982).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal materials

An isolate of Gigaspora margarita Becker & Hall MAFF 520054 (Ministry of Agriculture, Forestry and Fisheries Genebank, Tsukuba, Japan) was propagated by pot culture with white clover (Trifolium repens L.) in sterilized soil in a glasshouse. Spores were extracted from the soil by a wet-sieving and decanting method (Gerdemann & Nicolson, 1963) and were used directly or surface sterilized according to Bécard & Fortin (1988). Germ tubes were obtained from spores placed in the centre of cellulose acetate membranes (10 × 15 mm, 0.2 µm pore size) arranged on 1% agar containing soil in a plastic Petri dish (9 cm diameter) (Uetake et al., 2002). For DAPI staining and enzyme-affinity labelling, sterilized spores were incubated on the membrane combined with 1% agar containing Long Ashton nutrient solution (5 mm NH4NO3, 1.33 mm NaH2PO4, 2 mm K2SO4, 4 mm CaCl2, 1.5 mm MgSO4, pH 5.8). The plates were inverted and incubated in the dark at 25×C for 10 d. Germ tubes on the membrane were used throughout this study.

Rapid freezing and freeze-substitution

Each membrane with germ tubes was removed carefully from the Petri dish and transferred into a drop of distilled water on a glass slide. Under a dissecting microscope, the germ tubes were transferred carefully from the membrane to the slide, using needles. A glass slide was dipped in 1% formvar in dichloroethane, and was air-dried to make a formvar membrane. The membrane on the glass slide was cut into small squares (3 × 6 mm), and pieces of the membrane were removed from the slide by floating on water. The germ tubes were then transferred onto a formvar membrane formed around a copper loop with a short handle (3 mm diameter, 15 mm long). The mycelium on the loop was further covered by a formvar membrane, and excess water on the loop was removed by a piece of filter paper and quickly plunged into liquid propane cooled with liquid N. The frozen mycelium was stored in liquid N temporarily, then transferred to a substitution medium of 100% dry acetone containing Molecular Sieves 4A 1/16 (Wako, Osaka, Japan) with or without 2% osmium tetroxide. The samples were substituted at –80°C for 3 d and warmed at –20°C for 2 h, 4°C for 2 h, and room temperature for 2 h. The samples were immersed twice in 100% dry acetone for 10 min before being infiltrated in Spurr's resin (Nisshin EM, Tokyo, Japan) or Quetol 812-araldite resin (Nisshin EM) mixed with acetone (25%, 50%, 75% resin; 12 h for each step), then infiltrated with pure resin for 2 d (resin was replaced once at 24 h). Samples were polymerized at 70°C overnight. Embedded materials were sectioned with an ultramicrotome (Leica, Vienna, Austria). Sections approx. 30 or 70 nm thick for electron microscopy were cut with a diamond knife and picked up on copper or nickel grids. Semithin sections (to 300 nm) were cut with a glass knife and collected on aminosilane-coated glass slides (Matsunami, Osaka, Japan).

Electron microscopy

Ultrathin sections of germ tubes, fixed with acetone containing 2% osmium tetroxide, were stained with a saturated uranyl acetate : acetone solution (1 : 1) for 10 min, followed by lead citrate for 2 min, and observed with a TEM (model H-7100 Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV.

Energy-filter TEM (EF-TEM)

Elemental analysis of G. margarita germ tubes was performed with an EF-TEM, LEO912AB Omega (Carl Zeiss, Jena, Germany) at an acceleration voltage of 100 kV. Ultrathin sections (30 or 80 nm thick) of germ tubes, fixed with freezing techniques and substituted in acetone with or without osmium tetroxide, were picked up on 600-mesh hexagonal copper grids. The microscope was operated in the ESI mode for element mapping, and PEELS was performed for spectrum registration with the aid of esi-vision software (Soft Imagining Systems, Münster, Germany). At ESI, high-contrast images were taken at 0 and 250 eV, and P and N mappings were taken at three and two-windows methods, respectively, with an energy-selecting slit width as 15 eV. With PEELS, to detect the P–L2,3 edge at 132.2 eV, an energy range between 110 and 200 kV was taken at ×100 000 magnification on vacuoles, electron-opaque body type a (EOBa) and resin, and the backgrounds of the spectra were subtracted using the ‘power low’ mode of the software. PEELS at an energy range between 338 and 426 kV was conducted to detect the N–K edge at 399 eV and the Ca–L2,3 edge at 350 kV simultaneously on each organelle at ×100 000 magnification.

Poly P labelling using affinity of the poly P-binding domain (PPBD) of Escherichia coli exopolyphosphatase

Poly P in germ tubes of G. margarita was observed using the affinity of PPBD of E. coli exopolyphosphatase, as described previously (Saito et al., 2005, 2006). Soon after the blocks where materials were embedded had been trimmed and sectioned, labelling of poly P was conducted on the ultrathin sections. Ultrathin sections were picked up on a 200-mesh nickel grid covered with collodion film and then coated with a thin layer of carbon. The sections were immersed in methanol containing 10% H2O2 for 10 min at room temperature. After washes in distilled water, the sections were blocked for 10 min with Tris-buffered saline pH 8.3 (TBS) containing 1% bovine serum albumin (BSA). The sections were incubated at room temperature overnight in a mixture of 20 µg ml−1 PPBD, 10 µg ml−1 mouse anti-Xpress epitope antibody (Invitrogen, Bannockburn, CA, USA), TBS, and 1% BSA. Sections were washed with TBS containing 0.05% Triton X-100 followed by TBS. The ultrathin sections were incubated for 2 h at room temperature with a goat anti-mouse IgG antibody conjugated with 12-nm colloidal gold (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), diluted 1 : 100 in TBS containing 1% BSA. After labelling, sections were stained with uranyl acetate followed by lead citrate and observed using a TEM (JEM-1200EX II, Jeol) at an accelerating voltage of 80 kV. Digital images were captured with a digital camera (Gatan, Pleasanton, CA, USA) and analysed using DigitalMicrograph (Gatan). Negative controls were prepared by incubating sections without PPBD and mouse anti-Xpress antibody.

DAPI staining

Semithin sections or living germ tubes were stained with DAPI. Semithin sections on glass slides were immersed in 10% H2O2/methanol or 0.2% NaOH/ethanol for 10 min at room temperature, followed by washes in distilled water. Semithin sections and living germ tubes on glass slides were incubated with 5 mm DAPI (Molecular Probes, Eugene, OR, USA) for > 4 h and with 10 mm DAPI for 10 min at room temperature, respectively. Specimens were washed with distilled water and covered with cover slips. The cover slips were sealed with nail polish to prevent samples from drying. Fluorescence microscopy was performed with an epifluorescence microscope Axioplan2 (Carl Zeiss) or BX50 (Olympus). Fluorescence of DAPI was excited with UV, and emitted fluorescence was detected with long-pass filter, LP420. Digital images were captured with a digital CCD camera AxioCam (Carl Zeiss) operated with AxioVisio (Carl Zeiss) or with a digital CCD camera DP70 (Olympus).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ultrastructure of rapidly frozen and freeze-substituted germ tubes of G. margarita

Rapid freezing and freeze-substitution methods resulted in good preservation, as indicated by the smooth membranes of organelles in germ tubes (Figs 1–3). Most of the hyphal volume was occupied by vacuoles showing a variety of forms, including circular, ellipsoid, and elongate ellipsoid (Figs 1, 2). Vacuole forms in a hyphal section were often uniformly circular or were intermingled with circular to elongate ellipsoid; in the latter case often circular vacuoles were located in the centre and elongate ellipsoid vacuoles in the periphery of cells (Fig. 1a,b). Examples of arrays of vacuolar membranes distributed parallel to the long axis were also observed (Fig. 2e). These vacuoles were often filled with fine (Figs 1, 2e–g) or dotted (Fig. 2c,d) electron-opaque materials. Densities of the vacuolar contents varied from being nearly translucent (Fig. 2a,b) to very electron-opaque (Fig. 2f,g). Sometimes, small aggregates (Figs 1b, 2a, double arrowheads) were found in vacuoles along with finely dispersed materials.

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Figure 1. Transmission electron micrographs of rapidly frozen and freeze-substituted germ tubes of Gigaspora margarita. Elongate ellipsoid vacuoles were located mostly towards the periphery of the cell, whereas circular vacuoles tended to be located centrally. l, Lipid body; n, nucleus; v, vacuole; arrow, electron-opaque body type a (EOBa); arrowhead, electron-opaque body type b (EOBb); double arrowhead, electron-opaque aggregate in vacuole. Bars, 1 µm.

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Figure 2. Transmission electron micrographs of rapidly frozen and freeze-substituted germ tubes of Gigaspora margarita showing variable vacuole shape and contents. (a) Circular vacuoles with low-density contents. The cytoplasm is rich in cell organelles. (b) Elongate ellipsoid vacuoles with low-density contents. (c) Circular vacuoles with dispersed electron-opaque materials. (d) Circular vacuoles with dotted electron-opaque materials. (e) Longitudinal array of vacuoles with medium-density contents. (f) Circular vacuoles with high-density contents. (g) Elongate ellipsoid vacuoles with high-density contents. b, Endobacterium; l, lipid body; m, mitochondrion; n, nucleus; v, vacuole; arrow, electron-opaque body type a (EOBa); arrowhead, electron-opaque body type b (EOBb); double arrowhead, electron-opaque aggregate in vacuole. Bars, 1 µm.

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Figure 3. Transmission electron micrographs of rapidly frozen and freeze-substituted germ tubes and auxiliary cells of Gigaspora margarita showing variety of cell organelles. (a) Auxiliary cell with electron-lucent lipid bodies surrounded by electron-opaque body type a (EOBa). Vacuoles have medium-density contents. (b) Endobacteria forming in tandem with a bundle of endoplasmic reticulum. (c,d) EOBa associated with microtubules. (e) EOBa within a cell and also in the cell wall. (f) Microbody with crystalloid inclusion. (g) Virus-like particle. (h) Endobacterium showing binary fission. (i) Golgi apparatus in auxiliary cell. (j) Multivesicular bodies and a bundle of microtubules along cell wall. (k) Electron-opaque body type b (EOBb) along with microtubules. b, Endobacterium; er, endoplasmic reticulum; l, lipid body; m, mitochondrion; mb, microbody; mt, microtubule; mv, multivesicular body; n, nucleus; v, vacuole; vp, virus-like particle; arrow, EOBa; arrowhead, EOBb; double arrowhead, Golgi apparatus. Bars: (a–c,e) 1 µm; (d,f–j) 0.5 µm.

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Endobacteria, which were distinguished from other fungal organelle profiles (such as mitochondria) by their thicker electron-opaque cell wall, were observed in the cytosol, and sometimes a constriction in the equatorial plane of endobacteria was evident (Fig. 3h). Linearly arrayed bacteria were found within an endoplasmic reticulum (ER) bundle (Fig. 3b). Organelles most frequently observed were electron-opaque bodies (EOB) that were categorized into two types based on their structure. One type had an ellipsoid electron-opaque core, with a size of 0.45 × 0.2 µm, closely surrounded by membrane (EOBa; Fig. 3d,k, arrows). These were sometimes observed external to the plasma membrane and, on a few occasions, within the cell wall (Fig. 3e). The second type of EOB was a circular vesicle surrounded by membrane, having an electron-opaque core within an electron-lucent region where electron-opaque, cottony materials are scattered (EOBb; Figs 2b, 3k, arrowheads). The EOBb was approx. 0.5 µm in diameter and slightly larger than the EOBa. Hexagonal virus-like particles, approx. 170 nm in diameter and containing an electron-opaque core (Fig. 3g), were present in the cytosol. Microtubules were observed, often in the vicinity of the plasma membrane (Fig. 3j) and sometimes also within the cytosol (Fig. 3c,d,k). Multivesicular bodies were observed often colocalized with microtubules (Fig. 3c,d), which were sometimes arrayed linearly (Fig. 3j). Mitochondria with distinct cristae were observed as circular-to-ellipsoid forms (Fig. 3a,c,d). A few microbodies containing crystal inclusions were also found in germ tubes (Fig. 3f). Lipid bodies were often seen as electron-lucent areas (Figs 1a, 2c, 3a). Nuclei were elongated ellipsoid in longitudinal sections, with an electron-opaque area inside (Figs 1b, 3a). Rough ER was scarce in germ tubes.

In G. margarita, auxiliary cells were formed on germ tubes (Fig. 3a,i). The cells were occupied mostly with circular vacuoles and lipid bodies. Organelles were generally the same as found in the germ tubes, but some differences were noted. There were few vacuoles and more lipid, ER and Golgi bodies, the latter rarely observed in germ tubes. Golgi bodies and EOBa often surrounded the lipid bodies or vacuoles (Fig. 3a).

Elemental analyses using EF-TEM

Elemental mapping and spectrum analyses were conducted on rapidly frozen and freeze-substituted ultrathin sections of G. margarita germ tubes using ESI and PEELS modes, respectively, on an EF-TEM. Phosphorus was detected in vacuoles both in PEELS (Fig. 4e–g) and ESI that showed over all distribution of the element in vacuoles in a three-window method (Fig. 4c). Electron-opaque structures, such as EOBa, EOBb and cell wall, were P-negative in the three-windows method of ESI (Fig. 4c) and PEELS analyses (Fig. 4e–g). Chromatin regions of nuclei were positive in P. Nitrogen and calcium were detected in both ESI and PEELS in EOBas, but at very low levels, or not at all in vacuoles (Fig. 4d,h).

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Figure 4. Elemental analysis of Gigaspora margarita germ tubes performed with an energy-filter transmission electron microscope at acceleration voltage 100 kV. Germ tubes were rapidly frozen and freeze-substituted in 2% osmium acetone. (a–d) Electron spectroscopic images taken at magnification of ×4000 and energy selecting slit width of 15 eV. Bars, 0.5 µm. (a,b) High-contrast images at (a) 0; (b) 250 eV. Most of cell volume was occupied by vacuoles and a few lipid bodies. (c) Phosphorus mapping taken at a three-windows method. (d) Nitrogen mapping taken at a two-windows method. (e–h) Parallel electron energy-loss spectroscopy taken at ×100 000 magnification. (e–g) P–L2,3 edge at 132 eV (arrow) on: (e) resin region beside hypha, (f) vacuole, (g) EOBa. Backgrounds were subtracted by ‘power low’. Phosphorus was detected in vacuole but not in EOBa nor in resin. (h) N–K edge at 399 eV (double arrowhead) and Ca–L2,3 edge at 350 eV (arrowhead) of vacuole, EOBa and resin. N and Ca were detected in EOBa, but less in vacuole. l, Lipid body; v, vacuole; arrow, electron-opaque body type a (EOBa).

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Poly P-labelling using the affinity of PPBD

Most vacuoles were labelled with colloidal gold (Fig. 5a), indicating that poly P was present. Labelling was distributed over the vacuoles; however, the distribution was heterogeneous and associated with electron-opaque amorphous deposits. Cell walls were composed of three layers, although these were sometimes not clear and were different in thickness: from the inside, a thick, electron-opaque fibrillar layer (inner cell wall, iw); an electron-light thinner layer (middle cell wall, mw); and an electron-opaque amorphous layer with protrusions outside (outer cell wall, ow). The outermost two cell wall layers were labelled with colloidal gold (Fig. 5b). Some labelling was found infrequently in EOBas (Fig. 5b). The outer cell wall of auxiliary cells was also labelled (Fig. 5c). Sometimes, electron-opaque vesicles embedded in the cell wall were labelled (Fig. 5c). Negative control without PPBD–anti-Xpress complex did not show any labelling (Fig. 5d).

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Figure 5. Immuno-gold enzyme-affinity labelling of polyphosphate (poly P) in rapidly frozen and freeze-substituted germ tubes and auxiliary cells of Gigaspora margarita. Poly P is localized by incubating poly P-binding domain (PPBD)–anti-Xpress antibody complex followed by anti-mouse IgG antibody conjugated with 12 nm colloidal gold. (a) Poly P distributed dispersedly in vacuoles. (b) Some labelling, perhaps poly P occurs in electron-opaque body (EOBa). Outer and middle cell wall is labelled. (c) Outer cell wall of auxiliary cell is labelled. EOBa embedded in cell wall is labelled. (d) Control without PPBD–anti-Xpress antibody complex. iw, Inner cell wall; mw, middle cell wall; n, nucleus; ow, outer cell wall; v, vacuole; arrow, EOBa. Bars, 0.5 µm.

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DAPI staining on resin-embedded sections of germ tubes

Resin-embedded sections of rapidly frozen and freeze-substituted G. margarita germ tubes were stained with DAPI (Fig. 6a–h). Although yellow fluorescence of DAPI is not specific only to poly P, vacuolar profiles of yellow fluorescence in the semithin sections agreed with the results of direct labelling of poly P using the enzyme-affinity method and EF-TEM, indicating that yellow-fluorescing vacuoles in germ tubes probably reveal poly P localization. Using a long-pass filter and stronger intensity setting of UV light, fungal cell walls showed blue autofluorescence (data not shown), which silhouetted the hyphal profile. Within the hyphal profile, flattened and/or round-to-oval nuclei (Fig. 6a–c) and an amorphous blue mass, probably chromatin, caused by a disruption of the nuclear envelope (Fig. 6d,f), were often observed. Frequently, blue dots, presumably endobacteria, were stained blue with DAPI (Fig. 6c). In addition to the cell components fluorescing blue, hyphae often contained materials that emitted yellow fluorescence characteristic of DAPI binding with poly P. The yellow fluorescence faded quickly, and great care was necessary (e.g. reduction of intensity for excitation and minimum time exposure) for observation and capturing images.

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Figure 6. Semithin sections of rapidly frozen and freeze-substituted germ tubes and auxiliary cells of Gigaspora margarita stained with a high concentration of DAPI after H2O2 etching (a–f) or NaOH etching (g–i). Germ tubes are classified into intact hyphae (a–c,g–i) and degraded hyphae (d–f) based on shape and organization of nuclei. (a) An intact germ tube with round nuclei and beaded arrays of vacuoles. Yellow fluorescence with DAPI is dispersed in vacuoles. Large portion of the hypha is occupied by vacuoles stained with DAPI. (b) The germ tube shows intracellular features similar to hypha in (a). Simultaneously, a few granules fluorescing bright yellow (arrowhead) are observed. (c) Intact germ tube not showing yellow fluorescence. Round nuclei are present. Arrowheads indicate either mitochondria or endobacteria stained blue. (d) Degraded germ tube with chromatin strands released from collapsed nucleus. Yellow fluorescence is observed throughout the disorganized cytoplasm. (e) The germ tube shows intracellular features similar to hypha in (d). Simultaneously, a few granules fluorescing bright yellow (arrowheads) are observed. (f) Degraded hypha with a few granules fluorescing bright yellow (arrowheads); nuclei fluoresce blue. (g) Hyphal section treated with NaOH etching. An intact hypha with round nuclei and beaded arrays of vacuoles. Vacuolar features stained with DAPI are similar to that of the hyphal section treated with H2O2 etching. (h) Highly magnified image of hyphal section treated with NaOH etching. Vacuolar content stained with DAPI is heterogeneous. Intense yellow regions (arrows) in vacuoles are distinguished from a brightly fluorescent granule (arrowhead). Circular structures not stained with DAPI are presumably lipid body. (i) Auxiliary cells stained with DAPI. There are many vacuoles showing yellow fluorescence. Nuclei fluoresce blue. l, Lipid body; n, nucleus; v, vacuole; w, cell wall. Bars: (a–g,i) 5.0 µm; (h) 2.0 µm.

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Yellow fluorescence profiles within cells varied considerably in their forms and amounts among hyphal sections: the form was from dots (Fig. 6f) to circular or ellipsoid (Fig. 6a,b), and the amount was from none (Fig. 6c) to abundant, covering an entire hyphal profile (Fig. 6d,e). Examination at high magnification revealed that vacuolar contents stained with DAPI were heterogeneous with intense and faint yellow fluorescent regions in vacuoles (Fig. 6h). Auxiliary cells also possessed a large number of vacuoles that fluoresced yellow following DAPI staining (Fig. 6i). Two etching treatments with H2O2 (Fig. 6a–f) and NaOH (Fig. 6g–i) before DAPI staining revealed similar features of yellow fluorescent vacuoles along hyphae.

The relationship between poly P forms and hyphal integrity was evaluated (Table 1). Germ tube profiles that did not contain nuclei were excluded, and the remaining profiles were classified into two groups based on whether nucleus shape and organization were round or oval (Fig. 6a–c), or not (Fig. 6d–f).

Table 1.  Relative number of germ tubes of Gigaspora margarita stained with a high concentration of DAPI in each cellular state deduced by integrity of nuclei
Cellular stateaNumber of germ tubes examinedRelative number of germ tubes in each categoryb (%)
(1) Vacuoles stained evenly(2) Vacuoles stained evenly + small granules(3) Stained throughout hyphae(4) Stained throughout hyphae + small granules(5) Small granules only(6) Not stained
  • a

    Germ tubes with round or oval nuclei were classified as intact hyphae; germ tubes with irregularly shaped nuclei or strands of chromatin as degraded hyphae.

  • b

    Germ tubes stained with DAPI were categorized into six classes: (1) vacuoles evenly stained with DAPI; (2) evenly stained vacuoles and brightly stained small granules in the same germ tube; (3) yellow fluorescence observed throughout hyphae; (4) evenly stained cytoplasm and brightly stained small granules in the same germ tube; (5) small granules stained brightly; (6) no DAPI stain.

Intact405830 0 0 310
Degraded31 0 729233210

In intact hyphae with round or oval nuclei, yellow fluorescence in a dispersed form was most frequently observed in evenly stained circular vacuoles, which sometimes were arrayed linearly (Table 1; Fig. 6a,g). In these cells, the vacuoles often occupied a majority of the hypha. Some intact hyphae possessed a few small granules fluorescing markedly bright yellow in vacuoles also containing a dispersed form of yellow fluorescence (Fig. 6b). There were few intact hyphae having the bright fluorescent granules only, and there were a few intact hyphae having no yellow fluorescence (Table 1; Fig. 6c).

In hyphae with degraded nuclei, yellow fluorescence was most frequently observed throughout the cytoplasm with (Fig. 6e) or without (Fig. 6d) bright yellow granules. Some degraded hyphae had only a few granules fluorescing bright yellow (Fig. 6f) or no intracellular structures stained with DAPI (Table 1).

DAPI staining of live germ tubes

Live germ tubes were stained with DAPI. To visualize vacuolar poly P in live cells, it was necessary to use a much higher concentration than required to see blue fluorescence of nuclei in the same cell. Cell walls fluoresced slightly over the entire hyphae, but bright fluorescence was observed at the apical region of germ tubes (Fig. 7a) and the branching points of hyphae (Fig. 7b).

image

Figure 7. Living germ tubes of Gigaspora margarita stained with 10 mm DAPI. (a) Hyphal tip fluoresces an intense yellow. Cell wall in regions of hypha basipetal to tip faintly stains yellow. Nuclei (arrowhead) appear blue. (b) Cell wall in apical region of branching hypha (arrow) fluoresces intense yellow. Bars, 10 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This is the first report of the ultrastructure, including the nature of poly P in vacuoles, of germ tubes of an AM fungus using material processed by rapid freezing and freeze-substitution. To date, TEM studies of AM fungi in the free-living state applying cryotechniques have been limited to spores of G. margarita (Bonfante et al., 1994; Cruz, 2004; Lumini et al., 2007), partly because of the difficulty in obtaining germ tubes and extraradical hyphae for efficient rapid freezing and freeze substitution. Previously, in G. margarita spores, lipid droplets, protein-like bodies within specialized vacuoles and glycogen deposits were reported as storage substances, and rough ER, Golgi equivalents and vacuoles were reported as organelles. The vacuoles in the spores were described as having finely granular or homogeneously electron-opaque contents, and often bacteria were observed in specialized vacuoles. In this study, Golgi bodies were rarely observed in germ tubes but were present in auxiliary cells of G. margarita, suggesting differences in functions between these two fungal structures indicative of a high synthetic activity of proteins in auxiliary cells. The observation of electron-opaque bodies (EOBa) incorporated into the cell wall of germ tubes agrees with that reported for spores (Bonfante et al., 1994). However, protein-like bodies observed in vacuoles of frozen spores (Bonfante et al., 1994; Cruz, 2004) were not observed in germ tubes of G. margarita. This may be caused by different organs and/or different growth stages, as Bonfante et al. (1994) observed that the number and size of the protein-like bodies appeared to decrease in germinated spores.

The ultrastructure of G. margarita germ tubes confirmed the extensive tubular vacuole system observed in live cells of germ tubes, as well as intra- and extraradical hyphae stained with the fluorescent probe DFFDA (Uetake et al., 2002). In the live germ tubes, the tubular and spherical vacuoles, when they were observed simultaneously with cytoplasmic streaming, often tended to localize at the periphery and centre, respectively. The vacuolar profiles observed with TEM were mostly circular, but elongate ellipsoid forms located mostly in the cell periphery were also present; these corresponded with spherical and tubular vacuoles, respectively, in live germ tubes. One characteristic of the ultrastructure of relatively thick aseptate hyphae of G. margarita germ tubes was the prevalence of vacuoles correlated with less cytosol in the cells. Fungal hyphae are differentiated into a tip region and a more basal vacuolated region; in the former region vacuoles are more tubular, and in the latter area vacuoles consist of tubules and large globules (Ashford et al., 2001). At the ultrastructural level of such vacuolated hyphae, however, other organelles, such as tubular mitochondria and ER bundles, are evident in addition to vacuoles (Cole et al., 1998). In a few ultrastructural studies on germ tubes (Sward, 1981; Peterson & Bonfante, 1994) and extraradical hyphae of AM fungi (Bago et al., 1998), fewer vacuoles and a higher density of organelles have been reported compared with observations on germ tubes in this study. This might be caused by differences in fixation methods and in hyphal positions observed. In a study of P metabolism in the AM fungus Glomus intraradices using 31P nuclear magnetic resonance, Rasmussen et al. (2000) indicated that external hyphae showed very weak cytoplasmic Pi signal suggesting a low cytoplasmic volume, which may also be the case in germ tubes.

The localization of P or poly P in dispersed form in vacuoles was, for the first time, revealed on resin-embedded G. margarita germ tubes prepared by cryotechniques using two detection methods: elemental analysis by EF-TEM, and poly P direct labelling visualized by TEM. This agrees with results for other fungal taxa such as the Basidiomycete P. tinctorius (Orlovich & Ashford, 1993; Ashford et al., 1999), budding yeast (Saito et al., 2005) and the Ascomycete P. fortinii (Saito et al., 2006) when rapid freezing was used. The poly P in vacuoles in the resin-embedded germ tubes was also revealed as a dispersed form of yellow fluorescence with DAPI staining under fluorescence microscopy. Dispersed poly P may accumulate heterogeneously among germ tubes, as intensities of poly P among hyphal profiles were variable even in one section. The amount of poly P appeared to have no relationship between vacuole shapes, whether circular or elongate ellipsoid, which might be related to a translocation status of poly P in the hypha. A hypothesis that fungal tubular vacuoles act as an internal distribution system was tested by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker, and the modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimetres to centimetres (Darrah et al., 2006).

Direct labelling of poly P on ultrathin sections in the present study showed distribution of poly P in the outer cell wall as well as vacuoles in germ tubes, which corresponded with the faint fluorescence shown in live cells with DAPI staining. The DAPI staining was especially strong in the apical region of hyphae; this phenomenon could not be confirmed by the staining on semithin sections in this study because of the very sparse growth pattern of the germ tubes causing the technical limitation of embedding and sectioning of the hyphal tip. Werner et al. (2007) showed that the cell wall of the AM fungus G. intraradices contained poly P, by staining live cells with specific poly P-binding proteins, and that hyphal tips and young hyphae in Zygomycete fungi, Conidiobolus thromboides and Basidiobolus microsporus, revealed a stronger labelling of poly P than older mycelia. A major source of poly P in the cell wall may be EOBa, where some labelling of poly P was observed. However, the distribution in cell walls and EOBa was not supported by P detection by PEELS, which may be caused by a low poly P concentration in the structures or, as recognized by their different appearances, by poly P forms different from that in vacuoles that influenced the detection of P on EF-TEM. Ultrastructural evidence suggests that, as reported in spores (Bonfante et al., 1994), EOBas are exocytosed outside the plasma membrane and are moved into the cell wall region. The poly P in cell walls of AM fungi might suggest its involvement in P transfer from the mycorrhizal fungi to host plants.

Acidic vesicles have been reported in G. margarita live germ tubes and extraradical hyphae using the fluorescent acidotropic probe LysoTracker (Saito et al., 2004). The vesicles were approx. 0.3–0.7 µm in diameter, abundant in the cells, and moved by cytoplasmic streaming. Because internal acidity has been discussed as a motive force of poly P synthesis in membrane-bound structures (Ogawa et al., 2000; Ezawa et al., 2002), it is important to determine which organelle observed in the present study corresponds to the acidic vesicle, and whether the organelle contains poly P. Candidate organelles are the two electron-opaque bodies (EOBa and EOBb) observed in this study, and multivesicular bodies. Among these three, EOBa were most ubiquitous and abundant in germ tubes, and may correspond to the acidic vesicles. As mentioned above, although some specific labelling of poly P was observed on EOBa, elemental analyses of EOBa using EF-TEM were positive for N and Ca, but negative for P. Similar results of elemental analyses on presumably equivalent organelles, ‘granules included in small cytoplasmic vesicles’ and ‘less dense and often diffuse vacuolar granules’ were reported by Dexheimer et al. (1996), where the same analysis methods were used on chemically fixed Glomus mosseae hyphae within Allium cepa roots. Therefore EOBa and EOBb appear not to be responsible for a mass long-distance translocation of P within hyphae. Functions of EOBa and EOBb in AM fungi, and relationships between these organelles and acidic vesicles, need to be clarified in the future.

This study shows clearly that the vacuoles in germ tubes of an AM fungus, G. margarita, are predominant organelles occupying most of the cell volume and therefore have the potential for accumulation of poly P in dispersed form. In future, it is necessary to address whether the accumulated poly P in the vacuoles can be translocated within this compartment, and if so, the mechanism involved. The methods used in this study will be powerful tools to analyse in situ dynamics of poly P in fungal hyphae. Biophysical and/or molecular information on poly P metabolism in eukaryotic cells will advance interpretations of the structures involving P storage, which will lead to a better understanding of P transport mechanisms in mycorrhizal symbioses.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr William Allaway and his coworkers for their technical advice on freezing techniques. We also thank Dr Hiro-o Hamaguchi and Dr Yasuaki Naito for helpful discussions. This work was supported in part by the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of the Bio-oriented Technology Research Advancement Institution of Japan to M.S., the Ministry of Education, Culture, Sports, Science and Technology, Grant-in-Aids for Scientific Research (B) to Y.K. and for Young Scientists (Start-up) to K.S., and funding from the Natural Sciences and Engineering Research Council of Canada to R.L.P.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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