- Top of page
- Materials and Methods
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).
- Top of page
- Materials and Methods
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.