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Exploring structural definitions of mycorrhizas, with emphasis on nutrient-exchange interfaces1 R. Larry Peterson and Hugues B. Massicotte
Abstract: The roots or other subterranean organs of most plants develop symbioses, mycorrhizas, with fungal symbionts. Historically, mycorrhizas have been placed into seven categories based primarily on structural characteristics. A new category has been proposed for symbiotic associations of some leafy liverworts. An important feature of mycorrhizas is the interface involved in nutrient exchange between the symbionts. With the exception of ectomycorrhizas, in which fungal hyphae remain external to plant cell walls, all mycorrhizas are characterized by fungal hyphae breaching cell walls but remaining separated from the cell cytoplasm by a plant-derived membrane and an interfacial matrix that forms an apoplastic compartment. The chemical composition of the interfacial matrix varies in complexity. In arbuscular mycorrhizas (both Arum-type and Paris-type), molecules typical of plant primary cell walls (i.e., cellulose, pectins, β-1,3-glucans, hydroxyproline-rich glycoproteins) are present. In ericoid mycorrhizas, only rhamnogalacturonans occur in the interfacial matrix surrounding intracellular hyphal complexes. The matrix around intracellular hyphal complexes in orchid mycorrhizas lacks plant cell wall compounds until hyphae begin to senesce, then molecules similar to those found in primary cell walls are deposited. The interfacial matrix has not been studied in arbutoid mycorrhizas and ectendomycorrhizas. In ectomycorrhizas, the apoplastic interface consists of plant cell wall and fungal cell wall; alterations in these may enhance nutrient transfer. In all mycorrhizas, nutrients must pass into the symplast of both partners at some point, and therefore current research is exploring the nature of the opposing membranes, particularly in relation to phosphorus and sugar transporters. Key words: interface, apoplastic compartment, Hartig net, arbuscule, intracellular complex, nutrient exchange. Résumé : Les racines et autres organes souterrains de la plupart des plantes développent des symbioses avec des champignons, les mycorhizes. Historiquement, on a placé les mycorhizes dans sept catégories, basées surtout sur des caractéristiques structurales. Une nouvelle catégorie a été proposée pour les associations symbiotiques de certaines hépatiques foliacées. Une importante caractéristique des mycorhizes est l’interface impliqué dans les échanges de nutriments entre les symbiotes. À l’exception des ectomycorhizes, dans lesquelles les champignons demeurent à l’extérieur des parois cellulaires des plantes, toutes les mycorhizes se caractérisent par des hyphes fongiques traversant les parois cellulaires, mais demeurant séparés du cytoplasme par une membrane dérivée de la plante et une matrice interfaciale formant un compartiment apoplastique. La composition chimique de la matrice interfaciale varie en complexité. Chez les mycorhizes arbusculaires (de type Arum aussi bien que Paris), on retrouve des molécules typiques des parois cellulaires primaires (c.-à-d., cellulose, pectines, β-1,3-glucans, glycoprotéines riches en hydroxyproline). Chez les mycorhizes éricoïdes, il n’y a que des rhamnogalacturones dans la matrice interfaciale entourant les complexes d’hyphes intracellulaires. La matrice entourant les complexes intracellulaires ne comporte pas de matériel pariétal de la plante chez les mycorhizes des orchidées, jusqu’à ce que les hyphes deviennent sénescentes; on retrouve alors des molécules similaires à celles des parois primaires. La matrice interfaciale n’a pas été étudiée chez les mycorhizes arbutoïdes et les ectendomycorhizes. Chez les ectomycorhizes, l’interface apoplastique est constituée de paroi cellulaire végétale et de paroi cellulaire fongique; une altération de ces parois peut augmenter le transfert de nutriments. Chez toutes les mycorhizes, les nutriments doivent passer par le symplaste des deux partenaires à un point donné, et c’est pourquoi les recherches actuelles portent sur la nature des membranes opposées, surtout en relation avec les transporteurs de sucre et de phosphore. Mots clés : interface, compartiment apoplastique, réseau de Hartig, arbuscule, complexe intracellulaire, échange de nutriments. [Traduit par la Rédaction]
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Received 29 September 2003. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 18 August 2004. R.L. Peterson. Department of Botany, University of Guelph, Guelph, ON N1G 2W1, Canada. H.B. Massicotte.2 Ecosystem Science & Management Program, College of Science and Management, University of Northern British Columbia, 3333 University Way, Prince George, B.C. Canada. 1
This article is one of a selection of papers published in the Special Issue on Mycorrhizae and was presented at the Fourth International Conference on Mycorrhizae. 2 Corresponding author (e-mail:
[email protected]). Can. J. Bot. 82: 1074–1088 (2004)
doi: 10.1139/B04-071
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Introduction Mycorrhizas are usually defined as mutualistic symbioses between fungi and plants in which both partners can benefit from the association (Smith and Read 1997). It is recognized, however, that it is often difficult to assess the benefits derived by each of the symbionts in mutualistic associations, since these must be considered in terms of the costs to each partner (Ahmadjian and Paracer 1986). There is considerable discussion as to what constitutes a benefit to each of the partners in mycorrhizas as environmental conditions change. In mycorrhizas, the main benefit to the plant is largely nutritional in nature, but protection from root pathogens and improved water relations may also accrue. The term “balanced mycorrhizas” has been proposed to denote situations in which both organisms receive essential materials through reciprocal exchange (Brundrett 2002, 2004). An important step in the evolution of balanced mycorrhizas was, therefore, the development of a specialized interface zone for bidirectional nutrient exchange (Brundrett 2002). The term “exploitive mycorrhizal associations” has been suggested for those situations in which there is a unidirectional flow of nutrients, with the main benefit usually going to the plant partner (Brundrett 2002, 2004). Mycoheterotrophic plant species without photosynthetic ability, and green plants in situations where the fungus seems to gain little from the association, could be considered as examples of exploitive mycorrhizas (Brundrett 2002, 2004). It should be emphasized, however, that whether balanced or exploitive, mycorrhizal associations are the norm for almost all vascular plants and a few nonvascular plants (Smith and Read 1997; Read et al. 2000). Mycorrhizal symbioses can, therefore, be distinguished from interactions between pathogenic fungi and roots, in which nutrient flow is always to the fungi. Brundrett (2004) suggests that the nutrient-exchange interface established between the symbionts is unlikely to function in the same manner for balanced and exploitive mycorrhizal associations. To evaluate this suggestion, it is important to consider the structural aspects of interfaces for all categories of mycorrhizas and to assess the evidence that these sites function in nutrient exchange. In parallel with the evolution of nutrient-exchange interfaces, the extraradical mycelium of successful symbioses developed as an efficient soil–fungal interface to allow for exploration and extraction of nutrients and water from the substrate. This topic is discussed by Leake et al. (2004) in this Special Issue. In this review, we will use the seven traditional categories of vascular plant mycorrhizas based on structural characteristics identified by light and transmission electron microscopy (Scannerini and Bonfante-Fasolo 1983; BonfanteFasolo and Scannerini 1992; Peterson and Farquhar 1994; Smith and Read 1997; Peterson et al. 2004), recognizing that there is some discussion as to new ways of classifying mycorrhiza types (Brundrett 2004). These categories are arbuscular mycorrhizas (AM), ectomycorrhizas, ectendomycorrhizas, ericoid mycorrhizas, arbutoid mycorrhizas, monotropoid mycorrhizas, and orchid mycorrhizas. We will also include a brief discussion of mycorrhizas occurring with
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nonvascular plants, since these are receiving more attention in the literature and may provide insight into the evolution of symbioses between plants and fungi (Read et al. 2000; Kottke et al. 2003). The main objectives of this review are, therefore, to discuss what is known about the interfaces in all categories of mycorrhizas, to evaluate the evidence for nutrient transfer at these sites, to suggest future research that is needed, and finally to comment on possible modifications in defining categories of mycorrhizas.
Categories of vascular plant mycorrhizas Arbuscular mycorrhizas Arbuscular mycorrhizas, the symbiotic associations between the majority of vascular plant species and fungi in the new phylum Glomeromycota (Schüßler et al. 2001), can be subdivided into two main types, the Arum-type and the Paris-type (Gallaud 1905; Smith and Smith 1997). In the ontogeny of interface development in the Arum-type (Fig. 1), usually one arbuscule develops through repeated branching of a hypha (trunk hypha) that penetrates through the cortical cell wall (Bonfante and Perotto 1995). Paired arbuscules in adjacent cortical cells arising from the same intercellular hypha but developing at different rates have recently been reported by Dickson et al. (2003) in Linum usitatissimum. Figure 1 also illustrates paired arbuscules in cortical cells of a Pisum sativum root, illustrating that this feature may be more common than reported. In the Paris-type, penetration of the cortical cell wall by a single hypha is followed by extensive coiling of this hypha (Fig. 2) from which lateral branches are initiated to form arbusculate coils (Whitbread et al. 1996; Cavagnaro et al. 2001; also see Fig. 3). In the Arum-type nutrient-exchange interface, the fungus develops fine, thin-walled arbuscule branches within plant cortical cells, the walls of which are modified by becoming very thin (Bonfante-Fasolo and Grippiolo 1982; Bonfante-Fasolo et al. 1990) and which have an amorphous chitin deposition (Bonfante-Fasolo 1982; Bonfante-Fasolo et al. 1990). In the Paris-type, both hyphal coils and arbusculate coils may be involved in nutrient exchange, an idea suggested by calculations that show that the surface area of hyphal coils may be equal to that of arbuscules in Arum-type mycorrhizas (Dickson and Kolesik 1999) as well as by the presence of a membrane and interfacial matrix around hyphal coils and arbusculate coils (Armstrong and Peterson 2002). Arbuscules and arbusculate coils are separated from the corticalcell cytoplasm by a periarbuscular membrane and interfacial matrix material, both derived from the plant symbiont (Bonfante and Perotto 1995; Armstrong and Peterson 2002). The interfacial matrix, situated between the fungal cell wall and the periarbuscular membrane, is an apoplastic compartment composed of plant cell wall constituents, including cellulose, pectins, β-1,3-glucans, and hydroxyproline-rich glycoproteins, as demonstrated by the use of various affinity probes (Bonfante and Perotto 1995; Armstrong and Peterson 2002) and in situ mRNA probes (Blee and Anderson 2000). Bonfante and Perotto (1995) claim that the development of this apoplastic compartment between fungus and plant cell is the most important event marking successful colonization. With the development of the interfacial matrix, any transfer © 2004 NRC Canada
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Figs. 1–4. Interfaces of arbuscular mycorrhizas. Fig. 1. Arbuscules of Glomus aggregatum in a Pisum sativum root. Material stained with acid fuchsin and viewed with scanning laser confocal microscopy. Arbuscules are of the Arum type. Photo courtesy of Ryan Geil. Scale bar = 25 µm. Fig. 2. Intracellular hyphal coils of Glomus intraradices in a Panax quinquefolius root. These coils are typical of Paris-type arbuscular mycorrhizas. Material stained with acid fuchsin and viewed with scanning laser confocal microscopy. Scale bar = 25 µm. Fig. 3. Arbusculate coils of Glomus intraradices in a root of Panax quinquefolius. Arrowheads indicate fine branches formed on hyphal coils. Material cleared, stained with chlorazol black E, and examined by light microscopy. Scale bar = 25 µm. Fig. 4. Diagram of arbuscule illustrating the components of the interface. The periarbuscular membrane (arrowheads), interfacial matrix (*), fungal cell wall (arrows), and fungal plasma membrane (double arrowheads) are evident. T, trunk hypha; I, intercellular hypha. Scale bar = 25 µm.
of nutrients from the plant cell to the fungus would involve the periarbuscular membrane, the interfacial matrix, the fungal wall, and the fungal plasma membrane (Fig. 4). The reverse would also be true as nutrients pass from the fungus to the plant cell (Smith and Smith 1990). Since there is a bidirectional exchange of nutrients, with phosphate (P), nitrate, and trace elements moving from the fungus to the plant and sugars moving to the fungus (Franken et al. 2002), this would be an example of a balanced relationship (Brundrett 2002, 2004). H+-ATPase activity has been demonstrated cytochemically in the fungal plasma membrane in the interface (GianinazziPearson et al. 1991, 2000), and Saito (2000) has provided a detailed account of the localization of various phosphatases in host and fungal membranes of AM associations, evidence that active transport is likely involved in nutrient transfer. Ferrol et al. (2002) summarize the evidence for the upregulation of host plasma membrane ATPase genes in mycorrhizal roots of a number of plant species. Also, there
is a difference in plasma membrane proteins, as shown by 2D-PAGE analysis, between mycorrhizal and nonmycorrhizal tomato (Lycopersicon esculentum) roots (Benabdellah et al. 2000), but whether these are related to the nutrient-exchange interface is not known. Currently there is considerable interest in characterizing transporters in both the periarbuscular membrane and the fungal membrane, since these would be involved in the active transport across this interface (Harrison 1999a, 1999b; Smith et al. 2001; Ferrol et al. 2002). The recent review by Ferrol et al. (2002) summarizes the information on P and carbon transporters in AMs and points out the lack of information concerning the presence and activity of these in the nutrient-exchange interface. Other changes that occur in cortical cells containing arbuscules include alterations in expression of genes encoding enzymes involved in sucrose metabolism (Blee and Anderson 2002), leading to the establishment of a sink for sucrose transport from the phloem to the arbuscules. © 2004 NRC Canada
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The deposition of the interfacial matrix and the formation of the periarbuscular membrane must involve considerable cellular activity, and it has been suggested that alterations observed in the plant-cell cytoskeleton during arbuscule development may play a role in these processes (Genre and Bonfante 1997, 1998; Peterson et al. 2000; Armstrong and Peterson 2002; Timonen and Peterson 2002). Methods combining GFP (green fluorescent protein) tagging of cell components such as endoplasmic reticulum and Golgi bodies, in addition to cytoskeletal elements, and scanning laser confocal microscopy (Takemoto et al. 2003) could add additional information as to how interfaces are formed between symbionts. Intercellular hyphae, although not surrounded by either plant-derived membrane or interfacial matrix, interface with the apoplast (intercellular space system) of roots (Ferrol et al. 2002). They have the potential to be involved in the transfer of nutrients, and it has been suggested that this might be a pathway for carbon acquisition by the fungus (Smith and Smith 1990). In this case, only the fungal plasma membrane would be involved in the uptake of carbon compounds. Ectomycorrhizas Ectomycorrhizas, symbioses formed between both gymnosperm and angiosperm species and many basidiomycete fungi and fewer ascomycete fungi, are characterized primarily by the presence of a mantle (sheath) and an Hartig net consisting of modified fungal hyphae that develop between root cells (Smith and Read 1997). Generally, species belonging to the angiosperms have a paraepidermal Hartig net (i.e., confined to the root epidermis, as shown in Fig. 5), whereas species belonging to the gymnosperms have an Hartig net that develops around epidermal and cortical cells (Fig. 6). There are exceptions to this in that a few angiosperm genera (e.g., Dryas) possess an Hartig net that surrounds epidermal and cortical cells (Melville et al. 1988). In some species the Hartig net may reach the endodermis, although this is variable. Because of the intimate association of Hartig net hyphae with root cell walls (Fig. 7), the labyrinthine branching (Fig. 8) of these hyphae (Kottke and Oberwinkler 1986, 1987), and the presence of numerous mitochondria and other organelles in Hartig net hyphae (Massicotte et al. 1986, 1990), it has been concluded that this is the main interface involved in bidirectional transfer of nutrients (Kottke and Oberwinkler 1986; Massicotte et al. 1987, 1989; Smith and Read 1997). Inner mantle hyphae that are not part of the Hartig net but are positioned immediately adjacent to the outer tangential wall of root epidermal cells provide a second possible interface for nutrient exchange in ectomycorrhizas (Dexheimer and Gérard 1994) (Figs. 9, 10). These hyphae may also become highly branched, and in the few cases in which an Hartig net does not develop (e.g., in Pisonia grandis), it is the only interface for nutrient exchange (Ashford and Allaway 1982). More direct evidence that the Hartig net is involved in the transfer of P and carbon compounds comes from the use of radioactive tracers followed by microautoradiography of rapidly frozen and freeze-substituted poplar (Populus tremuloides × Populus alba) ectomycorrhizas (Bücking and Heyser 2001). In this study, 33Pi accumulated rapidly in the mantle and later was localized in the Hartig net and cortical
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cells; the authors interpret the results to support the concept that P moves across the mantle through the symplast and not the apoplast. Also, mantle hyphae and Hartig net hyphae in the median region of ectomycorrhizal roots were more highly labelled than in either the apical or basal region, indicating that there are some spatial differences in uptake and translocation of P along the root axis. Similar results were obtained for the distribution of labelled carbohydrates. This supports the observation that there is a gradient of host-cell response to fungi forming the Hartig net along the long axis of a root (Massicotte et al. 1986). It is known, also, that ectomycorrhizas of different ages affect the absorption and translocation of P (Cairney and Alexander 1992a) and carbon compounds (Cairney and Alexander 1992b). In some mycorrhizas, such as those formed in P. grandis (Ashford and Allaway 1982) and between Alnus crispa and Alpova diplophloeus (Massicotte et al. 1986), epidermal cells contiguous with mantle hyphae and Hartig net hyphae, if present, develop wall ingrowths (Fig. 10) that are enveloped by plasma membrane and assume the structure of transfer cells. Transfer-cell development is also found in several other ectomycorrhizas (Dexheimer and Gérard 1994). The increase in surface area of wall and membrane of transfer cells in P. grandis, although less than provided by the development of an Hartig net in other ectomycorrhizas, may be adequate for transfer of nutrients in the environment in which this species is found (Allaway et al. 1985). Regardless of whether root cells differentiate as transfer cells, or whether Hartig net or inner mantle hyphae are involved, the interface between symbionts consists of contiguous root cell walls and fungal hyphal walls with material (sometimes called cement) deposited between them, creating an apoplastic compartment. Plasma membranes of each symbiont would be involved during the bidirectional transfer of nutrients (see Figs. 9, 10). It is important to ascertain, therefore, whether changes that would enhance nutrient exchange occur in root cell walls and hyphal walls as an ectomycorrhiza is formed. Features of root-cell plasma membranes and fungal plasma membranes in the interface region may also enhance nutrient exchange. Both plant cell walls and fungal cell walls play an integral role during all stages in the establishment and functioning of ectomycorrhizas, and considerable progress has been made in determining changes in both walls at the molecular level (Duplessis et al. 2002; Tagu et al. 2002). However, only information related to the walls of the symbionts in the nutrient-exchange interface will be considered here. One change in host cell walls observed at the ultrastructural level is the apparent continuity between the wall and the intercellular matrix adjacent to Hartig net hyphae, suggesting that the cell wall, in particular, is modified in the interface (Dexheimer and Gérard 1994). Also, in two gymnosperm ectomycorrhizal associations, large amounts of acid polysaccharides (pectins) are present in cortical cell walls as well as in the matrix material separating these walls and Hartig net hyphae (Nylund 1987). This author concludes that this supports the concept that there is a “mycorrhizal infection zone” in roots, in which changes to host walls enable the development of the intercellular Hartig net; no comment was made in terms of nutrient exchange in this zone. Using a variety of affinity techniques, Balestrini et al. (1996) showed © 2004 NRC Canada
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Figs. 5–10. Features of interfaces in ectomycorrhizas. Fig. 5. Longitudinal section of a root tip of Alnus crispa colonized by Alpova diplophloeus. A thick mantle (M) and a paraepidermal Hartig net (arrowheads) are evident. Material embedded in resin, stained with toluidine blue O, and examined with light microscopy. Scale bar = 50 µm. Fig. 6. Longitudinal section of Pinus strobus root colonized by Pisolithus tinctorius showing dichomtomy of apex, a thin mantle (M), and Hartig net hyphae (arrowheads) interfaced with epidermal and cortical cells. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 7. Higher magnification of Hartig net region from material similar to that in Fig. 5 showing Hartig net hyphae (arrowheads) interfaced with epidermal (E) cells. M, mantle. Scale bar = 50 µm. Fig. 8. Inner mantle (M) and branched Hartig net hyphae (arrowheads) in a Betula alleghaniensis – Laccaria bicolor mycorrhiza. Material prepared as in Fig. 5. Bar = 50 µm. Fig. 9. Diagram illustrating the interface between fungus and root cells in ectomycorrhizas. The root cell wall (CW) abuts on the cell wall of branched inner mantle (arrowhead) and branched Hartig net (double arrowheads) hyphae. Nutrient exchange would also involve the plasma membrane of root cells (large arrows) and the plasma membrane of fungal hyphae (small arrows). Scale bar = 50 µm. Fig. 10. The interface region of some ectomycorrhizas may involve wall ingrowths (arrowheads) in root cells, which increases the surface area for nutrient exchange. Scale bar = 50 µm.
that in ectomycorrhizas formed between Corylus avellana and Tuber magnatum, there were no qualitative changes in the chemical nature of cortical cell walls of colonized roots compared with uncolonized roots. However, there was an apparent swelling of some cortical cell walls and the appearance of electron-lucent areas in these walls in the region of the Hartig net. They concluded that their observations support the premise that ectomycorrhizal fungi secrete hydrolytic enzymes to facilitate penetration of hyphae between root cells (Cairney and Burke 1994). What needs to be determined is whether modifications to host cell walls that are contiguous with Hartig net and inner mantle hyphae result in increased permeability to inorganic ions and organic molecules compared with cell walls not adjacent to fungal hyphae. Modifications of Hartig net fungal walls have been demonstrated using the Gomori–Swift cytochemical method for cystein-containing proteins (Paris et al. 1993). In these hyphal walls, only one poorly reactive layer was present, compared with walls of mantle hyphae, in which both a highly reactive and a poorly reactive layer were present. These authors suggest that the simpler organization of the Hartig net hyphal walls may enhance nutrient exchange, but no physiological evidence for this was provided. In ectomycorrhizas formed between Eucalyptus globulus and Pisolithus tinctorius, a group of symbiosis-related acidic polypeptides are enhanced during the colonization process (Burgess et al. 1995), and these have been immunolocalized to the hyphal cell wall, including the walls of mantle and Hartig net hyphae (Laurent et al. 1999; Tagu et al. 2000, 2002). Since symbiosis-related acidic polypeptides are present in hyphal walls at all stages of mycorrhiza development, their significance in enhancing nutrient exchange is uncertain. One characteristic of host cell walls in the Hartig net region is the presence of acid invertases that convert sucrose to fructose and glucose in the apoplastic compartment; these sugars can then be taken up by Hartig net hyphae (Salzer and Hager 1993; Nehls et al. 2000, 2001b). There has been and continues to be considerable interest in determining the involvement of the host and fungal plasma membranes in nutrient exchange in ectomycorrhizas. Earlier research demonstrated cytochemically that acid phosphatase (Dexheimer et al. 1986) and H+-ATPase activity (Lei and Dexheimer 1988) are localized along the plasma membranes of both symbionts in the Hartig net region, indicating that a bidirectional, active transport mechanism is likely involved in nutrient exchange.
Although the movement of P from Hartig net hyphae into the apoplastic interface is likely driven by a concentration gradient (Bücking and Heyser 2000), the involvement of P transporters in the fungal plasma membrane in active transport has not been determined (Chalot et al. 2002). Likewise, the existence of P transporters in host-cell plasma membranes for P uptake from the apoplastic compartment in the exchange interface zone has not been documented (Chalot et al. 2002). Although the movement of carbon compounds in the form of sucrose is usually from root cells to the apoplastic compartment, where, as noted above, acid invertase converts sucrose to glucose and fructose that can be taken up by the fungal hyphae, there is no direct evidence that hexose transporters are involved. However, Nehls et al. (2001a) have shown that, in Amanita–Populus ectomycorrhizas, expression of the gene encoding a monosaccharide transporter (AmMst1) was enhanced six-fold in Hartig net hyphae compared with mantle hyphae. Ectomycorrhizal fungal hyphae do possess various transporters, including Pi transporters and hexose transporters (see Chalot et al. 2002); it is important now to show their involvement in the nutrient-exchange interface. In gymnosperm and those angiosperm ectomycorrhizas in which most of the cortical cells are interfaced with Hartig net hyphae, there is a question as to whether these cells maintain plasmodesmata connections subsequent to the formation of the Hartig net. In two systems, one a gymnosperm (Nylund 1980) and the other an angiosperm (Melville et al. 1988), cortical cells do retain plasmodesmata, potentially providing a symplastic continuity from the phloem to all layers of cortical cells. Research is needed, however, to determine the extent of symplastic continuity from the phloem to the epidermis in ectomycorrhizas. Ectendomycorrhizas There is some debate as to whether ectendomycorrhizas should be placed in a separate category, or considered either as a modified ectomycorrhiza (Egger and Fortin 1988) or a fungal morphotype (Brundrett 2004). For this discussion, however, a separate category is maintained to emphasize the fact that there appears to be only a few species of ascomycete fungi that colonize mostly Pinus spp. and Larix spp. roots to form a unique structural relationship (Yu et al. 2001). Most of the structural work on ectendomycorrhizas has involved pine species associated with one member of the Pezizales, Wilcoxina mikolae; hence the full range of struc© 2004 NRC Canada
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tural characteristics has yet to be determined. In those ectendomycorrhizas studied, a mantle and Hartig net forms as in ectomycorrhizas, but in addition, intracellular hyphal complexes develop within epidermal and cortical cells (Figs. 11, 12). As a result, three potential sites for nutrient exchange are formed: the inner mantle – epidermal cell interface, the Hartig net epidermal – cortical cell interface, and the intracellular hyphal complex – root-cell cytoplasm. Hartig net hyphae are branched, a perifungal membrane develops
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around the intracellular hyphal complex (Fig. 13), and root cells remain alive, as judged by the presence of a nucleus and other organelles (Scales and Peterson 1991). When sections are viewed with transmission electron microscopy an interfacial matrix is apparent between the perifungal membrane and the hyphal wall; however, its composition has not been determined (Scales and Peterson 1991). There has been no experimental work to determine if there is an exchange of nutrients between the symbionts at any interface in this my© 2004 NRC Canada
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Figs. 11–13. Interfaces in ectendomycorrhizas. Fig. 11. Longitudinal section of Pinus banksiana root colonized by Wilcoxina mikolae var. mikolae. A thin mantle (arrows), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowhead) are present. Material processed as in Fig. 5. Scale bar = 50 µm. Fig. 12. Higher magnification of portion of root shown in Fig. 11 showing Hartig net hyphae (arrowheads) and intracellular hyphal complexes (double arrowheads) surrounding cortical cell nuclei (N). Scale bar = 50 µm. Fig. 13. Diagram showing the interface between Hartig net hyphae (HN) and the intracellular hyphal complex (*) with root cells. The latter interface involves the perifungal membrane (arrowheads), interfacial material (double arrowheads), fungal cell wall (small arrows), and the fungal plasma membrane (large arrow). P, plasmodesmata between cells. Scale bar = 50 µm. Figs. 14–16. Interface in ericoid and epacrid mycorrhizas. Fig. 14. An intracellular hyphal complex within an epidermal cell of a hair root of Kalmia angustifolia. Material cleared, stained with acid fuchsin, and examined by differential interference contrast microscopy. Scale bar = 25 µm. Fig. 15. Epidermal cells of Gaultheria procumbens with intracellular hyphal complexes. Narrow hyphae (arrowheads) join hyphal complexes (*) of adjacent epidermal cells. Material cleared, stained with acid fuchsin, and examined with scanning laser confocal microscopy. Scale bar = 25 µm. Fig. 16. Diagram of intracellular hyphal complexes in epidermal cells of typical ericoid and epacrid mycorrhizas. The outer tangential wall (*) of epidermal cells is thickened. The interface between the epidermal cell cytoplasm and fungus is similar to that illustrated in Fig. 13, with perifungal membrane (arrowheads) and interfacial matrix material (double arrowheads) separating the fungus from the epidermal cell cytoplasm. Scale bar = 25 µm.
corrhiza category, and nothing is known about the molecular aspects of root colonization and the development of the nutrient-exchange interface. Ericoid mycorrhizas There is considerable specificity shown in the formation of ericoid mycorrhizas in that few fungal species, primarily ascomycetes, form symbioses with members of the families Ericaceae and Epacridaceae (Smith and Read 1997). Although relatively few plant species in the Ericaceae and Epacridaceae have been investigated intensively in terms of their mycorrhizas, the colonization process is very similar among those examined, regardless of the particular fungal species involved (Perotto et al. 1995). Fungal hyphae contact the thickened epidermal cell walls of the very fine roots (hair roots), penetrate through these walls, and form intracellular hyphal complexes within epidermal cells (Figs. 14– 16). Although, in most instances, each epidermal cell is colonized by a separate “unit”, we have observed some exceptions to this in that fine hyphae can pass from epidermal cell to epidermal cell (Fig. 15). In any event, the intracellular hyphal coil is separated from the host cytoplasm by a perifungal membrane and interfacial matrix material (BonfanteFasolo and Gianinazzi-Pearson 1979; Bonfante-Fasolo and Perotto 1988; Perotto et al. 1995). This is shown in Fig. 16. The interfacial matrix differs in composition from that in AM in that immunolabelling shows the presence of only rhamnogalacturonan as a wall constituent and a low level of labelling for the enzyme polygalacturonase (Perotto et al. 1995). As in other mycorrhizas, an apoplastic compartment is present between the symbionts, and bidirectional transport would have to occur across this interface (Fig. 16). Little research has been conducted on this interface, although ATPase activity has been localized on the perifungal membrane (Gianinazzi-Pearson et al. 1984). There is considerable scope in extending observations made with ericoid mycorrhizas to mycorrhizas formed with epacrid plant species, since there is limited structural work with this mycorrhiza category (Cairney and Ashford 2002). Arbutoid mycorrhizas Arbutoid mycorrhizas occur in a group of species belonging to the Ericales (Smith and Read 1997). Structurally, they resemble ectendomycorrhizas in that a mantle, Hartig net,
and intracellular hyphal complexes form (Fusconi and Bonfante-Fasolo 1984; Münzenberger et al. 1992; Massicotte et al. 1993), but they differ in that the intracellular hyphal complexes are confined to the epidermis (Figs. 17, 19). In Arbutus menziesii, the outer row of cortical cells develops suberin lamellae in their walls; these may limit the development of the Hartig net to the epidermis (Massicotte et al. 1993). The intracellular hyphal complexes in Arbutus unedo mycorrhizas are surrounded by epidermal-cell plasma membrane and an interfacial matrix material (Fusconi and Bonfante-Fasolo 1984; Münzenberger et al. 1992; Filippi et al. 1995). As in ectendomycorrhizas, there are three possible sites of nutrient exchange: the interface between inner mantle hyphae and the tangential wall of epidermal cells, the interface between Hartig net hyphae and epidermal cells, and the interface between hyphal complexes and epidermal cell cytoplasm (Figs. 18, 19). Although Münzenberger et al. (1992) have shown that vesicles containing osmiophilic inclusions fuse with the membrane surrounding intracellular hyphae, experimental evidence is lacking as to the transfer of nutrients between symbionts. Monotropoid mycorrhizas Monotropoid mycorrhizas occur in several genera belonging to the subfamily Monotropoideae (family Ericaceae). All plant hosts in this category are myco-heterotrophic and, therefore, depend on adjacent autotrophic plant species for their source of carbon (Leake 1994). Plants within the Monotropoideae form close associations with specific fungal symbionts (Bidartondo and Bruns 2001, 2002; Young et al. 2002). These fungi form hyphal links between the roots of heterotrophic plants and the roots of autotrophic plants, through which carbon compounds can be transported. Currently, there is no evidence that the fungus is receiving nutrients from the heterotrophic host, therefore, according to Brundrett (2002, 2004), this would be an example of an exploitive mycorrhiza. Structural features of monotropoid mycorrhizas are unique (Figs. 20–22). A mantle and paraepidermal Hartig net form but, in addition, fungal hyphae penetrate epidermal cells to form “fungal pegs”, one per epidermal cell (Figs. 20, 21). In Monotropa species, fungal pegs originate from inner mantle hyphae that penetrate the outer tangential wall of epidermal cells (Lutz and Sjolund 1973; Duddridge and Read © 2004 NRC Canada
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1982; Snetselaar and Whitney 1990). Details of this are shown in Figs. 20 and 22. In Pterospora andromedea and Sarcodes sanguinea, however, fungal pegs have their origin from Hartig net hyphae that penetrate radial walls of epidermal cells (Robertson and Robertson 1982; Massicotte et al. 2004; and see Fig. 21). Each fungal peg becomes encased in a complex of finger-like projections of wall material surrounded by a membrane (Fig. 22). This interface consists of fungal plasma membrane, fungal wall, a plant-derived wall, and a plant-derived membrane (Fig. 22). The amplification of wall and surrounding plasma membrane surface area is similar to what occurs in transfer cells that are found where
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there is rapid flux of materials (Gunning and Pate 1969). However, there is no experimental evidence for bidirectional transfer or, indeed, unidirectional transfer of nutrients at this interface. A complicating feature of the “fungal peg” is that the plant and fungal wall at the tip apparently break down, resulting in a membranous sac extending into the epidermal cell (Duddridge and Read 1982; Robertson and Robertson 1982; Dexheimer and Gérard 1993). Although it has been suggested that nutrients might be transferred to the epidermal cell at this point (Francke 1934), there is no evidence for this. As pointed out by Robertson and Robertson (1982), © 2004 NRC Canada
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Figs. 17–19. Interfaces in arbutoid mycorrhizas. Fig. 17. Longitudinal section of Arbutus menziesii – Pisolithus tinctorius mycorrhiza. A thick mantle (M), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowheads) are present. Material embedded in LR White resin, stained with acriflavine HCl, and viewed with blue light using epifluorescence microscopy. Scale bar = 100 µm. Fig. 18. Portion of an Arbutus menziesii – Piloderma bicolor mycorrhiza showing a thin mantle (M), paraepidermal Hartig net hyphae (arrowheads), and sectioned intracellular hyphal complexes (double arrowheads) within epidermal cells. Material embedded in LR White resin, stained with toluidine blue O, and viewed with light microscopy. Scale bar = 25 µm. Fig. 19. Diagram of interfaces. Mantle (M) and paraepidermal Hartig net (HN) hyphae contact the wall of epidermal cells. Intracellular hyphae are separated from the epidermal cell cytoplasm by a perifungal membrane (arrowheads) and interfacial matrix material (double arrowheads). Scale bar = 25 µm. Figs. 20–22. Interfaces in monotropoid mycorrhizas. Fig. 20. Mantle (M), paraepidermal Hartig net (arrow), and fungal pegs (arrowheads) in portion of Monotropa uniflora root. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 21. Portion of a Pterospora andromedea root showing mantle (M), paraepidermal Hartig net (arrows), and fungal peg (arrowhead). Material prepared as in Fig. 5. Scale bar = 25 µm. Fig. 22. Diagram showing the interfaces in monotropoid mycorrhizas. Mantle (M) and Hartig net (arrows) hyphae contact epidermal cell walls. In addition, hyphal pegs (*) are enveloped by epidermal-cell-derived wall material (arrowheads) and plasma membrane (double arrowheads). Scale bar = 25 µm.
the contents of this sac do not resemble those of the fungal peg. The function of this structure remains enigmatic. Other interfaces that are potentially involved in bidirectional transfer of nutrients include inner mantle and Hartig net hyphae that are contiguous with epidermal cells, but, as yet, this has not been shown. Orchid mycorrhizas All orchid species have minute seeds that usually lack an endosperm and have limited storage of reserves in the undifferentiated embryo (Rasmussen 1995). Subsequent development of the embryo into a protocorm is dependent on the colonization of the germinating seed by soilborne fungi that are able to convert complex carbon compounds in the substrate into simple sugars, some of which are transported to the developing protocorm (Peterson et al. 1998). There may be some specificity in the fungal species that associate with particular orchid species at the seed germination stage. For example, the successful colonization of germinating seeds of Neottia nidus-avis in nature is dependent on a specific Sebacina-like fungus (McKendrick et al. 2002). To sustain growth, the developing protocorm and seedling receive carbon compounds via fungal hyphae (McKendrick et al. 2000; Alexander and Hadley 1985; Smith and Read 1997), but there is no evidence that the fungal symbiont receives nutrients from the plant in exchange. At this early stage of orchid plant development, therefore, this is an example of an exploitive association (Brundrett 2002, 2004). Both autotrophic and myco-heterotrophic species occur in the family, Orchidaceae. Roots of autotrophic orchids become colonized by fungi, and these mainly supply the host with mineral nutrients such as nitrogen and P (Alexander et al. 1984; Smith and Read 1997), although there is some evidence that plants may also receive some carbon compounds via the fungal hyphae (Smith and Read 1997). Myco-heterotrophic species are associated with fungi that provide hyphal links to neighbouring autotrophic plant species, through which they obtain photosynthates (Leake 1994). There appears to be some specificity in fungi involved with particular myco-heterotrophic orchid species. For example, Cephalanthera austinae is associated with mycobionts belonging to the Thelephoraceae (Taylor and Bruns 1997). Regardless of whether fungi associate with developing
protocorms, roots of autotrophic species, or roots of mycoheterotrophic species, the colonization process is similar in that hyphae within parenchyma cells of protocorms (Fig. 23) and roots (Fig. 24) form hyphal complexes called pelotons (Fig. 25). These are surrounded by a plant-derived membrane and an interfacial matrix (Fig. 26). These plant cell – fungus interfaces are assumed to be the sites of nutrient exchange in most orchid mycorrhizas. This mode of nutrient exchange has been termed tolypophagy (Burgeff 1909; Rasmussen 2002). Pelotons undergo degradation (Fig. 23) and, subsequent to this, cells containing the remains of the peloton can be recolonized. The interfacial matrix changes in composition, depending on the age of the peloton (Peterson et al. 1996). Early in the colonization process, the interfacial matrix lacks pectins, cellulose, and β-1,3-glucans, as determined by using various affinity probes; these wall components are, however, present following peloton senescence, and these encase the degenerating hyphae (Peterson et al. 1996). There is some evidence suggesting that the perifungal membrane has different physiological characteristics from that of the peripheral plasma membrane. For example, in protocorms of Spiranthes sinensis colonized by Ceratobasidium cornigerum, the perifungal membrane failed to react for adenylate cyclase activity when cytochemical methods were used, whereas the peripheral plasma membrane did (Uetake and Ishizaka 1995); the significance of this in terms of the functioning of the interface is not clear. Also, certain ATPases are active on the perifungal membrane but not on the peripheral plasma membrane (Serrigny and Dexheimer 1985), and neutral and acid phosphatases have been localized on the perifungal membrane (Dexheimer et al. 1988). As with AMs, the plant cytoskeleton undergoes considerable reorganization in that both microtubules and actin filaments become closely associated with developing pelotons (Uetake et al. 1997; Uetake and Peterson 1997, 1998). The role of these components of the cytoskeleton in establishing the nutrient-exchange interface is unknown. In some orchid species, there appears to be a unique mode of nutrient acquisition termed ptyophagy, first described by Burgeff (1909) and more recently by Wang et al. (1997), in the orchid Gastrodia elata. In this type, degradation of hyphae within regions of roots containing “digestive cells” results in the release of nutrients that are then presumably © 2004 NRC Canada
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Figs. 23–26. Interface in orchid mycorrhizas. Fig. 23. Section of Goodyera repens protocorm colonized by Ceratobasidium cereale. Sections of intracellular complexes (pelotons) (arrowheads), as well as collapsed pelotons (double arrowheads), are present. Material prepared as in Fig. 17. Scale bar = 100 µm. Fig. 24. Transverse section of Cypripedium arietinum root showing sections of pelotons (arrowheads) and collapsed pelotons (double arrowheads). Material prepared as in Fig. 5. Photo courtesy of Carla Zelmer. Scale bar = 100 µm. Fig. 25. Peloton in root of Paphiopedilum sanderianum. Material was embedded in LR White resin, stained with sulfarhodamine, and viewed with scanning laser confocal microscopy. Photo courtesy of Carla Zelmer. Scale bar = 50 µm. Fig. 26. Diagram of peloton showing the interface between fungus and root cell. The fungus is separated from the root cytoplasm by a perifungal membrane (arrowheads), and interfacial matrix material (double arrowheads). Scale bar = 50 µm.
taken up by intact pelotons in adjacent cells. As pointed out by Rasmussen (2002), this appears to be a novel mycorrhiza type and deserves further study. Nonvascular plant mycorrhizas The Bryophytes consist of three divisions: Hepatophyta (liverworts), Anthocerophyta (hornworts), and Bryophyta (mosses). For reasons unknown, only the mosses have failed to establish associations with symbiotic fungi (Read et al. 2000). The majority of experimental and structural work has involved liverworts, perhaps because of the diversity in morphology of the dominant gametophyte phase and the fact that fungi involved in the symbioses include members of the three groups of mycorrhizal fungi (Read et al. 2000). It is beyond the scope of this review to consider in detail the research published on bryophytes, but examples are given to illustrate the variation in symbiotic associations and possible nutrient-exchange interfaces that develop. In associations with AM fungi, the thalli of the genus Pellia develop typical arbuscules (Read et al. 2000).
Schüßler (2000), using two isolates of Glomus claroideum, has also demonstrated arbuscule formation in the thallus of the hornwort Anthoceros punctatus. Collections of the primitive liverwort Haplomitrium showed a symbiotic association that resembled an AM association, but hyphae were restricted to the outermost layer of cells in the organs colonized (Carafa et al. 2003). In the latter example, fine hyphae resembled arbuscular branches in that they were surrounded by a periarbuscular membrane and interfacial matrix material, but collapsed fungal swellings were very unusual in that they were of wide diameter and were encased by very thick interfacial matrix material containing crystalline deposits. As indicated by Read et al. (2000), the functional aspect of these associations has yet to be explored. The rhizoids of some liverworts associate with Hymenoscyphus ericae, an ascomycete that forms ericoid mycorrhizas with some members of the Ericaceae (Duckett et al. 1991; Duckett and Read 1995; Read et al. 2000). Rhizoid tips become swollen as they are colonized by fungal hyphae and peg-like wall deposits occur around the penetrating © 2004 NRC Canada
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hyphae (Duckett et al. 1991). These authors compare this to the fungal pegs in monotropoid mycorrhizas, noting the absence of the elaborated branched wall structure, and conclude that this site is where nutrient exchange occurs. In a combined structural and molecular study, Kottke et al. (2003) examined the fungal associations of species in 12 families of leafy liverworts and found considerable variability in fungal associates. They concluded that both ascomycete and basidiomycete hyphae could become surrounded by host-derived wall material and agreed with Read et al. (2000) that these peg-like structures are probable sites of nutrient exchange. Kottke et al. (2003) have proposed the term “jungermannioid mycorrhiza” for symbiotic associations between members in one clade of leafy liverworts, the Jungermanniidae, and either basidiomycete or ascomycete fungal symbionts. Interestingly, it has been demonstrated that some leafy liverworts can form symbiotic associations with basidiomycete fungi that can also form mycorrhizas with adjacent tree species (Read et al. 2000). These develop a typical mantle and Hartig net in the tree roots but coiled hyphal complexes in the liverwort. This suggests that the same fungus can form divergent nutrient-exchange interfaces depending on the host and can potentially establish fungal bridges between vascular and nonvascular plants. Future work with liverworts should include a more indepth study of the nutrient-exchange interface between the symbionts.
Discussion As suggested by Brundrett (2004), any attempt to redefine and recategorize mycorrhizas must be based on understanding the variation that occurs in these associations. With respect to the symbionts in question, this is relevant at various levels, including the development of the interface that is established at the cellular level. From the literature and from our own observations on the development and functioning of interfaces between symbionts (Peterson et al. 2004), it is clear that most information available is for AMs and ectomycorrhizas. This reflects the fact that most plant species develop either one or the other of these mycorrhizas (Smith and Read 1997). It is also evident that, regardless of the mycorrhiza category, the interface between symbionts includes an apoplastic compartment and opposing plasma membranes of the symbionts (Smith and Smith 1990; Dexheimer and Pargney 1991). With the exception of ectomycorrhizas, in which the exchange interface is external to plant cell walls, interfaces of all other mycorrhizas involve the formation of an intracellular apoplastic compartment (Smith and Smith 1990), the chemical nature of which has only been studied for a few mycorrhiza categories. The evidence for transfer of nutrients is mostly indirect, relying on structural features of the interface and membrane properties of the symbionts forming this interface (Smith and Smith 1990). Characterization of the membranes in terms of activation of various transporters and other features needs to be extended beyond the studies that have primarily involved AMs (Harrison 1999a, 1999b; Saito 2000) and ectomycorrhizas (Chalot et al. 2002). More direct approaches, such as microautoradiography, as used by Bücking
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and Heyser (2001, 2003) in their study of transfer of labelled P and carbon compounds in ectomycorrhizas, could be used to confirm nutrient transfer through the interface of other mycorrhizas. Although it has been suggested that the interfaces between “balanced” mycorrhizas and “exploitive” mycorrhizas are likely to differ, with lysis of fungal hyphae in exploitive mycorrhizas being more important in the release of nutrients (Brundrett 2004), further research such as that by Imhof (2003) on a range of mycorrhizal associations could verify this. Considerable interest exists in exploring the interface characteristics of symbiotic associations of nonvascular plants, particularly the liverworts, because of the diversity that is being discovered in this primitive group of plants (Read et al. 2000; Carafa et al. 2003; Kottke et al. 2003). These systems may provide valuable clues with respect to the evolution of mycorrhizal symbioses (Kottke et al. 2003). We suggest that the present categories of mycorrhizas be retained for symbiotic associations with vascular plants but that consideration be given to erecting one or more categories for symbiotic associations with nonvascular plants. Kottke et al. (2003) have initiated this discussion. We also suggest that there is scope for further structural studies of mycorrhizal categories that have received limited attention to date.
Acknowledgements We are indebted to Lewis Melville for the drawings, Linda Tackaberry and two anonymous reviewers for valuable comments on the manuscript, and the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support.
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