Arbuscular mycorrhizal (AM) fungi form a root endosymbiosis with many agronomically important crop species. They enhance the ability of their host to obtain nutrients from the soil and increase the tolerance to biotic and abiotic stressors. However, AM fungal species can differ in the benefits they provide to their host plants. Here, we examined the putative molecular mechanisms involved in the regulation of the physiological response of Medicago truncatula to colonization by Rhizophagus irregularis or Glomus aggregatum , which have previously been characterized as high- and low-benefit AM fungal species, respectively. Colonization with R. irregularis led to greater growth and nutrient uptake than colonization with G. aggregatum . These benefits were linked to an elevated expression in the roots of strigolactone biosynthesis genes ( NSP1 , NSP2 , CCD7 , and MAX1a ), mycorrhiza-induced phosphate ( PT8 ), ammonium ( AMT2;3 ), and nitrate ( NPF4.12 ) transporters and the putative ammonium transporter NIP1;5 . R. irregularis also stimulated the expression of photosynthesis-related genes in the shoot and the upregulation of the sugar transporters SWEET1.2 , SWEET3.3 , and SWEET 12 and the lipid biosynthesis gene RAM2 in the roots. In contrast, G. aggregatum induced the expression of biotic stress defense response genes in the shoots, and several genes associated with abiotic stress in the roots. This suggests that either the host perceives colonization by G. aggregatum as pathogen attack or that G. aggregatum can prime host defense responses. Our findings highlight molecular mechanisms that host plants may use to regulate their association with high- and low-benefit arbuscular mycorrhizal symbionts.

Legumes form tripartite interactions with arbuscular mycorrhizal fungi and rhizobia, and both root symbionts exchange nutrients against carbon from their host. The carbon costs of these interactions are substantial, but our current understanding of how the host controls its carbon allocation to individual root symbionts is limited. We examined nutrient uptake and carbon allocation in tripartite interactions of Medicago truncatula under different nutrient supply conditions, and when the fungal partner had access to nitrogen, and followed the gene expression of several plant transporters of the Sucrose Uptake Transporter (SUT) and Sugars Will Eventually be Exported Transporter (SWEET) family. Tripartite interactions led to synergistic growth responses and stimulated the phosphate and nitrogen uptake of the plant. Plant nutrient demand but also fungal access to nutrients played an important role for the carbon transport to different root symbionts, and the plant allocated more carbon to rhizobia under nitrogen demand, but more carbon to the fungal partner when nitrogen was available. These changes in carbon allocation were consistent with changes in the SUT and SWEET expression. Our study provides important insights into how the host plant controls its carbon allocation under different nutrient supply conditions and changes its carbon allocation to different root symbionts to maximize its symbiotic benefits.

Common mycorrhizal networks (CMNs) of arbuscular mycorrhizal (AM) fungi in the soil simultaneously provide multiple host plants with nutrients, but the mechanisms by which the nutrient transport to individual host plants within one CMN is controlled are unknown. Using radioactive and stable isotopes, we followed the transport of phosphorus (P) and nitrogen (N) in the CMNs of two fungal species to plants that differed in their carbon (C) source strength, and correlated the transport to the expression of mycorrhiza-inducible plant P (MtPt4) and ammonium (1723.m00046) transporters in mycorrhizal roots. AM fungi discriminated between host plants that shared a CMN and preferentially allocated nutrients to high-quality (nonshaded) hosts. However, the fungus also supplied low-quality (shaded) hosts with nutrients and maintained a high colonization rate in these plants. Fungal P transport was correlated to the expression of MtPt4. The expression of the putative ammonium transporter 1723.m00046 was dependent on the fungal nutrient supply and was induced when the CMN had access to N. Biological market theory has emerged as a tool with which the strategic investment of competing partners in trading networks can be studied. Our work demonstrates how fungal partners are able to retain bargaining power, despite being obligately dependent on their hosts.

The arbuscular mycorrhizal (AM) symbiosis, which forms between plant hosts and ubiquitous soil fungi of the phylum Glomeromycota, plays a key role for the nutrient uptake of the majority of land plants, including many economically important crop species. AM fungi take up nutrients from the soil and exchange them for photosynthetically fixed carbon from the host. While our understanding of the exact mechanisms controlling carbon and nutrient exchange is still limited, we recently demonstrated that (i) carbon acts as an important trigger for fungal N uptake and transport, (ii) the fungus changes its strategy in response to an exogenous supply of carbon, and that (iii) both plants and fungi reciprocally reward resources to those partners providing more benefit. Here, we summarize recent research findings and discuss the implications of these results for fungal and plant control of resource exchange in the AM symbiosis.

The arbuscular mycorrhizal (AM) symbiosis, formed between the majority of land plants and ubiquitous soil fungi of the phylum Glomeromycota, is responsible for massive nutrient transfer and global carbon sequestration. AM fungi take up nutrients from the soil and exchange them against photosynthetically fixed carbon (C) from the host. Recent studies have demonstrated that reciprocal reward strategies by plant and fungal partners guarantee a “fair trade” of phosphorus against C between partners [Kiers ET, et al. (2011) Science 333:880–882], but whether a similar reward mechanism also controls nitrogen (N) flux in the AM symbiosis is not known. Using mycorrhizal root organ cultures, we manipulated the C supply to the host and fungus and followed the uptake and transport of N sources in the AM symbiosis, the enzymatic activities of arginase and urease, and fungal gene expression in the extraradical and intraradical mycelium. We found that the C supply of the host plant triggers the uptake and transport of N in the symbiosis, and that the increase in N transport is orchestrated by changes in fungal gene expression. N transport in the symbiosis is stimulated only when the C is delivered by the host across the mycorrhizal interface, not when C is supplied directly to the fungal extraradical mycelium in the form of acetate. These findings support the importance of C flux from the root to the fungus as a key trigger for N uptake and transport and provide insight into the N transport regulation in the AM symbiosis.

* Here, nitrogen (N) uptake and metabolism, and related gene expression, were analyzed in germinating spores of Glomus intraradices to examine the mechanisms and the regulation of N handling during presymbiotic growth. * The uptake and incorporation of organic and inorganic N sources into free amino acids were analyzed using stable and radioactive isotope labeling followed by high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid scintillation counting and the fungal gene expression was measured by quantitative polymerase chain reaction (Q-PCR). * Quiescent spores store Asp, Ala and Arg and can use these internal N resources during germination. Although not required for presymbiotic growth, exogenous N can also be utilized for the de novo biosynthesis of amino acids. Ammonium and urea are more rapidly assimilated than nitrate and amino acids. Root exudates do not stimulate the uptake and utilization of exogenous ammonium, but the expression of genes encoding a putative glutamate dehydrogenase (GDH), a urease accessory protein (UAP) and an ornithine aminotransferase (OAT) were stimulated by root exudates. The transcript levels of an ammonium transporter (AMT) and a glutamine synthetase (GS) were not affected. * Germinating spores can make effective use of different N sources and the ability to synthesize amino acids does not limit presymbiotic growth of arbuscular mycorrhizal (AM) spores.

* Root exudates play a key role during the presymbiotic growth phase and have been shown to stimulate hyphal branching and the catabolic metabolism of arbuscular mycorrhizal (AM) fungal spores. * Here, the effect of root exudates on presymbiotic growth, uptake of exogenous carbon and transcript levels for genes putatively involved in the carbon metabolism of germinating spores were determined. * Crude root exudates led to a slight acceleration of spore germination, increased germ tube branching and stimulated uptake and catabolic metabolism of acetate, and to a greater extent of glucose, but had no effect on gene expression. By contrast, partially purified root exudates increased the transcript levels of acyl-CoA dehydrogenase (ss-oxidation of fatty acids to acetyl-CoA), malate synthase (glyoxylate cycle) and glutamine-fructose-6-phosphate aminotransferase (chitin biosynthesis), but did not differ from crude root exudates in their effect on substrate uptake and respiration. The expression of glycogen synthase (glycogen biosynthesis), glucose-6-phosphate dehydrogenase (pentose phosphate pathway) and neutral trehalase (hydrolysis of trehalose) were only marginally or not affected by root exudates. * Root exudates have an effect on both membrane activity and gene expression and the results are discussed in relation to the catabolic and anabolic metabolism of spores during presymbiotic growth.

Arbuscular mycorrhizal (AM) fungi colonize the roots of the majority of crop plants, forming a symbiosis that potentially enhances nutrient uptake, pest resistance, water relations, and soil aggregation. Inoculation with effective isolates of AM fungi is one way of ensuring the potential benefits of the symbiosis for plant production. Although inocula are available commercially, on-farm production of AM fungus inoculum would save farmers the associated processing and shipping costs. In addition, farmers could produce locally adapted isolates and generate a taxonomically diverse inoculum. On-farm inoculum production methods entail increasing inoculated isolates or indigenous AM fungi in fumigated or unfumigated field soil, respectively, or transplanting pre-colonized host plants into compost-based substrates. Subsequent delivery of the inoculum with seed to the planting hole in the field presents technological barriers that make these methods more viable in labor-intensive small farms. However, a readily available method for utilization of these inocula is mixing them into potting media for growth of vegetable seedlings for transplant to the field. Direct application of these inocula to the field and transplant of seedlings precolonized by these inocula have resulted in enhanced crop growth and yield. Key words: AM fungi, sustainable agriculture, biofertilizer

Indirect root for nitrogen The discovery of a previously unknown mechanism of nitrogen transfer from the arbuscular mycorrhizal fungi found on the roots of most land plants, to the host plants suggests that this symbiotic relationship may be a much more important factor in the global nitrogen cycle than was thought. The mechanism involves uptake of inorganic nitrogen by the fungus outside the roots, conversion to amino acids within the fungus, then transfer as ammonium ions from the fungal mycelium into the plant. The first event in host recognition by arbuscular mycorrhizal fungi is thought to be hyphal branching. A strigolactone, 5-deoxy-strigol, isolated from Lotus japonicus has now been identified as an inducer of branching. Strigolactones are root metabolites, previously isolated as seed germination stimulants for root parasitic weeds. This finding highlights the close relationship between plant and fungus, and may provide a new strategy for the control of both beneficial fungal symbionts and destructive parasitic weeds in agriculture and natural ecosystems.

Arbuscular mycorrhizal [AM] fungi are potentially important tools in sustainable agriculture due to their roles in crop nutrient uptake, disease resistance, and water relations and in stabilizing soil aggregates. Inocula of these fungi can be effectively produced on-farm in mixtures of compost and vermiculite with a suitable plant host, such as bahiagrass (Paspalum notatum Flugge). Success of this method, however, depends upon utilizing the optimal compost and vermiculite mixture ratio. Experiments were conducted over two years utilizing a complete factorial design with three composts, four mixture ratios, and three AM fungi with the objective of producing regression equations to predict optimal mixture ratios using routine measures of compost nutrient analyses as independent variables. Growth of colonized P. notatum in yard clippings and dairy manure + leaf composts; which were high in N, low in P, with moderate K levels; produced more spores of AM fungi at mixture ratios of 1:2 to 1:4 [v/v compost: vermiculite] relative to higher dilutions. Dilution ratios of 1:19 and 1:49 were best for controlled microbial compost, which was high in P, low in N, and moderately high in K. Simple equations were developed which predict the optimal fraction of compost in the mixture for each of the three AM fungi studied (Glomus intraradices, Glomus mosseae, and Gigaspora rosea). Percent N, P, and K and N:P ratio were the significant independent variables. These equations allow a farmer to choose a mixture ratio for the on-farm propagation of AM fungi knowing only the nutrient analysis of the compost to be used.

Nitrogen (N) is known to be transferred from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis, yet its metabolism, storage and transport are poorly understood. In vitro mycorrhizas of Glomus intra-radices and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes. (15)N- and/or (13)C-labeled substrates were supplied to either the fungal compartment or to separate dishes containing uncolonized roots. The levels and labeling of free amino acids (AAs) in the extra-radical mycelium (ERM) in mycorrhizal roots and in uncolonized roots were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Arginine (Arg) was the predominant free AA in the ERM, and almost all Arg molecules became labeled within 3 wk of supplying (15)NH(4) (+) to the fungal compartment. Labeling in Arg represented > 90% of the total (15)N in the free AAs of the ERM. [Guanido-2-(15)N]Arg taken up by the ERM and transported to the intra-radical mycelium (IRM) gave rise to (15)N-labeled AAs. [U-(13)C]Arg added to the fungal compartment did not produce any (13)C labeling of other AAs in the mycorrhizal root. Arg is the major form of N synthesized and stored in the ERM and transported to the IRM. However, NH(4) (+) is the most likely form of N transferred to host cells following its generation from Arg breakdown.

Carbon transfer from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis has been reported, but its significance and even its existence have been called into question and the issue remains controversial. We investigated carbon movement from fungus to plant and from one mycorrhizal root system to another via a common AM fungal network in monoxenic cultures to avoid limitations of some previous studies.C-13 and C-14 labeled substrates were supplied to functioning in vitro AM mycorrhizas between Ri T-DNA transformed carrot (Daucus carota) roots and Glomus intraradices to follow carbon movement into and between host and fungal metabolite pools.Fungal triacylglycerol and trehalose were labeled when permeant substrates were supplied to the extraradical mycelium (ERM), but host-specific compounds in the roots did not become labeled. When labeled glucose was provided to a donor root system, label moved to recipient roots via a common AM fungal network but remained in fungal compounds.We conclude that carbon flow in the AM symbiosis is normally unidirectional from plant to fungus and that while carbon is translocated by the fungus from one metabolically active root system to another, it remains within the intraradical mycelium (IRM).

Methods for the simultaneous measurement of vacuolar and cytoplasmic pH in plant tissues currently have significant limitations. This study demonstrates the usefulness of methyl difluoro alanine (F(2)ALA) and methyl trifluoro alanine (F(3)ALA) with in-vivo F-19 NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the chemical shift of F(2)ALA and F(3)ALA is greater than either the commonly used P-31 NMR signal of inorganic phosphate or the C-13 NMR signals of trans-aconitic acid, which is also found in some plant cells. F(2)ALA and F(3)ALA were also able to detect changes over a greater range of pH. When maize root tissue was incubated in the presence of 0.35 mm F(2)ALA or F(3)ALA, these accumulated to significant concentrations in two compartments of different pH with no significant effect on growth rate of root tips. The time course of accumulation and the pH of the two compartments were consistent with one being the cytoplasm and the other the vacuole. The chemical shift of both C-2 of trans-aconitic acid and vacuolar F(3)ALA indicated that the mean vacuolar pH of maize root cells was 4.6 and that the pH gradient across the tonoplast membrane was about 2.8 units. Under a variety of conditions, there was considerable heterogeneity in the pH of the vacuoles in maize root tissue as indicated by the peak width of the signal from F(3)ALA. The significance of these values is discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after 13C1 glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using 13C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.

(13)C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored (13)C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the first study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius.

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.

Arbuscular mycorrhizal (AM) fungi are multinucleate, coenocytic, obligate symbionts with no known sexual stages and very wide host and habitat ranges. While contributing vitally to the growth of land plants they face unique challenges in metabolism, transport, growth and development. To provide clues to the strategies that AM fungi have adopted, random sequencing of cDNA's from Glomus intraradices was undertaken. Putative genes for enzymes, transporters, structural proteins and cell-cycle regulatory factors were discovered. Among the EST's of particular interest are sequences with homology to known trehalase, arsenite transporter, cysteine synthase, tubulins, actin, dynein, cell cycle regulatory proteins, and three meiosis-related proteins. The significance of these sequences is discussed in the context of what is known about AM metabolism, transport, growth and phylogeny.

Arbuscular mycorrhizal (AM) fungi take up nitrogen from the soil, translocate it and transfer it to the host plant (Jennings, 1995; Smith & Read, 1997), often in significant quantities. Depending on N availability and mobility this can represent an important benefit to the plant, and thus the mechanisms involved are a matter of considerable interest (Ames et al., 1983; Martin & Botton, 1993; Johansen et al., 1996; Smith & Read, 1997; Botton & Chalot, 1999). We know the forms of N taken up by different types of mycorrhizal fungi, and something of the fungal enzymes involved in assimilation, but almost nothing of the translocation and transfer to the roots of host plants. As a result there are few integrated models (Martin et al., 1986) of the movement of N through mycorrhizas – a situation that contrasts with a well developed picture of the flow of phosphorus (Ashford et al., 1994; Smith & Read, 1997; Rasmussen et al., 2000) and the emerging models for the flow of carbon (Hampp & Schaffer, 1999; Bago et al., 2000). Here we propose a simple model for N flow from soil to host via the fungus that involves the urea cycle and the translocation of arginine from extraradical to intraradical mycelium and then to the host plant. The mechanisms involved in N movement in mycorrhizas must transfer N efficiently from heterotroph to autotroph with minimal net transfer of C. For AM fungi in particular, the possible transport mechanisms are further constrained by the necessity for vectorial translocation along coenocytic (nonseptated) hyphae. The model postulated here could meet these demands and is consistent with previous observations of N metabolism in the AM symbiosis (Fig. 1). A proposed model by which the urea cycle together with arginine translocation could act to move nitrogen in the arbuscular mycorrhizal symbiosis. The flow of N is indicated in red, of phosphorus in blue, and of carbon in black; membrane transporters/channels and enzymes are shown in green. Individual steps in the figure labeled a–g refer to the same steps discussed in the text. N acquired from the soil (a) is metabolized to Gln in the extraradical hyphae, then to Arg by the anabolic arm of the urea cycle (b). The Arg is imported into vacuoles by means of a specific Arg tonoplast carrier (c). The cationic Arg binds to polyanionic polyphosphates within fungal vacuoles (d), and these move to the intraradical fungal mycelium by cytoplasmic streaming or a peristaltic tubular vacuolar system. Once within the root, Arg and P are released into the fungal cytoplasm (e). The N is further metabolized through the catabolic arm of the urea cycle producing urea and Orn, and finally ammonium which is then transferred to the host root (f). Resulting C skeletons in the intraradical fungus are re-incorporated into fungal C pools (g). Both nitrate (Tobar et al., 1994; Bago et al., 1996) and ammonium (Ames et al., 1983; Johansen et al., 1992, 1996; Frey & Schüepp, 1993) can be taken up and used by AM fungi. There is no direct evidence for mobilization and uptake of organic N (Smith & Read, 1997; Hodge et al., 2000), though dilution of exogenously supplied inorganic 15N label has been interpreted as suggesting it (Ames et al., 1984; Cliquet et al., 1997). Accordingly only NH4+ and NO3− are shown as being taken up in step (a) of Fig. 1. Nitrate reductase (NR) activity has been found in germinating AM spores (Ho & Trappe, 1975) and in mycorrhizal root extracts (Subramanian & Charest, 1998), and high levels of a transcript with close homology to fungal NR genes were found in arbuscules of the AM fungus Glomus intraradices (Kaldorf et al., 1998). Several lines of evidence (e.g. the inhibition of 15N labelling from ammonium by albizzin, an inhibitor of glutamate synthase) indicate that the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is responsble for ammonium assimilation in AM extraradical hyphae (Johansen et al., 1996). However, the involvement of glutamate dehydrogenase (GDH) has not been experimentally excluded. Indeed the primary amination step in filamentous fungi is often catalysed by NADPH-dependent GDH, with synthesis of glutamate (Glu) (Pateman & Kinghorn, 1975), although, depending on the plant–fungal system considered, GS/GOGAT may also play a role (Smith & Read, 1997; Botton & Chalot, 1999). The model, therefore, shows nitrate and ammonium being assimilated into glutamine (Gln), without specifying enzymes other than nitrate reductase. There is no direct evidence as to the chemical form in which N is transferred along AM fungal hyphae to the plant, although the finding of nitrate reductase (NR) activity in arbuscules led to the suggestion that at least some of the N is transferred as nitrate (no assay for NR activity was reported in this study for extraradical hyphae; Kaldorf et al., 1998). We think that amino acids, and specifically Arg, are more likely to be transferred because of the active assimilation of ammonium and nitrate into amino acids by AM roots (Johansen et al., 1996) and the observation of Arg at substantial levels in AM fungi (Bago et al., 1999). Thus, for example, HPLC analyses showed that Arg is the dominant free amino acid in external hyphae of the AM fungus G.claroideum (Johansen et al., 1996). The usual pathway of Arg synthesis appears to be active in G. intraradices (Bago et al., 1999), with synthesis of ornithine (Orn) from Glu, and of carbamoyl P with fixation of CO2; and conversion of these two compounds to Arg (Pateman & Kinghorn, 1975; Davis, 1986; Jennings, 1995). Also in favour of Arg as a candidate for translocation is the requirement for a substantial flow of cations from soil to AM roots to accompany the polyanionic polyphosphates (polyP) translocated by AM fungi. It is tempting to suggest that Arg acts in part as this counterion in the AM symbiosis and that anionic polyP is a counterion for the N, as has been suggested for other mycorrhizal symbioses (Smith & Read, 1997; Bücking & Heyser, 1999). This should not be taken to mean that the model requires a tight coupling or exact stoichiometry, as the amount of N transferred compared with P is very variable and other counterions are probably involved to different extents at different times. Finally, variable amounts of protein have been reported to be associated with polyP in ectomycorrhizal species (Kottke et al., 1995) especially under conditions of high external N (Wallenda & Kottke, 1998), and this may play a role in storage and/or detoxification of excess N. No clear role for this polyP-associated protein in translocation has yet emerged, nor has such a protein been reported in AM fungi, and it is accordingly omitted from the present model. The fungal vacuole contains most of the amino acids that are basic, while acidic amino acids are predominantly cytoplasmic (Pateman & Kinghorn, 1975; Jennings, 1995). For instance, 90% of Orn, Lys, His and Arg is located in the fungal vacuole; only 10% is cytoplasmic (Messenguy et al., 1980). In the case of Arg and Orn, concentrations in the vacuole reach at least 50 times those in the cytoplasm, though it has been reported that a 1000-fold concentration difference is possible (Jennings, 1995). A fungal tonoplast carrier for Arg has been identified (Paek & Weiss, 1989). The vacuolar accumulation of Arg in fungi has generally been seen as a storage mechanism and if the external medium is depleted of Arg the vacuolar fraction acts as an N reserve (Davis, 1986). The proposed translocation of vacuolar Arg may be in addition to a storage role. There are other considerations that make the vacuole an attractive candidate for N translocation. First, the other main translocation flows in AM fungi, P from extraradical to intraradical hyphae and C in the opposite direction, appear to be mediated by the movement of membrane-bound or at least aggregate bodies. Thus, polyPs in AM fungi are mainly large (Solaiman et al., 1999) and found in vacuoles (Rasmussen et al., 2000); and triacylglycerides in lipid bodies seem to be the main form of C moving along hyphae (Bago et al., 1999; Pfeffer et al., 1999; B. Bago et al., unpublished). Indeed in a coenocytic network of fine hyphae that extends many centimeters from the root, simple diffusion of soluble cytosolic metabolites is probably insufficient for efficient bulk transport (even with cytoplasmic streaming). In agreement with this, labelling studies show that trehalose in the intraradical and extraradical mycelia have different metabolic origins and there appears to be little or no transfer of this soluble (presumably cytosolic) carbohydrate by translocation (Pfeffer et al., 1999; Bago et al., 2000). Also, there is good evidence that N, and particularly Arg, is associated with vacuolar polyP in fungi (Dürr et al., 1979; Jennings, 1995), including ectomycorrhizal fungi (Martin et al., 1985; Bücking et al., 1998). A related possibility for compartmentation arises from the finding that long distance translocation of solutes in the mycelium of several filamentous fungi occurs by means of a tubular vacuolar system (Ashford et al., 1994, Smith & Read, 1997). Such a system interconnects vacuoles and other cytoplasmic cisternae, moves by peristaltic pulses independent of the cytoplasmic streaming, and seems to transfer material between different hyphal compartments (Shepherd et al., 1993a,b). Therefore, Fig. 1 shows vacuolar compartmentation without specifying whether vacuolar N moves by translocation of the vacuoles themselves or via a tubular vacuolar network. If N is translocated in vacuoles in association with polyP, then the breakdown of polyP in the intraradical hyphae, before release of Pi to the host, would tend simultaneously to release N to the fungal cytoplasm in the intraradical locations most suitable for transfer. Accordingly, Fig. 1 shows release of N from vacuoles associated with the breakdown of polyP. If N is transferred from the fungus to the host in an organic form such as amino acids, substantial amounts of C must move with it. Since the question of whether meaningful transfer of C occurs from fungus to host is not settled (Watkins et al., 1996; Graves et al., 1997; Fitter et al., 1998), it may be that organic N is in fact transferred. Indeed the transfer of Gln has been postulated in the ectomycorrhizal symbiosis (Martin et al., 1986; Jennings, 1995; Botton & Chalot, 1999). If organic N is transferred, a molecule with high N : C ratio, such as Gln, or still better Arg, which would be more efficient in this respect, might be involved. An amino acid permease has recently been identified in an ectomycorrhizal fungus (Nehls et al., 1999) and this or other such transporters might mediate the efflux of organic N at the AM symbiotic interface. However, recent findings indicate that fungal C does not move beyond the mycorrhizal root into host shoots (Fitter et al., 1998) (although this may not hold for the special case of achlorophyllous phycobionts). It follows that any C transferred as organic N would need to be returned very efficiently to the fungus or sequestered within the root without entering other host metabolic pools. We believe that the transfer of inorganic N, which would require the breakdown of organic N by the fungus before transfer, would be a less elaborate mechanism and would also be more metabolically efficient for both host and fungus. The usual Arg catabolic pathway in fungi involves, successively, the hydrolysis of Arg to urea and Orn (via arginase), the breakdown of urea to ammonia and CO2 (via urease) and the conversion of Orn to Glu-γ-semialdehyde (via ornithine-oxoacid transaminase) and then to Glu (Pateman & Kinghorn, 1975; Davis, 1986; Jennings, 1995). Arginase genes have been identified in yeast, Neurospora and Aspergillus (Sumrada & Cooper, 1984; van Huffel et al., 1994; Pan et al., 1995; Marathe et al., 1998). Urease is also found in fungi (Yu et al., 1997; Cox et al., 2000), and the observation that N supplied as 15N-urea to the extraradical mycelium results in labelling of host and fungal amino acids indicates the existence of urease activity in G. intraradices (Shachar-Hill et al., 1997; D. B. Rolin et al., unpublished). Moreover, Orn has been detected as a significant amino acid pool of Glomus mycorrhizal roots (Johansen et al., 1996). These findings are all compatible with the idea that the catabolic arm of the urea cycle, together with urease, could operate in the intraradical mycelium to release ammonium within the root. The presence of ammonium transport proteins in the symbiosome membrane of an N-fixing symbiosis (Tyerman et al., 1995; Kaiser et al., 1998) suggests that in that situation at least, N transfer is in the form of NH4+. The breakdown of Arg (or indeed of Gln via GDH or GOGAT, for example) releasing ammonium could, therefore, allow transfer of N while allowing the fungus to retain the fixed C. The C skeletons released in the breakdown of organic N would probably be in the form of organic acids or amino acids of lower N content that could re-enter fungal metabolism in a number of ways. Pathways include entry into the TCA cycle and thence to respiration, and/or incorporation into lipids or proteins, and/or return as organic acids to the extraradical mycelium. However, there appears to be little flux through gluconeogenesis in the intraradical mycelium (Pfeffer et al., 1999; Bago et al., 1999). No matter the route for re-use of C, the fungus would retain the benefit of this fixed C and the model does not specify the fate of the organic C released from Arg breakdown. The model proposed here is speculative, but we believe it is plausible in the light of what is known of N movement in the AM symbiosis and related systems. The value of any such model is in making testable predictions and highlighting areas of ignorance. This model requires the activity of various enzymes and transporters at specific places in the arbuscular mycorrhiza, in particular, finding an arginine transporter in the vacuolar membrane as well as arginase and urease within the intraradical mycelium would provide support for the hypothesis. Apart from enzyme and transport assays, there are a number of pulse-chase experiments that could test the model, particularly ones to assess the rates of turnover of C and N in different positions of the Arg molecule. Thus, if Arg were used by the fungus only as a storage molecule it would not turn over rapidly. Animals use the urea cycle to translocate nitrogen from source to sink without losing excessive quantities of carbon. Might it be that a similar mechanism, with a similar overall purpose, operates in AM fungi using the same metabolic steps? The evidence is far from complete, but we hope that this proposal will inspire further studies. We thank C. Azcón-Aguilar, A. Fitter and F. Martin for critically reading the manuscript and for their expert opinions on the subject of this article.

SummaryNuclear magnetic resonance (NMR) studies of mycorrhizal symbioses have illuminated a number of functional aspects of these complex associations. Here we review studies of the two main types of mycorrhiza (ectomycorrhizas and arbuscular mycorrhizas) to which NMR has been applied. Although the physiological questions addressed in each case are frequently the same, these two mutualistic symbioses are sufficiently different to justify separate discussion. In conjunction with isotopic labelling NMR is able to examine the transfer of substrates between the symbionts both in vivo and in vitro, as well as the production of secondary metabolites in response to colonization. In addition, this methodology is capable of determining the locations of the biosynthesis and translocations of storage compounds, such as polyphosphates, lipids and carbohydrates, in mycorrhizal fungi both in the free‐living and in the symbiotic stages of their life cycle. NMR has been useful in analysing metabolism, transport and energetics, and the results of such studies have practical and ecological significance. Models of transport and physiology to which NMR has contributed form the necessary foundation for functional genomic exploration.