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Keywords:

  • Ammonium/methylamine uptake;
  • Kinetic experiment;
  • Mycorrhiza;
  • Paxillus involutus

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

Using [14C]methylamine as an analogue of ammonium, the kinetics and the energetics of NH4+ transport were studied in the ectomycorrhizal fungus, Paxillus involutus (Batsch) Fr. The apparent half-saturation constant (Km) and the maximum uptake rate (Vmax) for the carrier-mediated transport derived from the Eadie-Hofstee transformation were 180 μM and 380 nmol (mg dry wt)−1 min−1, respectively. Both pH dependence and inhibition by protonophores indicate that methylamine transport in P. involutus was dependent on the electrochemical H+ gradient. Both long-term and short-term uptake experiments were consistent with regulation of ammonium/methylamine transport processes by the presence of an organic nitrogen source. Analysis of methylamine uptake by different P. involutus isolates revealed no obvious trend in the uptake capacities in relation to N deposition at the collection site. Kinetic parameters were determined in P. involutus/Betula pendula (Roth.) axenic association and in detached mycorrhizal roots isolated from forest sites. Enhanced methylamine uptake in the presence of the fungal symbiont was demonstrated. Homogeneous Vmax values were found for axenic and detached mycorrhizas, whereas Km values showed greater variations.


Abbreviations
CCCP

carbonyl-cyanide-m-chlorophenylhydrazone

DEPC

diethylpyrocarbonate

DNP

2,4-dinitrophenol

MSX

methionine sulfoximine

SHAM

salicylhydroxamic acid

1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

Ammonium is a major source of mineral nitrogen in forest soils [1] and the competition for this nutrient is intense. Plants have therefore developed strategies to increase their capacity for N mobilisation. One strategy is the symbiotic association with mycorrhizal fungi which is considered to have been responsible for the colonisation of land by the plant [2,3]. Numerous studies have demonstrated that the ectomycorrhizal fungal partner plays an integral role in ammonium metabolism in trees [4–7]. Although the process of ammonium uptake is often considered to be the rate-limiting step in its acquisition [8], the process has received relatively little attention. Previous investigations have supported the hypothesis that ammonia is transported as a small, uncharged and lipophilic compound across the plasma membrane, a process which does not require specific transporters [9]. However, the rates of diffusion do not seem to be sufficient to account for the requirements for plant growth [7]. In contradiction to this hypothesis, recent experiments with cyanobacteria, higher plants, yeasts and seaweeds [10–14] support the conclusion that specific systems for NH4+ transport exist.

Ammonium uptake in higher plants has been described as a combination of (i) a saturating component (HATS, high affinity transport system) operating in the low concentration range and (ii) a linear component (LATS, low affinity transport system) which operates at high NH4+ concentrations [15]. However, single saturable systems have been observed in wheat [16]. The HATS and to a lesser extent the LATS operate in an energy-dependent manner and are probably driven by the membrane potential. These kinetic studies, however, did not establish whether ammonium transport is mediated by a single transporter or whether it results from the activity of multiple transport proteins. Using the heterologous complementation of a yeast mutant deficient in the NH4+ transporter, the first plasma membrane permease for ammonium has been cloned in Arabidopsis thaliana[12]. Other genes encoding NH4+ transport proteins have also been cloned in Saccharomyces cerevisiae[13,17] and Synechocystis sp. [18].

The aims of the present study were (1) to define the kinetics and energetics of ammonium transport in the ectomycorrhizal fungus Paxillus involutus and ectomycorrhizas and (2) to study the regulation of ammonium transport when the fungus was grown under various nitrogen regimes. Ammonium uptake was estimated by using its analogue tracer methylamine ([14C]CH3NH3+), as previously described [10–14].

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

2.1Organisms and growth conditions

The ectomycorrhizal fungus was a strain of P. involutus (Batsch) Fr. (ATCC 200175), which was originally isolated from fruit bodies associated with 15–30-year-old Betula pendula (Roth.) trees growing on coal waste (8 kg ha−1 of anthropogenic N deposition) in Midlothian, Scotland, UK. Two other isolates, originating from sporocarp collection, were used: Pi 03, isolated from fruit bodies associated with a 100-year-old Pinus sylvestris forest in Münskogstjärn, Northern Sweden (1 kg ha−1 of anthropogenic N deposition), and Pi 04, isolated from a 75–125-year-old Picea abies-dominated forest (10 kg ha−1 of anthropogenic N deposition) near Billingen, Southern Sweden [19]. Isolates were grown on cellophane-covered agar medium containing modified Melin-Norkrans medium (MMN) from which malt extract was omitted. The MMN medium [20] contained 10 g glucose l−1 and (mg l−1): KH2PO4 (500), (NH4)2HPO4 (250), CaCl2 (50), NaCl (25), MgSO4·7H2O (150), thiamine hydrochloride (0.1), FeCl3·6H2O (1).

For axenic production of mycorrhizas, birch B. pendula seeds were surface-sterilised with 3.5% (w/v) calcium hypochlorite for 30 min, rinsed in several changes of sterilised distilled water, germinated aseptically on water agar for 1 week and placed on top of 10-day-old colonies of P. involutus growing on cellophane-covered agar medium [21] in large round Petri dishes (150×15 mm). Plates were incubated in a growth chamber under an 18-h photoperiod (150 μmol m−2 s−1 PAR), with a temperature and a relative humidity of 22/16°C and 85/65% (day/night), respectively.

For experiments with detached mycorrhizas, roots were collected in the ‘Forêt de Haye’ during the vegetative period under beech and hornbeam trees. The forest is a beech grove located 10 km west of Nancy (France) at an elevation of 390 m, dominated by Fagus sylvatica and Carpinus betulus species. The geological formation is a limestone tableland and the horizon from which mycorrhizal roots were collected (7–12 cm) is a mesomull-type organic horizon of silty-clayed texture [22]. The pH in the upper 10 cm of humus is 5.0. Sample preparation was performed as described by Kielland [23] and Wallenda and Read [24]. For uptake experiments, only the most common morphotypes, Lactarius subdulcis and Amanita muscaria, determined according to the classification of Agerer [25], were used.

2.2Uptake experiments with [14C]methylamine

In the standard assay, fungal discs were cut from the actively growing edge of 10-day-old colonies using a 25 mm diameter cork borer and incubated for 120 min in a solution containing 1 ml nitrogen-free MMN medium (pH 5.5) at 25°C supplemented with [14C]CH3NH3+ at 0.34 μM (54 mCi mmol−1). For plant material, about 10 mg dry wt of root (10–20 mycorrhizal or non-mycorrhizal roots) was incubated in the same conditions. At the end of the uptake period, the biological material was washed for 2 min with 0.1 mM CaSO4 and freeze-dried prior to analysis. When needed, the incubation time was varied between 30 and 300 min, the total methylamine concentration between 0.0024 and 20 mM and the pH between 4 and 8.

In experiments using metabolic inhibitors, discs were pre-incubated for 5 min with the inhibitor prepared in a nitrogen-free MMN medium (pH 5.5) before transfer to a solution containing labelled methylamine, as described above. The inhibitor concentrations were: arsenate 1 mM, salicylhydroxamic acid (SHAM) 1 mM, KCN 1 mM, NaF 1 mM, diethylpyrocarbonate (DEPC) 1 mM; cycloheximide 0.01 mM, carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) 0.01 mM, DNP 0.01 mM, NaN3 0.1 mM, rotenone 0.1 mM, gramicidin, 0.001 mM and methionine sulfoximine (MSX) 0.05 mM.

In double labelling experiments, fungal discs were incubated with both 0.34 μM [14C]CH3NH3+ and 0.34 μM [3H]leucine (150 Ci mmol−1). In the detector windows, 0–250 and 400–600 nm, emission spectra for [3H]leucine and [14C]CH3NH3+ respectively, did not overlap, which allowed simultaneous detection of the two isotopes. Control uptake experiments were performed with either [14C]CH3NH3+ or [3H]leucine alone. For the determination of radioactivity, biological tissues were solubilised with Soluene 350 (Packard, USA) overnight at 60°C, mixed with 3 ml scintillation solution (Ionic Fluor) and the radioactivity in the tissues measured by liquid scintillation spectroscopy.

2.3Uptake experiments with ammonium

Ammonium uptake by the fungus grown in pure culture was determined by measuring the depletion of NH4+ from the medium containing 3 mM of NH4Cl and various glutamate concentrations (0.1, 0.5 and 1 mM) in shaken condition (50 rpm), by capillary electrophoresis according to Prima Putra et al. [26].

2.4[15N]Ammonium uptake and translocation by intact mycorrhizal systems

Ectomycorrhizal associations were synthesised between B. pendula seedlings and P. involutus (ATCC 200175) according to Ek et al. [27] and transferred to Perspex microcosm systems after 6 weeks. Two infected birch seedlings were grown in each chamber and the mycelium was allowed to extend from the roots and to colonise the peat substrate for 2 months. After this period, seedlings on one side of the chambers were carefully lifted out to disconnect the root systems from the external mycelium and put back. Five ml of 15NH4Cl (99 atom % excess) was immediately added and evenly distributed on the peat surface at a concentration of 200 μg N ml−1 and the chambers were further incubated for 72 h. In this system, both the fungal mycelium and the plant roots had access to the 15N. Six replicate chambers were incubated, with plants growing horizontally, in growth cabinets with 200 μmol m−2 s−1 PAR, 80% relative humidity and a 18/6-h and 18°C/16°C day/night cycle. 15N analyses were performed on an ANCA-MS (Europa Scientific Ltd) at the Swedish University of Agricultural Sciences (Umeå, Sweden).

2.5Expression of results

The inhibition data are expressed as percentage of the control. All statistical analyses were performed using STATVIEW (version 4.02) for Macintosh.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

3.1[15N]Ammonium uptake and translocation by intact mycorrhizal systems

When the uptake of [15N]ammonium by intact or disrupted ectomycorrhizal birch systems was compared in Perspex microcosm boxes, it was found that the uptake of ammonium was significantly increased when birch seedlings were connected to the intact external mycelium of P. involutus (Fig. 1). Higher concentrations of 15N were found in both shoot and root parts of intact mycorrhizal systems when compared with disrupted systems. These results confirmed previous findings that the external mycelium generally increases nutrient uptake [27] and that it can therefore be considered a nutrient channel [2].

image

Figure 1. [15N]Ammonium content in roots and leaves of intact (grey bars) or disrupted (white bars) birch/P. involutus systems. After 6 weeks of growth in microcosms, birch seedlings on one side of the chambers were carefully lifted out to disconnect the root systems from the external mycelium (disrupted) and put back. The right side seedling was left intact. Five ml of 15NH4Cl (99 atom % excess) was immediately added and evenly distributed on the peat surface at a concentration of 200 μg N ml−1 and the chambers were further incubated for 72 h. Values are means±S.E.M. (n=6). The effect of disruption was tested by one-way ANOVA. Significant differences are indicated by *P<0.05.

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3.2Uptake of methylamine versus uptake of ammonium

Accumulation of [14C]CH3NH3+ occurred at a linear rate (r2=0.96) during the 5-h incubation period (Fig. 2). Addition of 0.05 mM or 0.5 mM NH4+ after 1 h of incubation reduced [14C]CH3NH3+ accumulation by 76% and 98%, respectively, when measured at the end of the incubation period (Fig. 2). A Dixon plot of the inhibition of methylamine uptake by ammonium showed that inhibition was competitive (Fig. 3). Addition of KCl instead of NH4Cl did not inhibit uptake of methylamine (data not shown). The similarity of Km values for methylamine uptake (0.18 mM) by P. involutus isolate 01 (Table 1) and the Ki value for the inhibition of methylamine uptake by ammonium (0.16 mM, Fig. 3) indicates that methylamine can be used as an analogue to study ammonium uptake, as showm in previous studies [10–13,23]. Methylamine provides two potential advantages over NH4+: it is not metabolised by the cells [28], avoiding interference between metabolism and transport in uptake experiments, and uptake experiments can be carried out over short periods during which efflux is considered to be negligible.

image

Figure 2. Inhibition of methylamine uptake by ammonium in P. involutus. Uptake of [14C]methylamine was performed in a standard assay (•) or in the presence of 0.05 (□) or 0.5 (◯) mM NH4Cl added after 1 h of incubation. Values are means±S.E.M. (n=4). The effect of ammonium addition was tested by one-way ANOVA. Significant differences are indicated by *P<0.05, **P<0.001.

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image

Figure 3. Dixon plots of the inhibition of methylamine uptake by NH4Cl. [14C]Methylamine concentrations were 0.0024 (•), 0.0096 (■) and 0.192 (◯) mM. NH4Cl was added at concentrations of 0.05 and 0.5 mM or omitted. Reciprocal methylamine uptake rates were plotted against competitor concentrations.

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Table 1.  Kinetic parameters Vmax [pmol (mg dry wt)−1 min−1] and Km (μM) for uptake of methylamine or ammoniuma
  1. aValues were estimated from the Eadie-Hofstee plots; the kinetic parameters derived from the Lineweaver-Burk plots are given in parentheses. The Km and Vmax values for P. involutus were obtained on 6–8 different occasions. Results are the mean±S.E.M. A. cycadae: Anabaena cycadae, A. aquae: Anabaena flos-aquae.

OrganismKmVmaxSubstrateReferences
P. involutus 01154±24 (180±10)380±11 (293±9)MApresent work
P. involutus 03110±16 (180±10)450±20 (360±12)MApresent work
P. involutus 04140±40 (143±22)390±30 (320±23)MApresent work
L. bicolor6363NH4Cl[7]
L. rufus35425NH4Cl[7]
L. hepaticus55799NH4Cl[7]
L. laccata250ndNH4Cl[7]
A. cycadae10ndMA[11]
S. cerevisiae1–10ndMA[13]
A. thaliana65ndMA[12]
A. aquae7740MA[10]
nd: not determined. MA: [14C]methylamine.

3.3Effect of pH on uptake of methylamine and ammonium

Uptake assays performed at pH values in the range 4–8 indicated an optimum pH for ammonium and methylamine uptake between 4 and 5.5. When the pH was raised from 5.5 to 8, the uptake of ammonium and methylamine was decreased by 30%, indicating strong sensitivity of ammonium and methylamine to the protonation state. This is in good agreement with results from Ek et al. [27], which demonstrated that high pH had a pronounced negative effect on the mycelial growth of P. involutus. However, these results contrast with those found in cyanobacteria [11] and yeasts [12], where the optimum pH for uptake varied between 8 and 11 and 6 and 8, respectively. The pKa for NH4+ (9.25) predicts an increase of NH3 from less than 0.1% of total (NH3+NH4+) at pH 6 to 30% at pH 8 [15]. The uptake systems of ectomycorrhizal fungi therefore seem to be adapted to the acidic pH of forest soil solution where formation of the protonated form of ammonium is favoured. Indeed, the uptake of 15N at pH 5.9 by non-mycorrhizal birch in microcosm systems was much lower than that by P. involutus mycorrhizal birch or pine [29].

3.4Kinetics of uptake

The rate of methylamine uptake by P. involutus increased with increasing methylamine concentration (in the concentration range 0.0024–20 mM), but no saturation was observed (Fig. 4a). The Eadie-Hofstee transformation (v against v/S) showed biphasic kinetics (Fig. 4b) indicating the existence of two components in the uptake of methylamine by P. involutus. At the highest concentrations, the uptake rate was directly proportional to the external methylamine concentration. In order to differentiate between simple diffusion through the lipid bilayer and carrier-mediated transport, uptake assays were performed in the presence of 10 μM of the protonophore CCCP. This compound depletes the proton-motive force by increasing H+ influx and thereby induces acidification of the cytosol [30]. Linear rates of transport as a function of methylamine concentration were observed in the presence of CCCP (Fig. 4a), indicating methylamine diffusion. Similar results were found for methylamine transport in cyanobacteria [10,11] and higher plants [12]. When this diffusive component was subtracted from the uptake occurring in the absence of CCCP for each methylamine concentration, a saturable mediated uptake was obtained, which conformed to simple Michaelis-Menten kinetics, (Fig. 4a) and was consistent with carrier-mediated transport. In the ammonium concentration range usually found in forest soil solutions (10–100 μM) [1], the uptake system occurring at the highest concentrations is likely to be negligible. In subsequent kinetic experiments, only the saturable mediated uptake was considered.

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Figure 4. Concentration-dependent uptake of methylamine in the absence or presence of CCCP by P. involutus. Uptake of [14C]methylamine from 0.0024–20 mM solutions was assayed alone or in the presence of 10 μM CCCP under the standard conditions. a: Uptake occurring in the presence of CCCP (□) was subtracted from uptake under non-inhibition conditions (•) for each methylamine concentration, to give the mediated transport (◯). b: Eadie-Hofstee plot of the concentration dependence of the uptake without CCCP. c: Lineweaver-Burk plot without CCCP (inset).

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Values for Michaelis-Menten parameters (Vmax, maximum uptake rate) and (Km, apparent half-saturation constant), for the mediated uptake of methylamine by P. involutus 01 estimated from the Eadie-Hofstee transformation (Fig. 4b), are given in Table 1. Values obtained from Lineweaver-Burk transformation (Fig. 4c) are included for comparative purposes. Similar Vmax and Km values were obtained with two other isolates of P. involutus, originating from forest soils with various N deposition levels. It seems therefore that there was no relationship between methylamine uptake capacity and the amount of deposited anthropogenic N at the collection site, in agreement with experiments of Wallander et al. [19]. However, given the small number of isolates, large populations from each site would need to be studied before such relationships could be identified. Similarly, no clear relationship was found between methylamine uptake of Betula nana and Salix pulchra mycorrhizal roots and the nitrogen concentration of the soil [23]. Wallenda and Read [24] also concluded that the availability of organic nitrogen sources was not correlated with the kinetic parameters measured on mycorrhizas from sampling sites along the European north/south gradient. Thus, the soil N concentration is not necessarily of primary selective value for the N uptake capacities of mycorrhizal roots.

The Vmax values found in the present study are comparable to those obtained with the ectomycorrhizal fungi Lactarius rufus and Laccaria bicolor[7], cultivated in the presence of ammonium (Table 1). Km values show greater differences between different ectomycorrhizal fungi and other organisms. These discrepancies might reflect interspecific variations in the uptake capacities between different ectomycorrhizal strains. However, differences in growth conditions, which in turn may influence the nitrogen status of the fungal tissue, may account for much of the variation between ectomycorrhizal fungi. Properties of plasma membrane transport may be closely associated with the ecological role of fungi and determine, at least in part, the competitive ability of a fungus in a specific habitat [8,31].

3.5Effect of metabolic inhibitors

In order to determine the energetic nature of methylamine transport, various metabolic inhibitors were used (Table 2). The protonophores CCCP and DNP strongly inhibited methylamine uptake, and NaN3, which reduces the internal pH by 1 or 2 units, gave moderate inhibition. These results indicate that methylamine transport in P. involutus was dependent on the electrochemical H+ gradient, as already demonstrated for amino acid, nitrate, peptide and glucose uptake in higher plants and ectomycorrhizal fungi (for reviews, see [32–34]). However, these results contrast with those found with membrane patches on isolated symbiosomes for which the ammonium transport is carried out by passive channel-mediated conductance [35].

Table 2.  Effect of metabolic inhibitors and ionophores on methylamine uptake
InhibitorConcentration (mM)Inhibition (%)
CCCP0.0198
DNP0.0198
DEPC195.5
NaN30.159
SHAM122
Rotenone0.110
Gramicidin0.0014
KCN19
MSX0.050
NaF10
Discs of fungal inoculum were pre-incubated for 5 min with the inhibitor prepared in a nitrogen-free MMN medium at pH 5.5 and then transferred to a solution containing the inhibitor and methylamine. [14C]Methylamine uptake was measured in standard conditions. Results are expressed in % of control, 100%=375 pmol (mg dry wt)−1 min−1

MSX had no effect on the uptake of methylamine (Table 2) whereas it strongly inhibited amino acid uptake [36]. The histidine-modifying agent DEPC strongly inhibited methylamine uptake, suggesting that a histidine residue may play an important role in the reaction sequence. This result has been observed for sucrose and lactose transport in Beta vulgaris[37] and Escherichia coli[38], respectively. Rotenone, SHAM and KCN, respectively inhibitors of complex I, alternative pathway and complex IV of the mitochondrial respiratory chain, did not affect methylamine uptake, nor did the mitochondrial ATPase inhibitor, arsenate, and glycolysis inhibitor, NaF, which indicates that the transport system was not directly under metabolic control. Gramicidin, a Na+/K+ ionophore, did not affect the transport of methylamine, indicating that these cations did not take part in the uptake.

3.6Regulation of ammonium and methylamine uptake

The uptake of 3 mM ammonium was assayed by depletion of the culture medium in the presence of increasing concentrations of glutamate for several days (Fig. 5). After 1 day, the presence of glutamate increased ammonium uptake 2–3-fold. This effect was reduced as the incubation time was prolonged. In this experiment, the nitrogen sources were metabolised by the fungus and the uptake measurements may therefore result from three physiological processes, uptake, assimilation and storage.

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Figure 5. Effect of glutamate on ammonium uptake by P. involutus. Ammonium (as 3 mM NH4Cl) uptake was measured over a 125-h period by depletion from the medium in the absence (•) or in the presence of 0.1 (■), 0.5 (◯), and 1.0 (□) mM glutamate. Values are means±S.E.M. (n=5).

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Concerning the assimilation component, it has been shown that glutamate increased ammonium assimilation in the ectomycorrhizal fungus L. laccata, glutamine synthetase (GS) being strongly activated [39]. The increased ammonium assimilation could partly explain the enhanced ammonium transport by glutamate in P. involutus.

To determine the importance of the storage component in uptake capacities, fungal colonies were pre-incubated for 3 days in standard liquid medium (MMN) without nitrogen or supplemented with 3 mM glutamate as the sole nitrogen source. Methylamine uptake was measured after this pre-incubation period for various lengths of time and compared to the standard assay, with (NH4)2HPO4 as the nitrogen source. N starvation or glutamate supply increased methylamine transport four and six times, respectively, after 120 min incubation (Fig. 6). It was found previously in L. bicolor that nitrogen starvation induced a dramatic depletion of intracellular glutamine and NH4+[40] and the present results therefore suggest that fungal cells reconstitute their N pools by increasing their uptake capacities. Similarly, N-starved plants usually showed a faster NH4+ net uptake than N-fed plants [41]. When fed with [14C]glutamate in short-term experiments, fungal cells rapidly synthesised glutamine by ammonium incorporation into glutamate through the activity of glutamine synthetase [42]. In the present work, longer-term glutamate feeding probably induced ammonium requirement for glutamine synthesis, which explains the increased methylamine uptake.

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Figure 6. Effect of N starvation or glutamate (supplied as the sole nitrogen source) on methylamine uptake by P. involutus. Fungal discs were incubated for 3 days in the standard MMN medium (•), without nitrogen (◯) or with 3 mM of glutamate (■) and further used for measuring [14C]methylamine uptake in standard assay conditions. Values are means±S.E.M. (n=4). The effect of N starvation or glutamate addition was tested by one-way ANOVA. Significant differences are indicated by *P<0.05, **P<0.001 (similar for both treatments).

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To rule out interference with assimilation and storage processes, [14C]CH3NH3+ and [3H]leucine tracers were used in combination in double-labelling and short-term (150 min) experiments (Fig. 7). [14C]CH3NH3+ was used because it is a non-metabolising tracer and leucine was chosen because it is a relatively inert amino acid [43], which is not involved in the glutamate/glutamine metabolic pathway, being present at less than 1% in the amino acid pool [42]. Methylamine uptake was stimulated 4.4-fold after 90 min incubation in the presence of leucine (Fig. 7a). Methylamine had a limited effect on [3H]leucine transport (Fig. 7b). A number of previous experiments have led to the conclusion that uptake processes for organic and inorganic nitrogen sources operate quite independently [42,44]. However, these studies (and the data in Fig. 7b) concerned the effect of inorganic N sources on the uptake of organic N sources. In the present work, both long-term (using ammonium) (Fig. 5) and short-term (using methylamine) (Fig. 7a) experiments are consistent with the fact that ammonium/methylamine transport processes are highly regulated by the presence of an organic nitrogen source. These considerations are of ecological interest because mixtures of both inorganic and organic nitrogen sources normally occur in the natural substrate [34,45]. Our results contrast with those from S. cerevisiae[43] and wheat [41] in which amino acids significantly reduced the uptake of ammonium.

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Figure 7. Interaction between leucine and methylamine uptake by P. involutus. Fungal discs were incubated with both 0.34 μM [14C]methylamine and 0.34 μM [3H]leucine (150 Ci mmol−1) and simultaneous detection of [14C]methylamine (a, ◯) and [3H]leucine (b, ◯) was performed. Control uptake experiments were performed either with [14C]methylamine alone (a, •) or with [3H]leucine alone (b, •). Values are means±S.E.M. (n=4). The effect of leucine on methylamine uptake (a) and the effect of methylamine on leucine uptake (b) were tested by one-way ANOVA. Significant differences are indicated by *P<0.05, **P<0.001.

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Regulation at the molecular level remains to be clarified. Most information on the regulation of the transport proteins is currently derived from studies with S. cerevisiae[13,17] and higher plants [12]. In S. cerevisiae, three ammonium transporter genes, Mep 1, 2 and 3, have been cloned and two of them (Mep 1 and 2) are subject to nitrogen catabolite repression mediated by two general positive GATA factors [46]. Similar approaches are being developed in our laboratory for ectomycorrhizal fungi.

3.7Methylamine uptake by mycorrhizas

The kinetic parameters (Vmax and Km calculated from Lineweaver-Burk transformations) for methylamine uptake were determined in the concentration range 0.0024–20 mM for non-mycorrhizal and mycorrhizal B. pendula associated with the ectomycorrhizal fungus P. involutus (Table 3). The fungus represented about 45% of the whole root biomass, when the seedlings were harvested for uptake measurements [47]. Methylamine uptake in mycorrhizal plants occurred at a higher rate and with an increased affinity for methylamine supplied in the external medium. Similar results (lower Km and higher Vmax) have been found for nitrate uptake by mycorrhizal roots of Pinus pinaster/Hebeloma cylindrosporum compared to non-mycorrhizal roots [48]. Our findings are in good agreement with those obtained with Eucalyptus globulus/Pisolithus tinctorius and Scleroderma verrucosum[6] as well as conifer/Hebeloma crustuliniforme[49] associations for which enhanced uptake of ammonium in the presence of the fungal symbiont was demonstrated.

Table 3.  Kinetic parameters Vmax [nmol (mg dry wt)−1 min−1] and Km (μM) for uptake of methylamine by axenic and field ectomycorrhizas
  1. aMycorrhizas from field.

  2. bFrom Wallenda and Read [24].

  3. cFrom Kielland [23].

AssociationKmVmax
Non-mycorrhizal roots1600.190
Betula pendula/Paxillus involutus1000.260
Carpinus betulus/Lactarius subdulcisa6780.248
Carpinus betulus/Amanita muscariaa1050.175
Fagus sylvatica/Lactarius subdulcisa8140.223
Fagus sylvatica/L. subdulcisb2200.810
Fagus sylvatica/Russula ochroleucab5550.465
Fagus sylvatica/Xerocomus chrysenteronb4410.680
Betula nana/fungus sp.c  
DH670.093
TT38212.380
ST6631.265
Community abbreviations are: DH, dry heath; TT, tussock tundra; ST, shrub tundra. The Km and Vmax values for field mycorrhizas were obtained on four different occasions.

Uptake of methylamine was also measured with detached mycorrhizal roots isolated under beech and hornbeam from a forest soil near Nancy (France) in mid-spring. Kinetic parameters obtained from Eadie-Hofstee plots are given in Table 3. Preliminary experiments revealed that methylamine uptake was linear for the incubation period used (data not shown). Vmax values for field mycorrhizas were homogeneous and in the same range as those found for axenic mycorrhizas, whereas Km values exhibited greater variation. Previous results with B. nana mycorrhizas have shown variation, depending on the type of soil from which mycorrhizal roots have been collected, for both Km (ratio of 1 to 57) and Vmax (ratio of 1 to 28) values (Table 3). Taken together, these results suggest that methylamine uptake capacities (Vmax) are influenced by the soil characteristics (mainly soil moisture, pH and organic mat thickness), rather than by the type of fungal/host combinations. Indeed, beech/L. subdulcis mycorrhizas collected from two different sampling sites (data from the present work, Table 3, and from Wallenda and Read [24]) showed great variation in both Km and Vmax. However, it is worth noting that variations in Km and Vmax parameters were found for mycorrhizas formed between different fungi and the same host (Table 3), which suggests that the fungal partner may have a role in controlling the uptake of methylamine. Other factors such as the nitrogen status of the fungal tissue and the viability of field collected mycorrhizas are probably more likely to influence uptake.

4Conclusions and perspectives

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

The transport of low-molecular-mass nutrients and metabolites through the plasma membrane has been investigated in less than 1% of the estimated number of fungal species [8]. Transport studies with mycorrhizal fungi may be of particular importance. Indeed, studies of nitrogen metabolism in ectomycorrhizas have demonstrated that the fungal symbiont plays a fully integrated role in plant root metabolism and participates actively in the assimilation and transfer of newly absorbed nitrogenous compounds [5,34].

In the present work the kinetics and energetics of ammonium/methylamine transport in P. involutus have been characterised and it has been shown that complex regulatory processes were involved when the trophic N situation changed. Intriguing questions concerning the number of genes encoding these transporters and their trophic regulation have not yet been answered. A molecular approach, based on the PCR cloning of these transport systems, is currently under development in our laboratory to elucidate these questions. The assumption that the properties of fungal plasma membranes are somehow related to the ecological role of fungi [8] highlights the need for exploring the role, the distribution and the structure of transport proteins in ectomycorrhizal fungi and ectomycorrhizas. The answers to most of these ecologically relevant questions will come from combining physiological, biochemical and molecular methods and techniques. The challenge for the future is to understand the ecological reality of nitrogen acquisition by trees and their symbionts in natural ecosystems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References

We thank the students of the master degree in Plant Physiology (years 1996–1998) at the University H. Poincaré (Nancy) for helping with the collection of mycorrhizal roots and the determination of kinetic constants (data from Table 3).

References

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  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions and perspectives
  7. Acknowledgements
  8. References
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