Kinetics of amino acid uptake by ectomycorrhizal roots

Authors


Thomas Wallenda Fax: 44 114 276 0159; e-mail: thomas.wallenda@altavista.net

Abstract

It is well established that ectomycorrhizal fungi can use amino acids as nitrogen and carbon sources, but data on the kinetic properties of amino acid uptake systems of ectomycorrhizal systems are scarce. Using 14C-labelled compounds we have determined the kinetics of uptake of amino acids by excised ectomycorrhizal roots for a range of distinct mycorrhizal types from three tree species, beech, spruce, and pine. All mycorrhizal types examined took up amino acids via high-affinity transport systems (KM values ranging from 19 to 233 mmol m–3). A comparative analysis of kinetic parameters for uptake of amino acids and the ammonium analogue methylammonium showed that ectomycorrhizal roots have similar or even higher affinities (lower KM values) for the amino acids, indicating that absorption of these organic forms of nitrogen (N) can contribute significantly to total N uptake by ectomycorrhizal plants. Analysis of amino acid uptake by ectomycorrhizal roots collected along a European north/south gradient of increasing mineral N pollution from northern Sweden to south Germany revealed no obvious trend in the uptake capabilities for amino acids by ectomycorrhizal roots in relation to the location of the sampling site on this gradient. Rather, the fungal species forming a particular morphotype was the factor determining uptake kinetics. It can therefore be deduced that the species composition of the fungal community will contribute significantly to the functional diversity of a population of mycorrhizal roots.

INTRODUCTION

Ectomycorrhizal trees dominate boreal and temperate forest ecosystems in which nitrogen (N) is generally accepted to be the most important growth-limiting nutrient (Alexander 1983; Tamm 1991). It has been proposed (Read 1991) that selection has favoured symbiosis between the roots of trees and ectomycorrhizal fungi in these environments because the fungi improve the plants’ access to N. Increasing awareness of this possibility is reflected in a recent shift of emphasis from studies of phosphorus nutrition towards those of N use by ectomycorrhizal plants (see Botton & Chalot 1995 and Smith & Read 1997 for reviews). However, although most ectomycorrhizal root tips are located in the superficial organic horizons of the forest soil profile (Persson 1980; Büttner & Leuschner 1994) where organic forms of N predominate (Kaye & Hart 1997; Näsholm et al. 1998), most studies have focused on the uptake and assimilation of inorganic N sources by ectomycorrhizal roots.

Recognition of the ability of many ectomycorrhizal fungi to degrade macromolecular forms of organic N (Abuzinadah & Read 1986a,b) and assimilate the amino acids released from them (Abuzinadah & Read 1988; Finlay, Frostegård & Sonnerfeldt 1992; Chalot et al. 1994a,b; Wallanderet al. 1997; Näsholm et al. 1998), thus circumventing the mineralization step in the N cycle of forest ecosystems, necessitates a more detailed analysis of the kinetics of absorption of these organic and inorganic N forms. The kinetic properties of inorganic N uptake systems have been determined for ectomycorrhizal fungi (e.g. Littke, Bledsoe & Edmonds 1984; Jongbloed, Clement & Borst-Pauwels 1991; Plassard et al. 1994) as well as for mycorrhizal (Plassard et al. 1994; Eltrop & Marschner 1996) and non-mycorrhizal (e.g. Kamminga-van Wijk & Prins 1993; Kronzucker, Siddiqi & Glass 1995, 1996; Bassirirad et al. 1996; Kreuzwieser et al. 1997) roots. In contrast, understanding of the kinetic properties of amino acid uptake processes in ectomycorrhizal systems is limited; to our knowledge data are available only for the ectomycorrhizal fungus Paxillus involutus (Chalot et al. 1996) and various arctic plant species (Kielland 1994).

The purpose of the present study was to carry out a comparative analysis of the kinetics of amino acid and ammonium (NH4+) absorption by determining the KM and Vmax values of ectomycorrhizal roots representative of a range of tree species, and geographic locations. The amino acids glycine and glutamine were selected as organic N sources representative of those known to occur in soil solutions (Németh et al. 1988; Kielland 1994; Raab, Lipson & Monson 1996) and methylammonium hydrochloride was used as a surrogate for ammonium, which is the main mineral N source of typically acidic ectomycorrhizal habitats. Major emphasis in our study was placed upon several quantitatively important mycorrhizal morphotypes of the temperate deciduous tree Fagus sylvatica L. (European beech) growing in a UK beech wood. Kinetic parameters for amino acid uptake by ectomycorrhizal roots of this species were examined with those of the coniferous species Pinus sylvestris L. (Scots pine) and Picea abies (L.) Karst. (Norway spruce) collected along a gradient of increasing pollutant mineral N deposition from Northern Sweden through Denmark to Germany as part of the European Community project CANIF (Carbon and Nitrogen Cycling in Forest Ecosystems). This enabled us to test the hypothesis that natural exposure of ectomycorrhizas to mineral N sources may influence the absorption kinetics of organic N.

MATERIALS AND METHODS

Sampling sites

Ectomycorrhizal roots of Fagus sylvatica were collected at intervals throughout 1997 from a pure stand of this tree species at Ridgeway Side Wood, Hathersage, Derbyshire, UK (NGR SK 227830). These were used to facilitate a comparative analysis of amino acid and NH4+ uptake using methylammonium as an analogue of NH4+. Ectomycorrhizal roots were also collected from the CANIF sites over the period August–October 1997 (for site descriptions see Table 1). N deposition at the study sites measured as N in rainfall outside the canopies ranged from 1 kg N ha–1 year–1–30 kg N ha–1 year–1 (see Table 1).

Table 1.  . Location, stand and soil characteristics of the sampling sites (data taken and supplemented from Högberg et al. 1996 and Department of the Environment 1994). CANIF sites are marked with an asterisk Thumbnail image of

Sample preparation

Samples of the organic soil layers of the forest floor containing ectomycorrhizal roots were collected and transported immediately to the nearest laboratory to the sampling site. All samples were kept at 4 °C during transport and storage.

Humus and soil particles were removed from the mycorrhizal root systems by rinsing subsamples on a 2 mm sieve with tap water. Cleaned subsamples were transferred into Petri dishes containing 0·5 mol m–3 ice-cold CaCl2 solution (Kielland 1994). When further cleaning was necessary, sharp forceps were used to remove detritus.

Mycorrhizal roots were detached from the root systems using forceps and a scalpel and temporarily collected in ice-cold 0·5 mol m–3 CaCl2 solution shortly before the uptake experiments were started.

Morphotypes were sorted according to type of ramification, colour and texture of the mantle and presence of rhizomorphs and when possible identified using published descriptions (Agerer 1987–98, Brand & Agerer 1986; Brand 1989; Brand 1991). The most common types at each site were used for analysis. These were Russula ochroleuca (Pers.) Fr. and Lactarius subdulcis Bull. ex Fr. together with Xerocomus chrysenteron (Bull. ex St. Amans) on beech at Ridgeway Wood and R. ochroleuca and L. subdulcis at Hillerød and Schacht (AFS Taylor, personal communication). Identified and unidentified morphotypes of beech, birch, pine and spruce collected from Åheden, Hillerød, Schacht and Waldstein were also examined (see Table 1).

Uptake experiments

Amino acids and methylammonium, in all cases labelled with 14C, were used as tracers for the uptake experiments (Chapin, Moilanen & Kielland 1993; Chalot et al. 1995). Methylammonium has often been used as a non-metabolizable ammonium analogue in uptake studies (Chapin et al. 1993; Kielland 1994). It allows sensitive determination of uptake rates even at low concentrations when, as was the case in this study, only limited amounts of experimental material were available.

The radioactive N sources used, their specific activities and their suppliers were as follows: [U-14C]glycine 4 GBq mmol–1 (NEN, Hounslow, UK), 3·85 GBq mmol–1 (Amersham, Little Chalfont, UK); L-[U-14C]glutamine 9·028 GBq mmol–1 (NEN), 10·2 GBq mmol–1 (Amersham); L-[U-14C]glutamic acid 9·5275 GBq mmol–1 (NEN); [14C]methylammonium hydrochloride 1·7279 GBq mmol–1 (Amersham).

The use of 14C-labelling methods to determine uptake rates over short exposure times can result in overestimation of compound influx because substrate attachment to cell-wall binding sites can be included in the calculation (Borstlap et al. 1986; Jongbloed et al. 1991; Mäck & Tischner 1994). An equilibration step before exposure of roots to 14C-labelled substrate in unlabelled substrate was therefore included to saturate binding sites. This procedure also enabled equilibration of mycorrhizal roots to the conditions of temperature and solution composition used during the uptake of labelled compounds. Using this approach linear amino acid uptake rates could be measured for up to 1 h (Fig. 1). To minimize the risk both of 14C loss by respiration of amino acids (Chalot et al. 1995) and of tracer efflux of the kind observed by Kronzucker et al. (1996) for 15NH4+, a short standard incubation time of 10 min was chosen.

Figure 1.

. Time-dependent uptake of 14C-labelled glutamine by excised Lactarius subdulcis/Fagus sylvatica mycorrhizas. Glutamine concentration was 0·5 mol m–3 at pH 4·0. Values are means ± SE (n≥ 4). Sampling site: Hathersage.

For the the uptake experiments, mycorrhizal roots were first equilibrated in 20 cm3 plastic scintillation vials in 3 cm3 0·5 mol m–3 CaCl2 supplemented with the respective unlabelled amino acid or methylammonium. After equilibration for 8 min, samples were transferred into scintillation vials containing the respective [U-14C]-labelled amino acids or 14C-methylammonium. Total concentration (labelled + unlabelled compound) were the same as in the respective equilibration solutions. All samples in uptake solutions were incubated at 20 °C on a shaking water bath (70 r.p.m.). 0·5 mol m–3 CaCl2 and uptake solutions had been adjusted to pH 4·0, which is near the pH value of the soil solution of the sample sites (see Table 1) and is close to the optimum pH for uptake of various amino acids in the ectomycorrhizal fungus Paxillus involutus (Chalot et al. 1995). After an uptake period of 10 min, mycorrhizas were washed for 5 min (Cram & Laties 1971) in 10 cm3 ice-cold 0·5 mol m–3 CaCl2, adhering liquid absorbed with filter paper, the samples immediately frozen in liquid nitrogen and stored at – 20 °C until freeze-drying.

As amino acid transport has been shown to be proton coupled in other systems (Bush 1993) the uncoupler 2,4-dinitrophenol (DNP) was added at a concentration of 0·5 mol m–3 to the unlabelled and labelled uptake solution in the standard protocol. This enabled analysis of the role of transmembrane proton motive force in amino acid uptake by mycorrhizal roots.

Radioisotope analysis

Freeze-dried roots were weighed and then combusted in a Sample Oxidizer (Model 307, Packard, Pangbourne, UK). 14CO2 was trapped in CARBO-SORB E (Packard) and total radioactivity contained in the mycorrhizal samples counted by liquid scintillation counting (Packard Tri-Carb 1600 TR) using Permafluor E+ (Packard) as scintillation cocktail. Apparent uptake rates were calculated using the specific activity (total activity/(amount 14C + amount 12C)) of the uptake solution and the total radioactivity per sample dry weight.

Data analysis

For every data point at least four independent samples were analysed. Five to 12 substrate concentrations of amino acids and methylammonium were used in the range 0·001–10 mol m–3.

Vmax and KM values were calculated by non-linear curve fitting of the experimental data to the Michaelis–Menten equation [v = (Vmax×[S]) / (KM + [S])]. Standard errors of the fitted parameters have been included in the results section (n = 16–28).

RESULTS

Kinetics of amino acid uptake by ectomycorrhizas

Increasing substrate concentration resulted in increased apparent uptake rates, with no saturation observed over a substrate concentration range of 0·001–10 mol m–3 (Fig. 2a). Correspondingly, typical Eadie–Hofstee plots (of apparent uptake rate against the ratio of this rate to substrate concentration) showed a biphasic kinetic (e.g. Fig. 2c). Addition of the metabolic inhibitor 2,4-dinitrophenol at a concentration of 0·5 mol m–3 to equilibration and labelled uptake solution resolved a linear, non-inhibitable component in the total apparent amino acid uptake, which contributed substantially to total amino acid uptake at concentrations > 0·5 mol m–3 (Fig. 2a). Here, apparent uptake displayed saturable kinetics conforming to Michaelis–Menten kinetics up to concentrations of 0·5 mol m–3. However, for several samples apparent uptake rates at a substrate concentration of 0·5 mol m–3 diverged from simple Michaelis–Menten kinetics (see, e.g. Figure 3). Therefore, KM values were calculated for a substrate concentration of 0·001–0·25 mol m–3 for all substrates and all morphotypes. In this concentration range the contribution by the linear, non-inhibitable uptake component to the total apparent uptake was negligible and calculation of apparent Vmax and KM values with or without consideration of uptake rates in the presence of dinitrophenol resulted in similar kinetic parameters (Table 2).

Figure 2.

. Uptake of 14C-labelled glutamine into excised Lactarius subdulcis/Fagus sylvatica mycorrhizas as a function of glutamine concentration: Glutamine uptake in the range of (a) 0–10 mol m–3 or (b) 0–0·5 mol m–3 in the absence (––) or presence (·····) of additional 0·5 mol m–3 2,4-dinitrophenol. Active uptake (––) was calculated as the difference of means.

Figure 3.

. Uptake of 14C-labelled glutamine by excised Lactarius subdulcis/Fagus sylvatica mycorrhizas as a function of glutamine concentration in the range of 0–0·5 mol m–3 at three sampling different sites. Curves representing measured data (––) or calculated data using kinetic parameters obtained by non-linear regression of the Michaelis-Menten equation (·····) in a substrate concentration range of 0·001–0·25 mol m–3. Values are means ± SE (n = 4).

Table 2.  . Kinetic parameters for amino acid uptake by mycorrhizal beech roots in the absence of 0·5 mol m–3 2,4-dinitrophenol (DNP) (total uptake) or after subtraction of uptake rates in the presence of 0·5 mol m–3 DNP (calculated active uptake). Values for Vmax (μmol g–1 DW h–1) and KM (mmol m–3) (± SE of the fitted parameters) were calculated by non-linear curve fitting of the experimental data to the Michaelis–Menten equation [v = (Vmax×[S])/(KM + [S])] in a substrate concentration range of 0·001–0·25 mol m–3. Sampling site: Hathersage Thumbnail image of

Kinetic parameters of amino acid versus methylammonium uptake

For all three mycorrhizal types studied at Hathersage apparent Vmax and KM values for methylammonium were higher than for the amino acids tested (Table 3). Russula ochroleuca/Fagus sylvatica mycorrhizas had the lowest apparent uptake rates for amino acids and methylammonium.

Table 3.  . Kinetic parameters for methylammonium and amino acid uptake by mycorrhizal beech roots calculated by nonlinear regression analysis in a substrate concentration range of 0·001–0·25 mol m–3. Values for Vmax (μmol g–1 DW h–1) and KM (mmol m–3) (± SE of the fitted parameters) were calculated by non-linear curve fitting of the experimental data to the Michaelis– Menten equation [v = (Vmax×[S]) / (KM + [S])] in a substrate concentration range of 0·001–0·25 mol m–3. Sampling site: Hathersage Thumbnail image of

Kinetic parameters of amino acid uptake along a European north/south gradient

Lactarius subdulcis/Fagus sylvatica mycorrhizas showed almost identical uptake kinetics for glutamine at the three beech sampling sites (Fig. 3, Table 4). Higher apparent Vmax and KM values were determined for glycine, with greater variability among the sites.

Table 4.  . Kinetic parameters for amino acid uptake by mycorrhizal roots collected along a European North/South gradient. Values for Vmax (μmol g–1 DW h–1) and KM (mmol m–3) (± SE of the fitted parameters) were calculated by non-linear curve fitting of the experimental data to the Michaelis–Menten equation [v = (Vmax×[S])/(KM + [S])] in a substrate concentration range of 0·001–0·25 mol m–3; n.a. = not available Thumbnail image of

The mycorrhizas of Russula ochroleuca had the lowest apparent Vmax values of all morphotypes independent of the sampling site, the host plant or the type of amino acid supplied. Their apparent KM values for glutamine were in the same range as those calculated for other morphotypes. The KM values for glycine in Russula ochroleuca roots were, however, more variable as they were also in roots of Lactarius subdulcis. At Hillerød, Russula ochroleuca mycorrhizas showed similar uptake kinetics for glycine independent of the host plant.

Several unidentified morphotypes showed apparent Vmax and KM values ranging from 7·7 up to 24·7 μmol g–1 DW h–1, 19–130 mmol m–3 (glutamine) and 8·3–34·1 μmol g–1 DW h–1, 21–113 mmol m–3 (glycine), respectively (Table 4).

No obvious relationships could be seen between the apparent Vmax and KM values and the location of the sampling site on the N deposition gradient (Table 4). Similarly, no apparent correlation existed between sample site location and apparent uptake rates of various ectomycorrhizal morphotypes at a single (40 mmol m–3) amino acid concentration (Fig. 4).

Figure 4.

. Uptake of amino acids by mycorrhizal roots collected along a European north/south gradient at a single substrate concentration (40 mmol m–3). Average uptake rates for glycine or glutamine were not significantly different between different sampling sites (ANOVA, P = 0·05). Figures on the ordinate represent unidentified mycorrhizal morphotypes: 1 = P. sylvestris morphotype, 2 = B. pubescens morphotype, 3–9 = F. sylvatica morphotypes.

DISCUSSION

Our kinetic data show that all the mycorrhizal types investigated were able to take up amino acids at rates similar to those observed for methylammonium in the present study and for NH4+ in other reports (see Table 5). KM values obtained were generally in the same range (9–377 mmol m–3) as those observed by Kielland (1994) for mycorrhizal fine roots of several arctic plant species. The lowest recorded KM was seen in an unidentified morphotype of F. sylvatica that produced rates of uptake similar to those calculated for pure cultures of the fungus Paxillus involutus by Chalot et al. (1996).

Table 5.  . Kinetic uptake parameters of nitrogen compounds for non-mycorrhizal and mycorrhizal tree roots Thumbnail image of

With the exception of ectomycorrhizas formed by Russula ochroleuca, values of Vmax were higher than those reported by Kielland (1994). This, however, may be attributable to the selection, in our study, of an incubation temperature of 20 °C, which was 6 °C higher than that used by Kielland. Clearly, in vivo uptake rates will be lower at naturally occurring soil temperatures but the relative rates of assimilation of organic and inorganic N sources are unlikely to be changed.

Our data clearly show that mycorrhizal roots possess high-affinity transport systems that should enable them effectively to take up amino acids from soil solutions where concentrations of these organic sources, which are reported to be in the range 10–100 mmol m–3, can often exceed those of inorganic N (Abuarghub & Read 1988a,b; Németh et al. 1988; Kielland 1994; Näsholm et al. 1998). Uptake capacities (Vmax) and substrate affinity (KM) of the investigated systems indicate the potential for a substantial uptake of organic N from the soil solution. These results add impetus to the emerging consensus that ectomycorrhizal roots can contribute significantly to total N uptake by absorbing those organic forms of this element that often predominate in forest ecosystems (Näsholm et al. 1998). In so doing they render their host plant at least partially independent of the activities of separate populations of decomposers for ammonium release by mineralization.

The curvilinear and upwardly concave nature of the Eadie–Hofstee plots necessitates a dual or multiphasic interpretation of the systems responsible for uptake. Similar curves have been observed for a wide range of substrates and non-mycorrhizal species (see Reinhold & Kaplan 1984; Nissen 1991 for review). The biphasic nature of the curves indicates that high- and low-affinity transport systems (HATS and LATS, respectively, Wang et al. 1993) are functioning. However, the high affinity system is likely to be by far the most important over the μmolar range of amino acid concentrations found in soil solutions of tundra and forest systems (Chapin et al. 1993; Kielland 1994). In this range, uptake of amino acids into ectomycorrhizal roots followed Michaelis–Menten kinetics, there being a deviation only at substrate concentration in excess of 0·25 mol m–3 (Figs 2, 3).

Chalot et al. (1996), working with Paxillus involutus, observed a linear amino acid uptake component that was independent of that following Michaelis–Menten kinetics and that was not inhibited by the uncoupler 2,4-dinitrophenol (DNP). Although a similar linear uptake system was observed in the present study in the presence of DNP, subtraction of these uptake rates from rates measured in the absence of DNP still resulted in slight deviation of saturable Michaelis–Menten kinetics at substrate concentrations ≥ 0·5 mol m–3, possibly indicating the contribution of further low-affinity uptake systems at higher substrate concentrations. However, if the low affinity uptake systems contribute to total uptake only at substrate concentrations > 0·5 mol m–3, they are unlikely to be of significance under natural conditions. They were not therefore further considered in this study.

Though 14C-labelled methylammonium has been widely used as a transport analogue for NH4+ in algae (Kleiner 1981) higher plants (Jackson & Bloom 1994; Kosola & Bloom 1994; Kielland 1994) and fungi (Kleiner 1981) some doubts have been expressed about the extent to which its kinetic properties are representative of those observed for NH4+. Kosola & Bloom (1994), for example, showed for tomato that although methylammonium and NH4+ share a common transport system, this system has a far greater affinity for NH4+, the KM for the latter being six times lower than for 14C-methylammonium. Their reported Vmax values for the two N sources were, however, identical. In view of these findings it may be that the KM values for methylammonium reported in the present study are an overestimate of those that would be found for NH4+. Despite this, they fall within the range reported for the uptake of NH4 in kinetic studies of mycorrhizal and non-mycorrhizal tree roots (Table 5). In addition, uptake rates for NH4+ measured for mycorrhizal beech roots (Gessler et al. 1998) at a single, ecologically important concentration (100 mmol m–3) are in the same range as those that we obtained for methylammonium uptake in three distinct mycorrhizal types (4·5 μmol g–1 DW h–1 at 20 °C compared to 4·1, 7·3, 14·6 μmol g–1 DW h–1 for Russula ochroleuca, Xerocomus chrysenteron and Lactarius subdulcis mycorrhizas, respectively).

We did not observe any effect of the location of the sampling site along a European north/south gradient on the uptake kinetics of amino acids by the same morphotypes, on kinetic parameters shown by a range of different morphotypes or on the uptake rates at a single concentration (Fig. 4). In a similar comparative approach Kielland (1994) determined uptake kinetics of fine roots of the same plant species collected from different plant communities. He observed considerable variation in kinetic parameters, especially KM values among different sites, which were, however, not related to the availability of amino acids at the respective sites.

The possibility that local conditions such as the availability of inorganic N in the soil solution may modulate the actual rate of amino acid uptake at the different sites cannot be excluded. Chalot et al. (1995), however, showed that neither NH4+ nor NO3 had any effect on amino acid uptake by Paxillus involutus when applied at concentrations usually found in forest soil solutions (0·05–0·5mol m–3), indicating that the processes involved in uptake of organic and inorganic N sources operate independently (Chalot & Brun 1998). Other factors that might be involved in regulation of amino acid uptake include inducible transport systems and feedback control. Effects of these kinds have so far been characterized only for inorganic N uptake in plants (Von Wirén, Gazzarini & Frommer 1997) and amino acid transporters of non-mycorrhizal lower eukaryotes (Sophianopoulou & Diallinas 1995).

Our data clearly indicate that within a single host species there can be considerable differences in the kinetic properties of amino acid uptake that are determined by the fungal partner. Therefore, the actual composition of the below-ground ectomycorrhizal community appears to determine the extent to which ectomycorrhizal plants can supplement their acquisition of N through the uptake of amino acids. Our studies along the gradient of N deposition suggest that increasing availability of inorganic N will have no effect on the abilities of these ectomycorrhizal types to take up amino acids. However, the possibility that long-term exposure to inorganic N deposition could change the structure of ectomycorrhizal fungal communities and thereby affect patterns of amino acid uptake should not be ignored (Wallenda & Kottke 1998).

High-affinity transport systems for amino acids described in this study are also a prerequisite for the ability of ectomycorrhizal fungi and roots to use polymeric peptides and proteins as N sources (Abuzinadah & Read 1986a,b) after cleavage into amino acids by extracellular proteases (Zhu, Guo & Dancik 1990). Further research on the diversity of ectomycorrhizal species in forest ecosystems and their physiological properties is necessary to evaluate the possible connection between the close association of organic substrates and ectomycorrhizal mycelium (Harley 1978; Bending & Read 1995) to gain more insight into the complex interactions involved in the acquisition of organic and inorganic N sources in N-limited forest ecosystems.

Acknowledgements

This project is a contribution to the EC project CANIF (EEC contract ENV4-CT95–0053). We thank P. Högberg (Umeå), A. Kjøller and S. Struwe (Copenhagen), E.-D. Schulze (Bayreuth) and their coworkers for the provision and setting up of laboratory space for the uptake experiments.We also thank A. Wingler for critical reading of the manuscript.

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