University Henri Poincaré Nancy I, Faculty of Sciences, Laboratory of Forest Biology, UA INRA 977, BP 239, 54506 Vanduvre Cedex, France1
Author for correspondence: Michel Chalot. Tel: +33 3 83 91 27 38. Fax: +33 3 83 91 22 43. e-mail: Michel.Chalot{at}scbiol.uhp-nancy.fr
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ABSTRACT |
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Keywords: Cd uptake and compartmentation, Cd desorption, ectomycorrhizal fungus, Paxillus involutus
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodiimide
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INTRODUCTION |
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Understanding heavy-metal tolerance very often first requires a knowledge of the basic mechanisms of metal absorption into the cells. Most studies on metal-ion transport in fungi have concerned K+ and Ca2+, largely because of their importance in fungal growth, metabolism and differentiation (Gadd, 1993 ). Most of our knowledge concerning metal-uptake and compartmentation mechanisms is based on studies with yeasts and filamentous fungi other than ectomycorrhizal species. Filamentous fungi live in very different habitats and it was recently highlighted by Burgstaller (1997)
that investigations with fungi that represent distinct habitats (mycorrhizal fungi, wood-decomposing fungi and the Achlya spp.) are urgently needed. Indeed, mycorrhizal fungi have a unique feature as compared with most of the fungi studied so far in that they are able to form a symbiotic structure with plant roots, providing a unique tool in phytoremediation programmes. Studies on the transport of toxic metal species across fungal membranes could be of interest to engineer ectomycorrhizal fungi with either enhanced or reduced uptake capacities, yielding the possibility of using mycorrhizal trees to phytoremediate polluted soils.
Cadmium is known to be a non-essential element, and can be toxic at very low concentrations. This metal is also ubiquitous in sewage sludges, industrial wastes and mining sites. Uptake of Cd2+ across the plasma membrane of root cells has been shown to occur via a concentration-dependent process exhibiting saturable kinetics (Cataldo et al., 1983 ; Mullins & Sommers, 1986
; Costa & Morel, 1993
). The saturable nature of Cd uptake in these studies suggests that Cd is taken up via a carrier-mediated system. Conversely, Cd uptake in barley is not under metabolic control but is primarily controlled by diffusion (Cutler & Rains, 1974
).
Paxillus involutus, which has become one of the most intensively studied ectomycorrhizal fungi (Chalot et al., 1996 ), is an abundant species in many forest ecosystems and has been one of the most widely found species on industrial wastes polluted by heavy metals. This fungus also plays important roles in limiting heavy-metal toxicity, and the molecular mechanisms involved in Cd tolerance are currently being studied in our laboratory. In this study, we used the absorption/desorption procedure, which has been widely applied to higher plants (Kochian & Lucas, 1982
; Rauser, 1987
; Godbold, 1991
; Lasat et al., 1998
) to characterize 109Cd uptake and subcellular compartmentation in P. involutus mycelium.
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METHODS |
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Cd-uptake experiments.
In the standard assay, discs of mycelium were cut from the actively growing edge of 10-d-old colonies using a 15 mm diameter cork borer. The discs were floated for 30 min in a solution (standard assay medium) containing 0·5 mM CaCl2, 2 mM MES and 0·05 µM Cd containing 3·8 nM 109Cd [4·3 mCi mmol-1 (15·9x107Bq mmol-1)] at pH 4·5 and 20 °C. Ca2+ was used to structurally stabilize membranes and cell walls (Marschner, 1995 ). Because the accumulation rate was linear from 3 to 60 min, a 30 min-uptake period was chosen to investigate uptake, whilst minimizing the possibility of 109Cd loss by efflux across the plasma membrane to the external solution. At the end of the incubation period, discs were briefly rinsed in fresh uptake solution from which Cd was omitted, to remove the surface film of radiolabelled solution, dried and weighed before gamma activity was determined. When needed, the pH was adjusted with HCl or NaOH or the temperature adjusted to 4 °C. For the determination of concentration-dependent kinetics, a Cd concentration range of 050 µM was used.
Time course of Cd2+ desorption from mycelia.
For the determination of desorption rates, discs were first exposed for 30 min or 12 h to the standard assay medium, then briefly rinsed in the uptake solution from which Cd was omitted, and finally floated on a desorption solution containing 5 mM CaCl2 in 2 mM MES (pH 4·5) at 4 °C to initiate Cd desorption. The use of a low temperature for Cd desorption prevented the reuptake of this metal, as suggested by Hart et al. (1998) . At various time intervals, 200 µl aliquots of the desorption solution were taken out and gamma activity was determined. Desorption of 109Cd from mycelia in the external solution was monitored for 10 h.
Mycelium cell-wall preparations.
Cell-wall preparations were obtained by immersing discs of intact P. involutus mycelia in a methanol/chloroform solution (2:1, v/v) for 3 d. In a preliminary experiment it was found that this treatment gave rise to lipid-free mycelium cell-wall preparations that generally maintained the same shape and size as intact mycelia. Small amounts of proteins were still present on such preparations. Following this treatment, cell-wall preparations were washed in a number of changes of deionized water for 2 d. Discs of either intact or methanol/chloroform-treated mycelia were then incubated in the standard assay medium for various lengths of time (360 min) and then either briefly rinsed in the uptake solution from which Cd was omitted (undesorbed), or desorbed at 4 °C in 2 ml desorption solution for 20 min. Subsequently, discs of mycelia or mycelium cell-wall preparations were dried, weighed and 109Cd was quantified by gamma detection.
Effect of metabolic inhibitors on Cd accumulation.
Mycelium discs were preincubated for 20 min at 20 °C in a solution containing 0·5 mM CaCl2, 2 mM MES (pH 4·5) in the presence of various metabolic inhibitors [100 µM verapamil, 10 µM A23187, 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 10 µM dicyclohexylcarbodiimide (DCCD) or 1 µM nonactin], and then incubated in the standard assay medium, supplemented with the inhibitor. Discs of mycelia were subsequently rinsed in the uptake solution from which Cd was omitted and floated at 4 °C in a desorption solution containing 5 mM CaCl2 in 2 mM MES (pH 4·5). At various time intervals, 200 µl aliquots of the desorption solution were taken out and the gamma activity was determined. Desorption of 109Cd from mycelia in the external solution was monitored for 7 h. Metabolic inhibitors were prepared in 95% ethanol. Control treatments consisting of the same concentration of ethanol but without inhibitors were included.
Because Cd and other cations may adhere to glass surfaces, plastic material was used for all preincubation, uptake and desorption solutions. Labelled cadmium (109Cd) was pur chased from Amersham Laboratories. Other chemicals were from Sigma.
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RESULTS |
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Cd accumulation by cell-wall preparations
The efficacy of the desorption regimen at removing cell-wall Cd was further tested by investigating the time course of Cd accumulation (Fig. 5) in living mycelia and cell-wall extracts prepared as described in Methods. In intact, undesorbed mycelia, Cd accumulation was linear with time (Fig. 5a
). In desorbed intact mycelia, Cd accumulation was also linear, but the slope of this line was lower than that of the undesorbed intact mycelia. Cd accumulation in undesorbed cell-wall preparations was linear (Fig. 5b
) and exceeded Cd accumulation in undesorbed intact mycelia (Fig. 5a
). After a 20 min desorption treatment, most of the accumulated Cd was removed (Fig. 5b
). Thus, it appears that most of the desorbed Cd in intact mycelia was due to the loss by cell walls. Consequently, in subsequent experiments using metabolic inhibitors, a 20 min desorption regimen was used following radioactive uptake to remove most of the mycelium cell-wall Cd (cell-wall-bound Cd) and to quantify Cd transport into the intracellular compartments (intracellular Cd).
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DISCUSSION |
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Kinetics of uptake
Linear, time-dependent accumulation of Cd in roots has been reported previously in experiments using low Cd concentrations (20500 nM) (Cataldo et al., 1983 ; Hardiman & Jacoby, 1984
; Homma & Hirata, 1984
), whereas saturable, time-dependent Cd accumulation was reported in barley (Cutler & Rains, 1974
). An increase of pH in the incubation medium led to an inhibition of Cd uptake. At high pH values, Cd2+ ions may be hydrolysed and possibly precipitate as sparingly soluble hydroxides (Kwan & Smith, 1991
). These lower Cd concentrations also induced a reduced uptake by fungal cells. Previous studies with radish and soybean showed that Cd uptake increased with decreasing soil pH (Lagerwerff, 1971
; Miller et al., 1976
).
The Km value of 32 nM for Cd uptake by fungal cells is consistent with those reported for various herbaceous species such as wheat (Hart et al., 1998 ), lupin (Costa & Morel, 1993
) and soybean (Cataldo et al., 1983
). The value found for maximum velocity (Vmax) is lower than those of Hart et al. (1998)
. As discussed by these authors, the linear-uptake kinetic component can be interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component is the result of carrier-mediated transport across the plasma membrane.
Cell-wall binding
The confounding effect of cell-wall binding can be eliminated to some degree by desorbing cell-wall-bound Cd from mycelia following radioactive treatment. Binding of Cd to the cell walls may represent a substantial fraction of the metal accumulated by mycelia and may also be part of the mechanisms by which mycorrhizal fungi tolerate high amounts of metals. Methanol/chloroform-treated mycelia further showed that cell walls may bind high amounts of Cd and that this fraction was removed very quickly with the desorption regimen used. However, this treatment also induced a higher recovery of Cd in cell-wall preparations compared with intact mycelia. In agreement with Hart et al. (1998) , we found residual proteins that may have contributed to higher levels of metal binding. Ting & Teo (1994)
showed that formaldehyde-treated yeast cells consistently showed significantly greater accumulation of Cd than did non-treated cells. It has been proved that chemical modification of the isolated cell walls modifies their capacity to accumulate Cu2+ cations. The blocking of amino, carboxyl or hydroxyl groups reduced the amount of Cu2+ accumulated, indicating that they may play a role in the binding of Cu2+. This in turn indicates that both the protein and the carbohydrate fractions of the cell walls are involved in metal binding (Brady & Duncan, 1994
). Cd was found to be bound to negatively charged sites associated with the cell-wall components such as chitin, cellulose, cellulose derivatives and melanins (Galli et al., 1994
). Turnau et al. (1994)
found that Cd could be bound to the outer pigmented layer of the cell wall. It has been previously suggested that tolerance to metal is associated with the formation of pigments and that the activity of tyrosinase, the melanin biosynthetic complex, is stimulated by metals in ectomycorrhizal fungi, probably to increase metal sequestration onto cell-wall pigments (Gruhn & Miller, 1991
). This is in good agreement with recent experiments performed in our laboratory, in which a cDNA encoding a tyrosinase was found to be differentially expressed in P. involutus mycelia exposed to Cd (C. Jacob, A. Brun, B. Botton & M. Chalot, unpublished results).
Intracellular uptake
A low-temperature treatment decreased total accumulation of 109Cd and its distribution in the different compartments, suggesting that transport across membranes is a metabolically mediated process. In filamentous fungi, yeasts and plants, energy-dependent transport of many divalent cations has been demonstrated (Fuhrmann & Rothstein, 1968 ; Norris & Kelly, 1977
; Cataldo et al., 1983
; Godbold, 1991
; Hart et al., 1998
). Divalent cation uptake may be energized by the H+ gradient, as found for Co2+ and Ni2+ uptake in yeast cytoplasm (Okorokov, 1985
). CCCP inhibited Cd uptake in P. involutus mycelia by up to 28%, which is in agreement with previous studies showing that transport of divalent cations was dependent on the membrane potential in yeast cells (Borst-Pauwels, 1981
; White & Gadd, 1987
) and that uptake was strongly inhibited by protonophoric uncouplers that depolarize the cell membrane (Gadd & White, 1989
; Tripathi et al., 1995
). Since CCCP (a protonophore) partially inhibits Cd uptake, H+ may be considered as a counter ion. However, the effect of CCCP is only partial, suggesting that other Cd-uptake mechanisms may play a role. The effect of CCCP may be partially hidden by the higher proportion of cell-wall-bound Cd.
From the present experimental results, the lack of inhibition by nonactin, a K+ ionophore, of Cd accumulation provides evidence that the transport system for Cd in P. involutus mycelia is not dependent on the K+ gradient. The lack of effect of DCCD on Cd uptake by P. involutus rules out the involvement of the H+/ATPase for H+ efflux during Cd uptake. The possibility of Cd uptake via calcium carriers was also tested in P. involutus. A23187, a Ca2+ ionophore, decreased Cd accumulation, indicating that Ca2+ carriers could play a role in Cd2+ transport across membranes. Non-essential heavy metals such as Cd are also most likely taken up via plant nutrient transporters or channels that are not completely selective (Clemens et al., 1998 ). It has been shown that the plant cDNA LCT1 mediates the uptake of both calcium and cadmium in yeast (Clemens et al., 1998
). The study on the interactions between Cd and Ca uptake will be further investigated in our laboratory. After being transported to the cytoplasm, metals may be bound to intracellular substances, the chemical nature of which is not clear. Animals and some fungi, such as Saccharomyces cerevisiae and Neurospora crassa, induce metallothionein synthesis, whereas plants and some other fungi, such as Candida glabrata and Schizosaccharomyces pombe, synthesize phytochelatins (small peptides that are not products of RNA translation) in order to complex cytoplasmic Cd (Ortiz et al., 1992
; Rauser, 1995
). The low-molecular-mass phytochelatin/Cd2+/S2- complexes would function as scavengers and carriers of cytoplasmic Cd (Ortiz et al., 1992
).
Using compartmentation experiments, we have found significant transport of Cd into the vacuoles. The accumulation of metals in vacuolar phosphate-rich material has been previously suggested in the ectomycorrhizal fungus Pisolithus arrhizus (Turnau et al., 1994 ) and the high nitrogen and sulphur concentrations associated with polyphosphate granules may indicate the occurrence of metalthiolate binding by metallothionein-like peptides (Galli et al., 1994
). Movement of Cd across the tonoplast of the fission yeast cells has been described as occurring by a phytochelatin/Cd transporter. Mutants lacking the ability to accumulate phytochelatin/Cd2+ complexes in the vacuole are Cd sensitive (Ortiz et al., 1992
). Whatever the mechanism of tonoplast Cd transport, vacuolar compartmentation of Cd would tend to limit symplastic movement of the metal.
Concluding remarks
Metal-uptake and compartmentation studies with symbiotic micro-organisms such as ectomycorrhizal fungi may be of particular importance for phytoremediation strategies. As suggested by Galli et al. (1994) , ectomycorrhizal symbiosis can play a crucial role in protecting plant roots from heavy metals. The present work demonstrates the ability of the ectomycorrhizal fungus P. involutus to take up and further accumulate Cd in different compartments. Binding of Cd onto cell walls and accumulation of Cd in the vacuolar compartment may be regarded as two essential detoxification mechanisms. The present investigation was performed with P. involutus cultivated separately from its host plant; therefore it remains to be established to what extent these uptake processes apply to the symbiotic relationship. These data represent a first step towards the understanding of the mechanisms underlying metal tolerance in both ectomycorrhizal fungi and ectomycorrhizal plants.
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ACKNOWLEDGEMENTS |
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Received 29 September 1999;
revised 17 January 2000;
accepted 14 February 2000.