Bernard Botton

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd2+-uptake kinetics were characterized by a smooth, non-saturating curve that could be dissected into linear and saturable components. The linear-uptake kinetic component was interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component was the result of carrier-mediated transport across the plasma membrane. Cell-wall-bound Cd was almost completely removed during desorption from cell-wall preparations. Cd2+ desorption from intact mycelium was found to be a function of time involving three compartments corresponding in series to cell wall (50%), cytoplasm (30%) and vacuole (20%), when mycelia were exposed to a 0.05 microM Cd concentration. At 4 degrees C, most of the Cd recovered was due to the cell-wall-bound fraction, suggesting that transport across the plasma membrane is a metabolically mediated process. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Cd accumulation in P. involutus mycelia by up to 28%, which indicates that transport of Cd2+ was partially dependent on the membrane potential. Cd2+ uptake into symplasm is linked to Ca2+ transport, as revealed by the inhibition of Cd accumulation by the Ca2+ ionophore A23187. 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 metal-detoxification mechanisms. These data represent a first step towards the understanding of metal-tolerance mechanisms in mycorrhizal fungi.

In vitro weathering of phlogopite by ectomycorrhizal fungi

Oxalate accumulation in external medium under hyphal mats of two ectomycorrhizal species is strongly stimulated (1.7 to 35 fold) by a simultaneous depletion of available K+ and Mg2+. Pisolithus tinctorius strain 441 accumulates oxalate both on NH4−N and on NO3−N whereas Paxillus involutus strain COU only accumulates oxalate on NO3−N. On NO3−N, under a simultaneous K+ and Mg2+ deficiency, P. involutus COU is a very active oxalate producer compared to P. tinctorius 441. The present results could explain the various mineralogical evolutions of a phlogopite mica previously recorded under P. involutus COU or P. tinctorius 441 and suggest a key role for fungal oxalic acid during mineral weathering in response to nutrient deficiency.

Differential responses of ectomycorrhizal fungi to heavy metals in vitro.

Thirty-nine ectomycorrhizal isolates of Paxillus involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and S. variegatus were tested on cadmium, copper, nickel and zinc amended media to determine their in vitro tolerance, measured as inhibition of biomass production. Twenty-one isolates were from heavy metal polluted sites, whereas the others were from non-contaminated soils. There was a strong interspecific variation in metal tolerance. S. luteus, S. variegatus and P. tinctorius were more tolerant of Cu, Cd and Zn when compared with P. involutus, whereas the reverse was true for Ni. A high intraspecific heterogeneity in metal tolerance was also found. EC 50 values for isolates originating from polluted sites were not statistically different from EC 50 values for isolates originating from non-contaminated sites. The findings are discussed in relation to the potential benefits of ectomycorrhizal fungi in protecting their host plants from metal contamination.

Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP‐dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N‐starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

An update on nutrient transport processes in ectomycorrhizas

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.

DNA polymorphism in morels: PCR/RFLP analysis of the ribosomal DNA spacers and microsatellite-primed PCR

As a part of investigations on heterokaryon formation in morels, a characterization of DNA polymorphism within this fungal group was attempted. In order to assess which discrimination level is necessary to trace nucleus populations in heterokaryons, but also in the context of the debatable species definition in morels, different taxa and strain types (monosporal and heterokaryons) were analysed with two polymerase chain reaction (PCR) techniques of distinct sensitivity: (i) PCR of the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal nuclear DNA coupled with restriction fragment length polymorphism (RFLP) analysis, (ii) microsatellite-primed PCR. The ITS and IGS PCR/RFLP appeared at first to be adequate to assess morel systematics. The microsatellite-primed PCR with the primer (GTG)5 revealed, however, that morels exhibit less intraspecific DNA polymorphism than other ascomycetes. Based upon these results, two strategies for investigating somatic strain interactions within morels using DNA analyses are proposed.

Variation in the ITS and IGS regions of ribosomal DNA among the biological species of European Armillaria

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA gene of isolates representing seven European species of Armillaria was examined by PCR, coupled with RFLP analysis and partial sequencing of the ITS region.

Release of complexing organic acids by rhizosphere fungi as a factor in Norway spruce yellowing in acidic soils

A field experiment in the Vosges (a low mountain range in north-eastern France) had shown that a CaMg amendment of an acidic forest soil reduced the total number of fungal propagules per gramme dry soil, while the number of species considerably increased. Some rhizosphere fungi isolated from the amended and/or control plots were grown in three media with ammonium or nitrate as the sole source of nitrogen, and with or without aluminium. Complexing organic acids released by the fungi and likely to interfere with tree nutrition were analyzed in the culture medium after fourteen days incubation. The largest amounts of complexing organic acids are released by fungi isolated from the acid control plot. These rhizospheric fungi take part in soil acidification, which results in the long-term decrease of soil mineral reserves in a forest soil subjected to permanent leaching. Therefore, we conclude from this work that fungi might contribute to the deficient mineral nutrition of the Norway spruce on acidic soils, by impoverishment of the root environment in essential cations. However, among the causes of the Norway spruce yellowing, rhizosphere deleterious fungi act as secondary agents or contributing factors rather than primary causes of decline.

Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum

Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus. External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed. The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae. Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells. The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Δ cells during pseudohyphal differentiation. Northern blot analysis in H. cylindrosporum showed that the HcAMTs were up‐regulated upon nitrogen deprivation and down‐regulated by ammonium.

Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves, The apparent K-m derived from the Eadie-Hofstee plots ranged from 7 mu M for alanine to 27 mu M for glutamate, Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide. vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both ph dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus, Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast, Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive, Tire physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed. (Less)