SummaryExternal 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 NH 4 + + + + . AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd 2M -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. Cd 2M 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 005 µM Cd concentration. At 4 SC, 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 Cd 2Mwas partially dependent on the membrane potential. Cd 2M uptake into symplasm is linked to Ca 2M transport, as revealed by the inhibition of Cd accumulation by the Ca 2M 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.

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SUMM.ARVIn order to investigate the relative contribution of glutamine synthetase and NADP-glutamate dehydrogenase to the assimilation of ammonium (NH^") by spruce ectomycorrhizas, changes in free amino acid content and kinetics of''^N incorporation into free amino acids were measured together with the effect of specific enzyme inhibitors. Exposure of detached ectomycorrhizas to ('''NH^).,SOj showed that tbe greatest flow of ^"N enters into tbe amido group of giutamine. Label was also detected in glutamic acid, alanine and y-aniinobutyric acid. Large amounts of alanine and glutamate accumulated in response to the addition of methionine sulfoximine (MSX) together witb a decrease in '^N incorporation into both amido-and amino-nitrogen of glutamine. Tbese results are consistent with a major role of glutamate debydrogenase and glutamine syntbetase in nitrogen assimilation in the symbiosis and do not suggest any significant role for glutamate synlhase in the synthesis of glutamate. A large accumulation of uniabelled asparagine in response to MSX and albizziine inhibition suggests the occurrence of an unlabelled NH,* pool in the host plant. The transfer of nitrogen compounds between tbe fungal cells and the host tissues is discussed.