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First direct visualization of amino acids in
ectomycorrhizal fungi by immunocytochemistry
poster
BRUN ANNICK1, M. CHALOT1, J.S.
MATHISEN2, P.O. OTTERSEN2 & B.
SÖDERSTRÖM3
1University Henri Poincaré-Nancy I, Faculty of
Sciences, Laboratory of Forest Biology, U.A. INRA 977, BP
239, F-54500 Vandoeuvre Cedex, France
2Anatomical Institute, University of Oslo, Karl
Johansgate 47, N-0162 Oslo 1, Norway
3Department of Microbial Ecology, University of
Lund, S-223 62 Lund, Sweden
ICOM1 Abstract
The localization of amino acids was investigated in the
ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. by
immunocytochemistry. Antibodies raised against glutamate
and glutamine have been produced which allow the
utilization of immunogold labelling techniques to explore
the cellular and tissular distribution of amino acids.
Classical fixation procedures with glutaraldehyde and
paraformaldehyde resulted in a loss of amino acids during
the incubation step as demonstrated with [14C]
labelled amino acids. Therefore we applied a freeze-
substitution technique to fungal cells which allow better
conservation of the vacuole content. Immunogold labelling
of freeze-substituted cells clearly showed a cytosolic
localization of glutamate and glutamine. However, there
was a great heterogeneity of labelling distribution
between different cells, with cells intensily labelled
whereas other cells were poorly labelled. When using a
double labeling protocole, with two particle sizes of 10
and 30 nm for glutamate and glutamate, respectively, it
appeared that glutamine amount was 3 to 5 fold higher than
glutamate amount, which is in good agreement with
quantification of amino acids by HPLC. This study
represents a first step towards the use of electron
microscopc immunocytochemistry as a tool to assess the
distribution and the absolute concentrations of amino
acids in fungal cells and symbiotic tissues.