Zinc and copper are essential nutrients required for many biological processes (e.g. cofactor in many proteins). Despite their importance, excess Zn and Cu are toxic. For this reason, organisms have evolved with mechanisms of metal homeostasis that tightly control the intracellular level of metals when extra-cellular concentrations change. Genetic adaptation towards higher Zn and Cu tolerance was found in ectomycorrhizal fungi of the genus Suillus, originating from highly metal contaminated areas. A better understanding of the sub-cellular compartmentalization of these metals in the fungal cells would give insight into the tolerance mechanism, since the intracellular localisation of the metals and the chemical form wherein they appear will determine the toxicity of the metal for the ectomycorrhizal fungus and eventually for its host plant. First, Zn and Cu will be localised with the commonly used techniques; electron microscopy in combination with energy dispersive X-ray microanalysis.
To characterize the species and spatial distribution of Zn-Cu within the fungus and plant, synchrotron based techniques will be used; including X-ray fluorescence and K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy, all performed to micrometer-scale resolution. Since we have no experience with the latter techniques we will collaborate with Dr Sarret (LGIT, Universite J.Fourier, Grenoble). She has a lot of experience in the structural determination of metals by EXAFS spectroscopy in plants and fungi. Bulk EXAFS measurements will take place at the European Synchrotron Radiation facility in Grenoble, on the French beam line FAME.
The subcellular localisation and speciation of Zn-Cu will be investigated at the micron scale by synchrotron-based x-ray microfluorescenc e and uEXAFS spectroscopy. Such technique is available on beam line 10.3.2 at the ALS synchrotron in Berkeley (USA), where the LGIT have access through a 3-year approved program.
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