Dispersed polyphosphate in fungal vacuoles in Eucalyptus pilularis/Pisolithus tinctorius ectomycorrhizas

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilul aris under axenic conditions were rapidly frozen, freeze-substituted in tet rahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microana lysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosph orus and potassium. They also contained sulfur and variable amounts of chlo rine. In anhydrously processed freeze-substituted mycorrhizas, dispersed el ectron-opaque material filled the fungal vacuoles. X-ray maps indicated tha t P was distributed evenly throughout the entire vacuole profile and was no t concentrated in spherical bodies or subregions of the vacuole, There were no electron-opaque granules surrounded by electron-lucent areas, such as a re commonly seen in chemically fixed material. The fungal vacuoles were als o rich in K, which similarly gave a signal from the entire vacuolar profile . Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig n et hyphae. Stained sections of ether-acrolein freeze-substituted mycorrhiza s also showed only dispersed material in the fungal vacuoles as, in most ca ses, did acetone-osmium freeze-substituted material. Precipitation of metac hromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as wel l as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exo dermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosp hate is indeed present in the vacuoles of the fungal cells of these ectomyc orrhizas, but that in vivo it is in a dispersed form, not in granules. (C) 1999 Academic Press.