Abstract
For the quantification of surface-bound phosphomonoesterase activity (SBPA) of fungi, roots, or mycorrhiza, a colorimetric method based on p-nitrophenyl phosphate (pNPP) is widely used. Unfortunately, this method does not reveal information about the localization of the surface-bound phosphomonoesterase (SBP). We introduce a method that localizes and quantifies SBPA in living hyphae of ectomycorrhizal fungi using confocal laser scanning microscopy of the hydrophilic substrate enzyme-labelled fluorescence (ELF-97) and compare it to the pNPP assay. ELF-97 turns into a strongly fluorescent precipitate upon activation by SBPA and forms bright fluorescent centres on the outer cell wall of the hyphae. Our data show that the enzymatic reaction is not substrate-limited during an incubation period of 15 min in fungal hyphae of Pisolithus tinctorius, Cenococcum geophilum, and Paxillus involutus. Image-processing routines determined the total intensity and the average number of the fluorescent ELF-97 centres per micrometre fungal hyphae of C. geophilum and Paxillus involutus. ELF-97 and pNPP detected similar variations of the SBPA at different pH values (3–7) during the measurement and different phosphorus (P) concentrations during the growth period of the fungi. The ELF-97 method revealed that C. geophilum and Paxillus involutus adapt in different ways to the variation of the P concentrations during the growth period by varying the number, the activity, or both properties of the SBP centres. The phosphatases show peak activities at different pH values, so the response of the fungal mycelium to varying P concentrations in soils is pH selective. In conclusion, ELF-97 is a promising substrate to reveal SBPA and adaptation strategies on a structural–physiological level.
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Acknowledgements
Maricel Alvarez is a postdoctoral fellow of Mecesup UCO 02–14 (Chile). This study is a contribution to Fondecyt 1040913 (Chile). Steffen Härtel is supported by Fondecyt 3030065 (Chile). Institutional support to the Centro de Estudios Científicos (CECS) from Empresas CMPC is gratefully acknowledged. CECS is a Millennium Science Institute and is funded in part by grants from Fundación Andes and the Tinker Foundation.
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Alvarez, M., Gieseke, A., Godoy, R. et al. Surface-bound phosphatase activity in ectomycorrhizal fungi: a comparative study between a colorimetric and a microscope-based method. Biol Fertil Soils 42, 561–568 (2006). https://doi.org/10.1007/s00374-005-0053-6
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DOI: https://doi.org/10.1007/s00374-005-0053-6