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Intracellular pH in Arbuscular Mycorrhizal Fungi1
A Symbiotic Physiological Marker

Mario Jolicoeur, Sophie Germette, Martin Gaudette, Michel Perrier, and Guillaume Bécard*

BIOPRO Research Center, École Polytechnique de Montréal, Chemical Engineering Department, 2900 Édouard-Montpetit, Montréal, Québec, Canada, C.P. 6079, H3C 3A7 (M.J., M.G., M.P.); and Laboratoire de Mycologie Végétale, Université Paul-Sabatier, 118 route de Narbonne, 31062 Toulouse, France (M.J., S.G., G.B.)

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0-2 µm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 µm), and decreased over the next 250 µm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

1   Scholarships to M.J. and M.G. supporting their stay in Toulouse were provided by the Fonds pour la formation de Chercheurs et l'Aide à la Recherche (contract no. 94-CI-0117), and the Conseil Régional de Midi-Pyrénée (contract no. 9300417) funded the spectrofluorometric microscope used in this study.
*   Corresponding author; e-mail becard{at}cict.fr; fax 33-5-61-55-62-10.

Plant Physiol. (1998) 116: 1279-1288
Copyright Clearance Center:   0032-0889/98/116/1279/10
© 1998 American Society of Plant Physiologists




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