In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

Christine Le Guernevé,Hervé Quiquampoix,Margarita Torres-Aquino,Adeline Becquer,Laurie K Amenc,Carlos Trives-Segura,Siobhan Staunton,Claude Plassard

doi:10.21769/bioprotoc.2973

Abstract

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

Highlights

[Background] The association between mycorrhizal fungi and plants improves the P nutrition of the host-plant (Smith and Read, 2008; Plassard and Dell, 2010; Cairney, 2011; Smith et al, 2015)
Absorbed Pi is partly incorporated into phosphorylated metabolites, phospholipids and nucleic acids, and partly condensed into polyphosphates (PolyP) where they constitute a storage pool in the vacuoles (Ashford et al, 1994). This protocol details a device that allows the study of Pi transport in fungal cell compartments and metabolism of PolyP by 31P-NMR spectroscopy
Mycelia are incubated without plants, with host plants or with non-host plants (Torres-Aquino et al, 2017)

Summary

Introduction

[Background] The association between mycorrhizal fungi and plants improves the P nutrition of the host-plant (Smith and Read, 2008; Plassard and Dell, 2010; Cairney, 2011; Smith et al, 2015). Int diam 3.17 mm (Dominique DUTSCHER, Silicone, catalog number: 4906600) 12. 10 mm diameter NMR tubes for in vitro studies (Norell, catalog number: 1008-UP-8) 26. Concentrated (30%) hydrogen peroxide (H2O2) solution (Sigma-Aldrich, catalog number: 216763-500ML-M) 32.

Results

Conclusion