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

In and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals. Bio-protocol [Abstract] We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance ( 31 P-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).


Culture conditions for Pinus pinaster
The procedure for disinfection of seeds, germination and culture conditions have been i. An external NMR tube (10 mm diameter).
ii. An internal glass cylinder (7 mm diameter) which can be screwed on the external tube.
iii. A plastic cap on the top of this device which is bored to allow 3 Teflon tubings to connect the NMR tube to a peristaltic pump permitting the circulation of the oxygenated interaction medium. The inlet tubing extends to the bottom of the NMR tube. The output tubing extends to the top of the mycelia in the NMR tube. The end of the safety output tubing is 3 cm above the mycelia.
A string is attached to this device to allow its manual introduction into the hole in the magnet of the NMR spectrometer without using the air-lift system, to avoid friction between the 3 PVC tubings and magnet walls. Copyright   ii. The inlet tubing supplies the interaction medium from a glass bottle (2,000 ml), in which oxygen is bubbled from a compressed O2 gas cylinder, to the NMR tube.
iii. The output tubing collects the medium which has oxygenated the mycelia to another originally empty glass bottle (2,000 ml).
iv. In case of dysfunction of the normal output system, a safety output tubing should avoid any leakage in the magnet hole. c. 20 sec inter-scan delay (5 times the spin-lattice relaxation time T1) giving a fully relaxed spectrum (Quiquampoix et al., 1993). This is a very important parameter for the quantitative analysis of the chemical metabolites identified in the 31P-NMR spectrum. A much lower inter-scan delay does not allow a real quantification.

Data analysis
NMR peak areas of Pi and polyP are expressed relative to that of the MDP capillary which is the same for all experiments and is arbitrarily fixed to 1. If you compare several treatments, test the normality of data using the Kolmogorov Smirnov test and, where necessary, transform the data either square root or log10 prior to analysis to meet the assumptions of the ANOVA. To compare the effect of treatments, use an ANOVA analysis followed by Tukey's honest significance difference.
For 31 P-NMR measurements, data in percentage were transformed (arcsin√ ) as described by Legendre and Legendre (1998) before ANOVA.

1.
As the growth of the fungus in liquid medium may vary, we prepare up to 4 additional culture flasks inoculated with the fungus to discard those with poor growth.
2. If you need the assistance of a scientist who is in charge of an NMR facility unit, you will probably have to convince them of the safety of the homemade perfusion NMR tube for in vivo studies. Launch several distinct experiments with this device outside the NMR spectrometer to check that no leakage of liquid occurs.
3. If an NMR spectrometer of lower magnetic field is used, the number of accumulations will have to be increased. 4. The flow of oxygenated liquid medium in the perfusion NMR tube is a compromise between two unwanted phenomena: (i) a too low flow will not be sufficient for a good oxygenation of the cells and (ii) a too high flow will lead to bubbles formation in the matrix of the biological sample which causes a decrease in the spectral resolution. The latter effect usually does not appear below 10 ml min -1 (Lee and Ratcliffe, 1983). Copyright