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Feb 2017
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In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals
利用31P-NMR技术体内外研究柱孢白桦在响应植物根组织信号后的磷酸盐转运和多聚磷酸盐代谢   

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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).

Keywords: 31P-NMR spectroscopy (31P-核磁共振波谱法), Phosphate compartmentation (磷酸盐间隔), Polyphosphate metabolism (多聚磷酸盐代谢), Ectomycorrhizal fungi (外生菌根真菌)

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). This positive effect has been attributed primarily to phosphate (Pi) uptake by the fungal hyphae exploring a large volume of soil beyond the depletion zone around actively absorbing roots (Smith and Read, 2008; Cairney, 2011; Smith et al., 2015) and to the secretion of extracellular phosphatases by the fungal cells (Quiquampoix and Mousain, 2005). 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). For the in vivo studies, this perfusion system permits the circulation of an oxygenated nutrient solution in an NMR tube so that the risk of liquid leakage in the NMR spectrometer is prevented. The in vitro studies are based on the perchloric acid extraction of the fungal mycelia. This protocol could be used for other fungal or plant species.

Materials and Reagents

  1. 50 ml Falcon® tube (Corning, catalog number: 352098 )
  2. Sterile plastic Petri dish, 90 mm (Dominique DUTSCHER, Gosselin, catalog number: 688302
  3. Sterile plastic Petri dish, 35 mm (Corning, Falcon®, catalog number: 351008 )
  4. Nichrome wire, stainless steel, round, 22 gauge, 0.64 mm diameter (suppliers for electronic cigarettes)
  5. Aluminum screw cap, 40 mm with rubber liner (VWR, SPV, catalog number: 215-2690
  6. Multi-Purpose Silicone for kitchen or bathroom, 280 ml (Castorama, Rubson)
  7. 60 ml Luer-lock syringes (B. Braun Melsungen, Omnifix®, catalog number: 4613503F )
  8. TeflonTM PTFE microtube, 1.15 mm and 1.75 mm for internal (int) and external (ext) diameter (diam), respectively (Dominique DUTSCHER, PTFE, catalog number: 091932 )
  9. Needles 18 G 0.9 x 40 mm (Dominique DUTSCHER, BD MicrolanceTM 3, catalog number: 301300 )
  10. Tubing, int diam 1.14 mm (Dominique DUTSCHER, Silicone, catalog number: 4906591 )
  11. Tubing, int diam 3.17 mm (Dominique DUTSCHER, Silicone, catalog number: 4906600 )
  12. Tubing, int diam 2.79 mm (Gilson, PVC, catalog number: F117948 )
  13. Microtubes, 1.5 ml (Dominique DUTSCHER, Eppendorf, catalog number: 033511 )
  14. Straight barbed reducing connectors (Cole-Parmer, catalog number: EW-50621-95 )
  15. Valve Luer polycarbonate one way (Cole-Parmer, catalog number: EW-30600-01 )
  16. Sterile syringe filters for air, 0.2 µm, 6.4 cm diam (Labomoderne, Midisart, catalog number: RS3320 )
  17. Autoclavable Polypropylene bag, 3 L, non-printed (Dominique DUTSCHER, Gosselin, catalog number: 140230 )
  18. Tips 1,200 µl for pipet (Dominique DUTSCHER, Sartorius, catalog number: 077200B
  19. Home-made syringe holder
  20. Home-made needle holder for aeration
  21. Folding skirted caps, 14.9 mm diam (Dominique DUTSCHER, Saint Gobain, catalog number: 110602 )
  22. Paper for sterilization (Dominique DUTSCHER, catalog number: 006950 )
  23. Surgical blade sterile No 21 (Dominique DUTSCHER, catalog number: 132521
  24. Borosilicate glass capillaries with filament for easy filling, 1.5 mm outer diameter, 0.86 mm internal diameter (Harvard Apparatus, catalog number: 30-0057 )
  25. 10 mm diameter NMR tubes for in vitro studies (Norell, catalog number: 1008-UP-8 )
  26. Specially homemade NMR tube for in vivo studies (see Figure 1)
  27. Silicone film
  28. Hebeloma cylindrosporum (ectomycorrhizal basidiomycete) (laboratory’s own collection, available upon request)
  29. Pinus pinaster
  30. Zea mays
  31. Concentrated (30%) hydrogen peroxide (H2O2) solution (Sigma-Aldrich, catalog number: 216763-500ML-M )
  32. Liquid nitrogen (Air Liquide)
  33. Calcium sulfate dihydrate (CaSO4•2H2O) (Merck, catalog number: 102161 )
  34. Thiamine hydrochloride –HCl (Sigma-Aldrich, catalog number: T4625-10G )
  35. Manganese (II) sulfate monohydrate (MnSO4•H2O) (Sigma-Aldrich, catalog number: M7899-500G )
  36. Zinc sulfate heptahydrate (ZnSO4•7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
  37. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768-500G )
  38. Copper (II) sulfate pentahydrate (CuSO4•5H2O) (Sigma-Aldrich, catalog number: C8027-500G )
  39. Sodium molybdate dihydrate (Na2MoO4•2H2O) (Sigma-Aldrich, catalog number: M1651-100G )
  40. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291-1KG )
  41. Sodium phosphate monobasic monohydrate (NaH2PO4•H2O) (Sigma-Aldrich, catalog number: 71504-250G-M )
  42. Magnesium sulfate heptahydrate (MgSO4•7H2O) (Sigma-Aldrich, catalog number: 63138-250G )
  43. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  44. Calcium chloride dihydrate (CaCl2•2H2O) (Sigma-Aldrich, catalog number: C5080-500G )
  45. Ferric ammonium citrate (Sigma-Aldrich, catalog number: RES20400-A702X )
  46. D-glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  47. Agar-agar (Sigma-Aldrich, catalog number: A7002-500G )
  48. MES (2-N-morpholino-ethanesulfonic acid, 4-morpholineethanesulfonic acid monohydrate) (Sigma-Aldrich, catalog number: 69892-500G )
  49. Tris(hydroxymethyl)aminomethane (TRIS) (Sigma-Aldrich, catalog number: T1378-500G )
  50. Methylenediphosphonic acid (MDP) (Sigma-Aldrich, catalog number: M9508-5G )
  51. 1 N sulfuric acid solution (Merck, catalog number: 109072 )
  52. Perchloric acid 70% (HClO4) (Sigma-Aldrich, catalog number: 311421-250ML )
  53. Sodium metavanadate (NaVO3) (Sigma-Aldrich, catalog number: 590088-25G )
  54. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 60339-500G )
  55. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758-500G )
  56. Potassium hydroxide 1 N (KOH) (Merck, catalog number: 105029 )
  57. 4-Morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: M1254-250G )
  58. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002-100G )
  59. Deuterium oxide (D2O) (Sigma-Aldrich, catalog number: 151882-100G )
  60. Trace elements (see Recipes)
  61. Mineral salt base solutions (see Recipes)
  62. Thiamine solution (see Recipes)
  63. N6 complete liquid solution (see Recipes)
  64. Solid N6 complete liquid solution (see Recipes)
  65. Interaction medium (IM) (see Recipes)

Equipment

  1. Sample bottles,120 ml (VWR, SPV, catalog number: SPVAGO2246 )
  2. Glass bottles, 1,000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046415
  3. Glass bottles, 2,000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046416
  4. Glass cylinder (diameter 7 mm)
  5. Graduated borosilicate glass beaker 1,000 ml (Dominique DUTSCHER, catalog number: 068942 )
  6. Polypropylene economical beaker 3,000 with moulded graduations (Dominique DUTSCHER, catalog number: 391134 )
  7. Two pairs of stainless steel straight tweezers Wironit, Brucelles type, 130 mm (Dominique DUTSCHER, catalog number: 491037
  8. A scalpel handle for blade 20 to 25 (Dominique DUTSCHER, catalog number: 3740004 )
  9. A standalone burner (Dominique DUTSCHER, catalog number: 071109 )
  10. Butane gas cartridge for the burner (Dominique DUTSCHER, catalog number: 060415 )
  11. A nail, a hammer, scissors and cutting pliers
  12. Incubator with controlled temperature set at 25 °C 
  13. Autoclave
  14. Laminar flow cabinet
  15. Cork-borer, 7.5 mm diameter (Dominique DUTSCHER, catalog number: 942783 )
  16. High pressure O2 gas cylinder (Air products)
  17. Centrifuge 5804 R, refrigerated, without rotor (Eppendorf, model: 5804 R , catalog number: 5805000017)
  18. Rotor F-34-6-38, for 6 × 85 ml tubes, incl. rotor lid (Eppendorf, catalog number: 5804727002 )
  19. Adapter for 50 ml Falcon tubes (Dominique DUTSCHER, catalog number: 033252 )
  20. Peristaltic pump (Gilson Minipuls3) 
  21. Varian UNITY INOVA 500 MHz NMR spectrometer equipped with a 10 mm broad band probe operating at 202.4 MHz for 31P

Software

  1. Microsoft Excel for calculations
  2. Statistica 7.1 (StatSoft Inc., Tulsa, OK, USA) for statistical analysis
  3. VnmrJ and ACDlabs for processing the 31P-NMR spectra

Procedure

  1. Preparation of fungal and plant cultures and subsequent plant-fungus incubation
    1. Culture conditions for Hebeloma cylindrosporum
      Mycelia were grown in glass jars according to the procedure previously described in Bio-protocol (Becquer et al., 2017a).
    2. Culture conditions for Pinus pinaster
      The procedure for disinfection of seeds, germination and culture conditions have been previously described in Bio-protocol (Becquer et al., 2017b).
    3. Culture conditions for Zea mays
      1. Sterilize the surface of seeds of maize (Zea mays L.) with a 6% H2O2 solution for 30 min and rinse with sterile distilled water. 
      2. For germination, put the seeds between two sterile sheets of filter paper previously wetted with deionized sterile water. Incubate for 6 days at 30 °C in the dark for germination. 
      3. Transfer the seedlings to the test tubes filled with a 0.2 mM CaSO4 solution (35 ml/tube) for 3 days in a growth chamber chamber under 16/8 h light/dark cycle at 25 °C/20 °C, 80%/100% RH, CO2 volume fraction in air of 350 x 10-6 and a photosynthetically active radiation of 400 μmol m-2 sec-1 (400-700 nm). 
      4. Then, take the test tubes under a flow laminar cabinet to replace the CaSO4 solution by 35 ml of interaction medium (see Recipe 6) in each test tube. Return the test tubes into the growth chamber for an acclimatization period of 24 h.
    4. To study the plant roots signals eliciting a specific response of the fungal mycelia, follow the incubation procedure previously described in Becquer et al. (2017b).

  2. Fungal sample preparation for in vivo NMR study
    1. Preparation of equipment
      1. A wooden skewer with a hook made with a nichrome wire at its end.
      2. A glass capillary for the reference of chemical shift of NMR spectra (16.38 ppm), filled with a solution of methylene diphosphonate (MDP) 50 mM in Tris buffer 30 mM at pH 8.9 (see Video 1 how to prepare the capillary).

        Video 1. Making the chemical shift reference with MDP solution

      3. A special homemade NMR tube composed of 3 parts (see Figure 1 and Video 2)
        1. An external NMR tube (10 mm diameter).
        2. An internal glass cylinder (7 mm diameter) which can be screwed on the external tube.
        3. 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.


        Figure 1. Scheme of the device used for in vivo 31P NMR measurements

        Video 2. Experimental set-up for in vivo NMR measurements

      4. A system that allows the circulation of the oxygenated interaction medium into the above-described tube, avoiding anoxia of the mycelia during the NMR data acquisition.
        It consists of:
        1. The 3 Teflon tubings extending from the NMR tube that are connected to a peristaltic pump through PVC tubings.
        2. 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.
        3. The output tubing collects the medium which has oxygenated the mycelia to another originally empty glass bottle (2,000 ml).
        4. In case of dysfunction of the normal output system, a safety output tubing should avoid any leakage in the magnet hole.
    2. Introduction of the fungal mycelia in the in vivo device NMR tube (see Video 2)
      1. Remove the fungal mycelia from the flask, or from the syringe containing interaction medium (see Becquer et al., 2017b), let the solution drip off gently and put it on a filter paper with the help of spatula or tweezers.
      2. Put the fungal mycelia at the bottom of the NMR tube, as gently as possible, using the spatula first and then the flat side of the wooden skewer. With Hebeloma cylindrosporum, three mycelial samples obtained in the above-described culture conditions are necessary for a reasonable signal to noise 31P-NMR of 1 h of fully relaxed data acquisition.
      3. Put the chemical shift reference capillary at the bottom center of the NMR tube with the help of the wooden skewer and the hook.
      4. Screw the internal tube on the NMR tube. If the reference capillary is not well positioned inside the internal tube, start again and move it with the hook to set it up in the middle of the internal tube.
      5. Roll a silicone film on the plastic cap to ensure that the oxygenated perfusion solution will not leak off the NMR tube.
    3. Starting the oxygenating perfusion system (adapted from Lee and Ratcliffe, 1983)
      1. Start the oxygen bubbling in the glass bottle containing the interaction medium connected to the NMR tube via the inlet tubing.
      2. Adjust the flow controlled by the peristaltic pump of the inlet, output and safety output tubings to 9 ml min-1 for each of them.
      3. Introduce manually and slowly the NMR tube with the 3 tubings in the magnet hole of the NMR spectrometer with the help of the string.

  3. Fungal sample preparation for in vitro NMR study (adapted from Roby et al., 1987)
    1. Freeze the mycelia with liquid nitrogen.
    2. Store them at -80 °C before extraction.
    3. For each 31P-NMR measurement, thaw 6 frozen mycelia at 4 °C and crush them with 1 ml of 70% (v/v) perchloric acid containing sodium vanadate 10 mM.
    4. Remove the particulate matter and neutralize the perchloric acid in the sample (adapted from Aubert et al., 1996):
      1. Centrifuge the thick suspension at 10,000 x g for 10 min to discard particulate matter in the pellet.
      2. Transfer the supernatant into a 50 ml Falcon tube, add a pH electrode and neutralize the solution by very slowly adding a solution of 2 M KHCO3, allowing foam to subside, stop when a pH of 6.5 is obtained.
      3. Centrifuge again at 10,000 x g for 10 min to remove KClO4 in the pellet.
      4. Freeze dry the supernatant and store it at -80 °C.
      5. For the NMR measurement, the freeze-dried material is redissolved in 2 ml MOPS 40 mM (non-adjusted pH). Then add 0.2 ml EDTA 100 mM. Adjust the pH to 7.8 with KOH 1 N.
      6. Add NaN3 to a final concentration of 100 mg ml-1 to avoid microbial degradation.
      7. Add D2O (final concentration 10%) for the field frequency lock of the NMR signal.

  4. 31P-NMR data acquisition
    1. Record NMR spectra with a Varian UNITY INOVA 500 MHz operating at 202.4 MHz for 31P equipped with a 10 mm broadband probe operating at 25 °C.
    2. For the in vivo experiments use the following parameters for acquisition conditions
      1. It was not necessary to optimize shims considering the broadness of 31P NMR signals in in vivo experiments. Carry out the experiments without a lock signal.
      2. 90° radio frequency pulses angle; spectral width = 20,000 Hz; acquisition time = 0.4 sec; 31P frequency offset = 3,900 Hz. No decoupling (not useful considering the broadness of 31P-NMR peaks in vivo NMR experiments: 1H decoupling does not affect the linewidths of peaks).
      3. 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.
      4. 180 scans giving a total accumulation time of 1 h.
      5. Apodization of the spectra using a 10 Hz exponential function and zero filling to 16 K points.
      6. Phase and baseline correction.
      Note: Examples of in vivo spectra obtained with this protocol can be seen in Torres-Aquino et al. (2017).
    3. For the in vitro experiments use the following parameters for acquisition conditions
      1. 10% of D2O in the mycelial sample as a lock signal.
      2. 90° radio frequency pulses angle; spectral width = 9,600 Hz; acquisition time = 0.84 sec; 1H decoupling during the acquisition time using a Waltz-16 pulse sequence at a field strength of 4 KHz. 31P frequency offset = 4,900 Hz; 1H frequency offset = 170 Hz.
      3. 4 sec inter-scan delay. In these experiments, the scope is the identification of the phosphorylated fungal metabolites based on the signal chemical shift, without the need of quantification. If quantification is required, use the 20 sec inter-scan delay as for in vivo studies.
      4. A sufficient number of accumulations for an acceptable signal-to-noise ratio.
      5. Apodization of the spectra using a 2 Hz exponential function and zero filling to 16 K points
      6. Phase and baseline correction.
      Note: Examples of in vitro spectra obtained with this protocol can be seen in Torres-Aquino et al. (2017).

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 31P-NMR measurements, data in percentage were transformed (arcsin√x) as described by Legendre and Legendre (1998) before ANOVA.

Notes

  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).

Recipes

  1. Trace elements (1,000 ml)
    MnSO4•H2O
    3.08 g
    ZnSO4•7H2O
    4.41 g
    H3BO3
    2.82 g
    CuSO4•5H2O
    0.98 g
    Na2MoO4•2H2O
    0.29 g
    Add ddH2O to 1,000 ml, store at 4 °C 
  2. Mineral salt base solutions (100 ml each)

  3. Thiamine solution (100 ml)
    Thiamine-HCl
    0.01 g
  4. N6 complete liquid solution (1,000 ml)
    1. Add in a 1 L-beaker approximately 500 ml of deionized water and the following volumes of mineral base solutions:
      KNO3
      6 ml
      NaH2PO4
      1 ml
      MgSO4
      1 ml
      KCl
      4 ml
      CaCl2
      0.5 ml
      Ferric ammonium citrate
      0.5 ml
    2. Add also trace elements 0.2 ml and thiamine solution 0.5 ml
    3. Complete the volume to 1 L and check the pH which should be adjusted to 5.5
    4. Finally, add 5 g of D-glucose and shake until complete dissolution
    5. Distribute this solution into glass jars
  5. Solid N6 complete liquid solution (1,000 ml)
    Take two 1 L glass bottles:
    1. Add 7.5 g of Agar-agar and pour 500 ml of liquid N6 medium in each bottle
    2. Sterilize the medium by autoclaving at 121 °C for 20 min
    3. Pour the cooled medium (55-60 °C) in Petri dishes 90 mm diameter
  6. Interaction medium (3,000 ml)
    1. Add in a 3 L-beaker approximately 1.5 L of deionized water and the following volumes of mineral base solutions: 
      MgSO4
      0.6 ml
      CaCl2
      1.5 ml
      MES (final concentration of 5 mM)
      3.2 g
      TRIS (final concentration of 5 mM)
      1.82 g
    2. Complete to 3 L with deionized water and adjust the pH to 5.5 with 1 N H2SO4
    3. Pour 1.5 L of medium into 2 L glass bottles and sterilize by autoclaving (20 min, 121 °C)

Acknowledgments

This research was supported by INRA (France) through a Young Scientist Contract granted to Adeline Becquer and by CONACYT (Mexico) through a Ph.D. fellowship granted to Margarita Torres-Aquino. The protocol is adapted from our previous work (Torres-Aquino et al., 2017).

Competing interests

The authors declare that they have no conflicts of interest.

References

  1. Ashford, A. E., Ryde, S. and Barrow, K. D. (1994). Demonstration of a short chain polyphosphate in Pisolithus tinctorius and the implications for phosphorus transport. New Phytologist 126(2): 239-247.
  2. Aubert, S., Gout, E., Bligny, R., Marty-Mazars, D., Barrieu, F., Alabouvette, J., Marty, F. and Douce, R. (1996). Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates. J Cell Biol 133(6): 1251-1263.
  3. Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L.K., Trives-Segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017a). A method for radioactive labelling of Hebeloma cylindrosporum to study plant-fungus interactions. Bio-Protocol 7(20): e2576
  4. Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L.K., Trives-Segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017b). Establishing a symbiotic interface between cultured ectomycorrhizal fungi and plants to follow fungal phosphate metabolism. Bio-Protocol 7(20): e2577
  5. Cairney, J. W. G. (2011). Ectomycorrhizal fungi: the symbiotic route to the root for phosphorus in forest soils. Plant Soil 344: 51-71.
  6. Lee, R. B. and Ratcliffe, R. G. (1983). Development of an aeration system for use in plant tissue NMR experiments. J Exp Bot 34(146): 1213-1221. 
  7. Legendre, P. and Legendre, L. (1998). In: Numerical Ecology. 2nd edition. pp. 33-46. Elsevier Science B. V. Amsterdam.
  8. Plassard, C. and Dell, B. (2010). Phosphorus nutrition of mycorrhizal trees. Tree Physiol 30(9): 1129-1139.
  9. Quiquampoix, H., Bačić, G., Loughman, B. C. and Ratcliffe, R. G. (1993). Quantitative aspects of the 31P-NMR detection of manganese in plant tissues. J Exp Bot 44(269): 1809-1818.
  10. Quiquampoix, H. and Mousain, D. (2005). Enzymatic hydrolysis of organic phosphorus. In: Organic Phosphorus in the Environment, pp. 89-112. In: Turner, B. L., Frossard, E. and Baldwin, D. (Eds.). CAB International. Wallingford.
  11. Roby, C., Martin, J. B., Bligny, R. and Douce, R. (1987). Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies. J Biol Chem 262(11): 5000-5007.
  12. Smith, S. E. and Read, D. J. (2008). Mycorrhizal symbiosis. 3rd edition. Academic Press, London.
  13. Smith, S. E., Anderson, I. C. and Smith, F. A. (2015). Mycorrhizal associations and phosphorus acquisition: from cells to ecosystems. Annual Plant Reviews 48: 409-440.
  14. Torres-Aquino, M., Becquer, A., Le Guerneve, C., Louche, J., Amenc, L. K., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31P NMR spectroscopy study. Plant Cell Environ 40(2): 190-202.

简介

我们使用体内和体外磷-31核磁共振( 31 P-NMR)光谱来跟踪运输,分区和 外生菌根真菌 Hebeloma cylindrosporum 中的磷酸盐代谢响应来自宿主( Pinus pinaster )或非宿主( Zea mays )的根信号植物。 开发了一种用于体内研究的装置,其允许连续氧化的矿物质溶液在含有菌丝体的NMR管中循环。 在几个连续的处理步骤(在液氮中冷冻;用高氯酸压碎;消除高氯酸;冷冻干燥;在适当的液体培养基中溶解)后,对真菌材料进行体外研究。

【背景】 菌根真菌和植物之间的关联改善了宿主植物的P营养(Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith 等人,,2015)。这种积极效应主要归因于真菌菌丝对磷酸盐(Pi)的吸收,探测了在活跃吸收根周围的耗竭区以外的大量土壤(Smith和Read,2008; Cairney,2011; Smith et al。< / em>,2015)和真菌细胞分泌细胞外磷酸酶(Quiquampoix和Mousain,2005)。吸收的Pi部分地掺入磷酸化的代谢物,磷脂和核酸中,并且部分地浓缩成多磷酸盐(PolyP),其中它们构成液泡中的储存池(Ashford 等人,,1994)。该协议详述了一种装置,该装置允许通过 31 P-NMR光谱研究真菌细胞区室中的Pi转运和PolyP的代谢。菌丝体在没有植物,宿主植物或非宿主植物的情况下孵育(Torres-Aquino 等人,,2017)。对于体内研究,该灌注系统允许氧合营养液在NMR管中循环,从而防止NMR光谱仪中液体泄漏的风险。 体外研究基于高氯酸提取真菌菌丝体。该方案可用于其他真菌或植物物种。

关键字31P-核磁共振波谱法, 磷酸盐间隔, 多聚磷酸盐代谢, 外生菌根真菌

材料和试剂

  1. 50毫升Falcon ®管(Corning,目录号:352098)
  2. 无菌塑料培养皿,90毫米(Dominique DUTSCHER,Gosselin,目录号:688302)&nbsp;
  3. 无菌塑料培养皿,35毫米(Corning,Falcon ®,目录号:351008)
  4. 镍铬合金线,不锈钢,圆形,22规格,直径0.64 mm(电子烟供应商)
  5. 铝制螺帽,40毫米带橡胶衬垫(VWR,SPV,目录号:215-2690)&nbsp;
  6. 用于厨房或浴室的多功能硅胶,280毫升(Castorama,Rubson)
  7. 60毫升Luer-lock注射器(B. Braun Melsungen,Omnifix ®,目录号:4613503F)
  8. Teflon TM PTFE微管,内径(int)和外径(ext)直径分别为1.15 mm和1.75 mm(Dominique DUTSCHER,PTFE,目录号:091932)
  9. 针18 G 0.9 x 40 mm(Dominique DUTSCHER,BD Microlance TM 3,目录号:301300)
  10. 油管,直径1.14 mm(Dominique DUTSCHER,Silicone,目录号:4906591)
  11. 油管,直径3.17 mm(Dominique DUTSCHER,Silicone,目录号:4906600)
  12. 油管,直径2.79 mm(Gilson,PVC,目录号:F117948)
  13. Microtubes,1.5 ml(Dominique DUTSCHER,Eppendorf,目录号:033511)
  14. 直带倒刺连接器(Cole-Parmer,目录号:EW-50621-95)
  15. Valve Luer聚碳酸酯单向(Cole-Parmer,目录号:EW-30600-01)
  16. 用于空气的无菌注射器过滤器,0.2μm,6.4 cm直径(Labomoderne,Midisart,目录号:RS3320)
  17. 可高压灭菌的聚丙烯袋,3 L,非印刷(Dominique DUTSCHER,Gosselin,目录号:140230)
  18. 提示1,200μl用于移液管(Dominique DUTSCHER,Sartorius,目录号:077200B)&nbsp;
  19. 自制注射器支架
  20. 用于曝气的自制针座
  21. 折叠裙边帽,直径14.9毫米(Dominique DUTSCHER,Saint Gobain,目录号:110602)
  22. 灭菌用纸(Dominique DUTSCHER,目录号:006950)
  23. 手术刀片无菌No 21(Dominique DUTSCHER,目录号:132521)&nbsp;
  24. 带有灯丝的硼硅酸盐玻璃毛细管,易于填充,外径1.5 mm,内径0.86 mm(哈佛电器,目录号:30-0057)
  25. 用于体外研究的10mm直径NMR管(Norell,目录号:1008-UP-8)
  26. 用于体内研究的特殊自制NMR管(见图1)
  27. 硅胶膜
  28. Hebeloma cylindrosporum (ectomycorrhizal basidiomycete)(实验室自己的系列,可根据要求提供)
  29. Pinus pinaster
  30. Zea mays
  31. 浓缩(30%)过氧化氢(H 2 O 2 )溶液(Sigma-Aldrich,目录号:216763-500ML-M)
  32. 液氮(液化空气)
  33. 硫酸钙二水合物(CaSO 4 •2H 2 O)(默克,目录号:102161)
  34. 盐酸硫胺素-HCl(Sigma-Aldrich,目录号:T4625-10G)
  35. 硫酸锰(II)一水合物(MnSO 4 •H 2 O)(Sigma-Aldrich,目录号:M7899-500G)
  36. 硫酸锌七水合物(ZnSO 4 •7H 2 O)(Sigma-Aldrich,目录号:Z0251-100G)
  37. 硼酸(H 3 BO 3 )(Sigma-Aldrich,目录号:B6768-500G)
  38. 硫酸铜(II)五水合物(CuSO 4 •5H 2 O)(Sigma-Aldrich,目录号:C8027-500G)
  39. 钼酸钠二水合物(Na 2 MoO 4 •2H 2 O)(Sigma-Aldrich,目录号:M1651-100G)
  40. 硝酸钾(KNO 3 )(Sigma-Aldrich,目录号:P8291-1KG)
  41. 磷酸二氢钠一水合物(NaH 2 PO 4 •H 2 O)(Sigma-Aldrich,目录号:71504-250G-M)
  42. 硫酸镁七水合物(MgSO 4 •7H 2 O)(Sigma-Aldrich,目录号:63138-250G)
  43. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  44. 氯化钙二水合物(CaCl 2 •2H 2 O)(Sigma-Aldrich,目录号:C5080-500G)
  45. 柠檬酸铁铵(Sigma-Aldrich,目录号:RES20400-A702X)
  46. D-葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
  47. 琼脂(Sigma-Aldrich,目录号:A7002-500G)
  48. MES(2-N-吗啉代 - 乙磺酸,4-吗啉基甲磺酸一水合物)(Sigma-Aldrich,目录号:69892-500G)
  49. 三(羟甲基)氨基甲烷(TRIS)(Sigma-Aldrich,目录号:T1378-500G)
  50. 亚甲基二膦酸(MDP)(Sigma-Aldrich,目录号:M9508-5G)
  51. 1 N硫酸溶液(Merck,目录号:109072)
  52. 高氯酸70%(HClO 4 )(Sigma-Aldrich,目录号:311421-250ML)
  53. 偏钒酸钠(NaVO 3 )(Sigma-Aldrich,目录号:590088-25G)
  54. 碳酸氢钾(KHCO 3 )(Sigma-Aldrich,目录号:60339-500G)
  55. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758-500G)
  56. 氢氧化钾1 N(KOH)(默克,目录号:105029)
  57. 4-吗啉丙磺酸(MOPS)(Sigma-Aldrich,目录号:M1254-250G)
  58. 叠氮化钠(NaN 3 )(Sigma-Aldrich,目录号:S2002-100G)
  59. 氧化氘(D 2 O)(Sigma-Aldrich,目录号:151882-100G)
  60. 微量元素(见食谱)
  61. 矿物盐基础解决方案(见食谱)
  62. 硫胺素溶液(见食谱)
  63. N6完全液体溶液(见食谱)
  64. 固体N6全液体溶液(见食谱)
  65. 交互媒介(IM)(见食谱)

设备

  1. 样品瓶,120 ml(VWR,SPV,目录号:SPVAGO2246)
  2. 玻璃瓶,1,000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046415)&nbsp;
  3. 玻璃瓶,2,000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046416)&nbsp;
  4. 玻璃缸(直径7毫米)
  5. 刻度硼硅酸盐玻璃烧杯1,000 ml(Dominique DUTSCHER,目录号:068942)
  6. 聚丙烯经济型烧杯3,000,带模压刻度(Dominique DUTSCHER,目录号:391134)
  7. 两对不锈钢直镊子Wironit,Brucelles型,130毫米(Dominique DUTSCHER,目录号:491037)&nbsp;
  8. 刀片20至25的手术刀手柄(Dominique DUTSCHER,目录号:3740004)
  9. 独立式燃烧器(Dominique DUTSCHER,目录号:071109)
  10. 用于燃烧器的丁烷气筒(Dominique DUTSCHER,目录号:060415)
  11. 钉子,锤子,剪刀和剪钳
  12. 温度控制在25°C的培养箱&nbsp;
  13. 高压灭菌器
  14. 层流柜
  15. 直径7.5毫米的软木钻孔机(Dominique DUTSCHER,目录号:942783)
  16. 高压O 2 气瓶(空气产品)
  17. 离心机5804 R,冷藏,无转子,(Eppendorf,型号:5804 R,目录号:5805000017)
  18. 转子F-34-6-38,适用于6×85毫升管,含转子盖(Eppendorf,目录号:5804727002)
  19. 50毫升Falcon管适配器(Dominique DUTSCHER,目录号:033252)
  20. 蠕动泵(Gilson Minipuls3)&nbsp;
  21. Varian UNITY INOVA 500 MHz核磁共振谱仪,配备10 mm宽带探头,工作频率为202.4 MHz, 31 P

软件

  1. 用于计算的Microsoft Excel
  2. Statistica 7.1(StatSoft Inc.,Tulsa,OK,USA)用于统计分析
  3. 用于处理 31 P-NMR谱的VnmrJ和ACDlabs

程序

  1. 真菌和植物培养物的制备以及随后的植物真菌培养
    1. Hebeloma cylindrosporum 的培养条件 根据先前在 Bio-protocol (Becquer 等人,2017a)中描述的程序,将菌丝体在玻璃罐中培养。
    2. Pinus pinaster 的培养条件
      种子消毒,发芽和培养条件的程序先前已在 Bio-protocol 中进行了描述(Becquer et al。,2017b)。
    3. Zea mays的培养条件
      1. 用6%H 2 O 2 溶液对玉米种子( Zea mays L.)表面进行灭菌30分钟,然后用无菌冲洗蒸馏水。&nbsp;
      2. 为了发芽,将种子放在预先用去离子无菌水润湿的两张无菌滤纸之间。在30°C下在黑暗中孵育6天以进行发芽。&nbsp;
      3. 将幼苗转移到装有0.2mM CaSO 4 溶液(35ml /管)的试管中,在生长室中,在16/8 h光/暗循环,25°C /空气中的20℃,80%/ 100%RH,CO 2 体积分数为350×10 -6 且光合有效辐射为400μmolm - 2 sec -1 (400-700 nm)。&nbsp;
      4. 然后,将试管置于流动层流柜下,用每个试管中35ml相互作用介质(见配方6)代替CaSO 4 溶液。将试管放回生长室中,使其适应24小时。
    4. 为了研究引起真菌菌丝体特异性反应的植物根部信号,遵循先前在Becquer 等中描述的孵育程序。 (2017b)。

  2. 用于体内 NMR研究的真菌样品制备
    1. 设备准备
      1. 带有钩子的木制扦子,在其末端用镍铬合金丝制成。
      2. 用于参考NMR光谱(16.38ppm)化学位移的玻璃毛细管,填充50mM亚甲基二膦酸盐(MDP)在30mM Tris缓冲液(pH8.9)中的溶液(参见视频1如何制备毛细管)。 />

        视频1.使用MDP解决方案制作化学位移参考

      3. 一种特殊的自制核磁共振管,由3部分组成(见图1和视频2)
        1. 外部NMR管(直径10mm)。
        2. 内部玻璃圆筒(直径7毫米),可拧在外管上。
        3. 在该装置顶部的塑料盖,其被钻孔以允许3个特氟隆管将NMR管连接到蠕动泵,从而允许氧合的相互作用介质循环。入口管延伸到NMR管的底部。输出管延伸到NMR管中的菌丝体顶部。安全输出管的末端位于菌丝体上方3厘米处。
        该装置附有一根绳子,可以在不使用气升系统的情况下手动将其引入核磁共振谱仪磁铁的孔中,以避免3根PVC管和磁铁壁之间的摩擦。


        图1.用于体内 31 P NMR测量的装置的方案


        视频2.用于体内NMR测量的实验装置

      4. 一种允许含氧相互作用介质循环到上述管中的系统,在NMR数据采集过程中避免菌丝体的缺氧。
        它包括:
        1. 从NMR管延伸的3个特氟龙管通过PVC管连接到蠕动泵。
        2. 入口管从玻璃瓶(2,000ml)供应相互作用介质,其中氧气从压缩的O 2 气瓶鼓泡到NMR管。
        3. 输出管将已经氧化菌丝体的培养基收集到另一个原来空的玻璃瓶(2,000ml)中。
        4. 如果正常输出系统功能失常,安全输出管应避免磁铁孔中的任何泄漏。
    2. 在体内装置NMR管中引入真菌菌丝体(见视频2)
      1. 从烧瓶中取出真菌菌丝体,或从含有相互作用介质的注射器中取出(参见Becquer et al。,2017b),轻轻滴下溶液,用刮刀将其放在滤纸上或镊子。
      2. 将真菌菌丝体尽可能温和地放在NMR管的底部,首先使用刮刀,然后使用木制叉的扁平侧。对于 Hebeloma cylindrosporum ,在上述培养条件下获得的三个菌丝体样品对于完全松弛的数据采集的1小时的合理信噪比<1> 1 P-NMR是必需的。
      3. 借助木制扦子和钩子将化学位移参考毛细管放在NMR管的底部中心。
      4. 将内管拧在NMR管上。如果参考毛细管没有很好地定位在内管内,请再次启动并用钩子移动它以将其设置在内管的中间。
      5. 在塑料盖上滚动硅胶膜以确保氧化灌注溶液不会从NMR管中泄漏。
    3. 启动充氧灌注系统(改编自Lee和Ratcliffe,1983)
      1. 在含有通过入口管连接到NMR管的相互作用介质的玻璃瓶中开始氧气鼓泡。
      2. 将进口管,输出管和安全输出管的蠕动泵控制的流量调节至9 ml min -1 。
      3. 在弦谱的帮助下,用NMR光谱仪的磁体孔中的3根管子手动和缓慢地引入NMR管。
  3. 体外 NMR研究的真菌样品制备(改编自Roby et al。,1987)
    1. 用液氮冷冻菌丝体。
    2. 在萃取前将它们储存在-80°C。
    3. 对于每次 31 P-NMR测量,在4℃下解冻6个冷冻菌丝体,并用1ml含有10mM钒酸钠的70%(v / v)高氯酸将其压碎。
    4. 去除颗粒物质并中和样品中的高氯酸(改编自Aubert et al。,1996):
      1. 将浓悬浮液在10,000 x g 下离心10分钟,以丢弃沉淀中的颗粒物质。
      2. 将上清液转移到50 ml Falcon试管中,加入pH电极并通过非常缓慢地加入2 M KCO 3 溶液中和溶液,使泡沫消退,当pH值为6.5时停止。
      3. 再次以10,000 x g 离心10分钟以除去沉淀中的KClO 4 。
      4. 冷冻干燥上清液并将其储存在-80°C。
      5. 对于NMR测量,将冷冻干燥的材料重新溶解在2ml MOPS 40mM(未调节的pH)中。然后加入0.2ml EDTA 100mM。用KOH 1 N调节pH至7.8。
      6. 加入NaN 3 至终浓度100mg / ml -1 以避免微生物降解。
      7. 添加D 2 O(终浓度10%)用于NMR信号的场频锁定。
  4. 31 P-NMR数据采集
    1. 记录核磁共振谱,Varian UNITY INOVA 500 MHz,工作频率为202.4 MHz, 31 P,配备10 mm宽带探头,工作温度为25°C。
    2. 对于体内实验,使用以下参数获取条件
      1. 考虑到体内实验中 31 P NMR信号的广泛性,没有必要优化垫片。在没有锁定信号的情况下进行实验。
      2. 90°射频脉冲角;光谱宽度= 20,000 Hz;采集时间= 0.4秒; 31 P频率偏移= 3,900 Hz。没有解耦(考虑到 31 P-NMR峰体内核磁共振实验的广泛性没有用: 1 H去耦不会影响峰的线宽)。
      3. 20秒的扫描间延迟(自旋晶格弛豫时间T1的5倍)给出完全弛豫的光谱(Quiquampoix 等人,,1993)。这是用于定量分析 31 P-NMR谱中鉴定的化学代谢物的非常重要的参数。更低的扫描间延迟不允许实际量化。
      4. 扫描180次,总累积时间为1小时。
      5. 使用10 Hz指数函数对零谱进行切趾,零填充至16 K点。
      6. 相位和基线校正。
      注意:使用该方案获得的体内光谱的实例可以在Torres-Aquino等人中看到。 (2017)。
    3. 对于体外实验,使用以下参数获取条件
      1. 菌丝体样品中10%的D 2 O作为锁定信号。
      2. 90°射频脉冲角;光谱宽度= 9,600 Hz;采集时间= 0.84秒; 1 H在采集时间内使用华尔兹-16脉冲序列在4KHz的场强下解耦。 31 P频率偏移= 4,900 Hz; 1 H频率偏移= 170 Hz。
      3. 4秒扫描间延迟。在这些实验中,范围是基于信号化学位移鉴定磷酸化的真菌代谢物,而无需定量。如果需要定量,则使用20秒扫描间延迟,如体内研究。
      4. 足够数量的累积以获得可接受的信噪比。
      5. 使用2 Hz指数函数对光谱进行切趾,并将零填充至16 K点
      6. 相位和基线校正。
      注意:使用该方案获得的体外光谱的实例可以在Torres-Aquino等人中看到。 (2017)。

数据分析

Pi和polyP的NMR峰面积相对于MDP毛细管的NMR峰面积表示,对于所有实验都是相同的,并且任意固定为1.如果比较几种处理,使用Kolmogorov Smirnov检验测试数据的正态性,并在必要时,在分析之前将数据转换为平方根或log10,以满足ANOVA的假设。为了比较治疗效果,使用ANOVA分析,然后使用Tukey的真实显着性差异。对于 31 P-NMR测量,如ANVA之前的Legendre和Legendre(1998)所述,转化百分比数据(arcsin√x)。

笔记

  1. 由于真菌在液体培养基中的生长可能会有所不同,我们准备多达4个额外的培养瓶接种真菌,以丢弃那些生长不良的培养瓶。&nbsp;
  2. 如果您需要负责核磁共振设施单位的科学家的帮助,您可能必须让他们相信自制灌注核磁共振管用于体内研究的安全性。在NMR光谱仪外用该装置启动几个不同的实验,以检查没有液体泄漏。
  3. 如果使用较低磁场的NMR光谱仪,则必须增加累积的数量。
  4. 灌注NMR管中的含氧液体介质的流动是两种不希望的现象之间的折衷:(i)太低的流量不足以使细胞充分氧化和(ii)过高的流量将导致气泡形成在生物样品的基质中导致光谱分辨率降低。后一种效应通常不会低于10 ml min -1 (Lee和Ratcliffe,1983)。

食谱

  1. 微量元素(1,000毫升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:250px;” border =“0”cellspacing =“0”cellpadding =“2”>MnSO 4 •H 2 O
    3.08克
    ZnSO 4 •7H 2 O
    4.41克
    H 3 BO 3
    2.82克
    CuSO 4 •5H 2 O
    0.98克
    Na 2 MoO 4 •2H 2 O
    0.29克
    将ddH 2 O加入1,000 ml,在4°C下储存&nbsp;
  2. 矿物盐基溶液(各100毫升)

  3. 硫胺素溶液(100毫升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:250px;” border =“0”cellspacing =“0”cellpadding =“2”>硫胺素-HCl
    0.01克
  4. N6完全液体溶液(1,000毫升)
    1. 在1L烧杯中加入约500ml去离子水和以下体积的矿物质基础溶液:
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:250px;” border =“0”cellspacing =“0”cellpadding =“2”>KNO 3
      6毫升
      NaH 2 PO 4
      1毫升
      MgSO 4
      1毫升
      KCl
      4毫升
      CaCl 2
      0.5毫升
      柠檬酸铁铵
      0.5毫升
    2. 加入0.2毫升微量元素和0.5毫升硫胺素溶液
    3. 将体积调至1L并检查应调节至5.5的pH值
    4. 最后,加入5克D-葡萄糖并摇匀直至完全溶解
    5. 将此解决方案分配到玻璃罐中
  5. 固体N6完全液体溶液(1,000毫升)
    取两个1升玻璃瓶:
    1. 加入7.5g琼脂,并在每个瓶中倒入500ml液体N6培养基
    2. 通过在121℃下高压灭菌20分钟对培养基灭菌
    3. 将冷却的培养基(55-60°C)倒入直径90 mm的培养皿中
  6. 相互作用介质(3,000毫升)
    1. 在3L烧杯中加入约1.5L去离子水和以下体积的矿物基础溶液:&nbsp;
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:350px;” border =“0”cellspacing =“0”cellpadding =“2”>MgSO 4
      0.6毫升
      CaCl 2
      1.5毫升
      MES(终浓度为5 mM)
      3.2克
      TRIS(终浓度为5 mM)
      1.82克
    2. 用去离子水完成3L并用1N H 2 SO 4 将pH调节至5.5
    3. 将1.5升培养基倒入2升玻璃瓶中,并通过高压灭菌消毒(20分钟,121℃)

致谢

这项研究由INRA(法国)通过授予Adeline Becquer和CONACYT(墨西哥)授予博士学位的青年科学家合同提供支持。玛格丽塔托雷斯 - 阿基诺获得奖学金。该协议改编自我们以前的工作(Torres-Aquino et al。,2017)。作者声明他们没有利益冲突。

参考

  1. Ashford,A.E.,Ryde,S。和Barrow,K.D。(1994)。 在 Pisolithus中展示短链多磷酸盐tinctorius 以及对磷转运的影响。 新植物学家 126(2):239-247。
  2. Aubert,S.,Gout,E.,Bligny,R.,Marty-Mazars,D.,Barrieu,F.,Alabouvette,J.,Marty,F。和Douce,R。(1996)。 受到碳剥夺的高等植物细胞中自噬的超微结构和生化特征:通过供应线粒体来控制有呼吸基质。 J Cell Biol 133(6):1251-1263。
  3. Becquer,A.,Torres-Aquino,M.,LeGuernevé,C.,Amenc,L.K.,Trives-Segura,C.,Staunton,S.,Quiquampoix,H。和Plassard,C。(2017a)。 放射性标记 Hebeloma cylindrosporum 以研究植物 - 真菌相互作用的方法。 生物协议 7(20):e2576
  4. Becquer,A.,Torres-Aquino,M.,LeGuernevé,C.,Amenc,L.K.,Trives-Segura,C.,Staunton,S.,Quiquampoix,H。和Plassard,C。(2017b)。 在培养的外生菌根真菌和植物之间建立共生界面,以跟踪真菌磷酸盐代谢。 Bio-议定书 7(20):e2577
  5. Cairney,J。W. G.(2011)。 外生菌根真菌:森林土壤中磷的共生途径。 植物土壤 344:51-71。
  6. Lee,R。B.和Ratcliffe,R。G.(1983)。 开发用于植物组织核磁共振实验的曝气系统。 实验植物学杂志 34(146): 1213至1221年群组。
  7. Legendre,P。和Legendre,L。(1998)。在:数字生态学。第2版。第33-46页。 Elsevier Science B. V.阿姆斯特丹。
  8. Plassard,C。和Dell,B。(2010)。 菌根树的磷营养。 Tree Physiol 30( 9):1129-1139。
  9. Quiquampoix,H.,Bačić,G.,Loughman,B.C。和Ratcliffe,R.G。(1993)。 31 P-NMR检测锰的定量方面植物组织。 J Exp Bot 44(269):1809-1818。
  10. Quiquampoix,H。和Mousain,D。(2005)。 有机磷的酶促水解。在:环境中的有机磷,第89-112页。在:Turner,B.L。,Frossard,E。和Baldwin,D。(编辑)。 CAB国际。沃灵福德。
  11. Roby,C.,Martin,J.B.,Bligny,R。和Douce,R。(1987)。 高等植物细胞中蔗糖剥夺期间的生化变化。磷-31核磁共振研究。 J Biol Chem 262(11):5000-5007。
  12. Smith,S.E。和Read,D.J。(2008)。 Mycorrhizal symbiosis。第3版。学术出版社,伦敦。
  13. Smith,S.E.,Anderson,I。C. and Smith,F.A。(2015)。 菌根协会和磷的获取:从细胞到生态系统。 年度植物评论 48:409-440。
  14. Torres-Aquino,M.,Becquer,A.,Le Guerneve,C.,Louche,J.,Amenc,L.K.,Staunton,S.,Quiquampoix,H。和Plassard,C。(2017)。 寄主植物 Pinus pinaster 对磷酸盐外排和多磷酸盐代谢产生特定影响外生菌根真菌 Hebeloma cylindrosporu m:放射性示踪剂,细胞学染色和 31 P核磁共振波谱研究。 植物细胞环境 40( 2):190-202。
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引用:Le Guerneve, C., Becquer, A., Torres-Aquino, M., Amenc, L. K., Trives-Segura, C., Staunton, S., Plassard, C. and Quiquampoix, H. (2018). In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals. Bio-protocol 8(16): e2973. DOI: 10.21769/BioProtoc.2973.
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