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Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi
脂肪酸同位素标记谱分析追踪脂质从寄主植物到丛枝菌根真菌的转运   

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eLIFE
Jul 2017

Abstract

Lipid transfer from host plants to arbuscular mycorrhiza fungi was hypothesized for several years because sequenced arbuscular mycorrhiza fungal genomes lack genes encoding cytosolic fatty acid synthase (Wewer et al., 2014; Rich et al., 2017). It was finally shown by two independent experimental approaches (Jiang et al., 2017; Keymer et al., 2017; Luginbuehl et al., 2017). One approach used a technique called isotopolog profiling (Keymer et al., 2017). Isotopologs are molecules, which differ only in their isotopic composition. For isotopolog profiling an organism is fed with a heavy isotope labelled precursor metabolite. Subsequently, the labelled isotopolog composition of metabolic products is analysed via mass spectrometry. The detected isotopolog pattern of the metabolite(s) of interest yields information about metabolic pathways and fluxes (Ahmed et al., 2014). The following protocol describes an experimental setup, which enables separate isotopolog profiling of fatty acids in plant roots colonized by arbuscular mycorrhiza fungi and their associated fungal extraradical mycelium, to elucidate fluxes between both symbiotic organisms. We predict that this strategy can also be used to study metabolite fluxes between other organisms if the two interacting organisms can be physically separated.

Keywords: Arbuscular mycorrhiza (丛枝菌根), Lotus japonicus (百脉根), Isotopolog profiling (同位素标记谱分析), Stable isotope labelling (稳定同位素标记), Rhizophagus irregularis (异形根孢囊霉), MSR medium (MSR 培养基), Inter-organismic lipid transfer (机体间脂质转运), Root organ culture (根器官培养), Nurse plant system (保护植物系统)

Background

Arbuscular mycorrhiza fungi are biotrophic organisms. As such, they cannot be cultivated independently but rely on interaction with host plants to stay alive and complete their life cycle. This characteristic makes it challenging to study the two symbiotic organisms and especially the fungus separately.

To cultivate, treat and harvest the fungus separately from the host root, a 2-compartmented Petri dish system was developed and used for labelling studies in previous work (Bécard and Fortin, 1988; Pfeffer et al., 1999; Trépanier et al., 2005). This system is composed of two compartments; one containing a Ri (root-inducing) T-DNA transformed carrot root (Mosse and Hepper, 1975), which hosts the fungus (‘carrot compartment’) and another one, which contains only the fungus (fungal compartment), because the extraradical mycelium has grown across the border, which divides the two compartments. Kuhn et al. (2010) have advanced this setup to colonize Medicago truncatula tester plants in the fungal compartment.

In this protocol, we used this previous knowledge and further modified the system to our needs. Combining this growth system with stable isotopolog labelling and profiling (Eisenreich et al., 2013) enables analysis of plant and fungal metabolites separately from each other to gain information about metabolite fluxes between the two symbionts.

Materials and Reagents

  1. Growth system setup
    1. Fine sandpaper (60 µm)
    2. 2-Compartmented Petri dish (diam. 9.4 cm) (Greiner Bio One International, catalog number: 635161 )
    3. Square Petri dishes (12 cm) (Greiner Bio One International, catalog number: 688161 )
    4. Pipette tips
    5. Scalpel
    6. Parafilm
    7. 2.0 ml Eppendorf tubes
    8. 1.5 ml Eppendorf tubes
    9. Black card-sheet paper (50 x 70 cm; 150 g/m2)
    10. Adhesive tape
    11. Sterile Rhizophagus irregularis spores (Agronutrition, Carbonne, France; LOT: 000300195)
    12. 1% NaClO solution
    13. Bacto agar (BD, BactoTM, catalog number: 214010 )
    14. EtOH
    15. [U13C6]-Glucose (Sigma-Aldrich, catalog number: 389374 )
    16. 10% KOH
    17. Ink & vinegar staining solutions (Vierheilig et al., 1998)
    18. Liquid nitrogen
    19. MSR plate with carrot root organ culture (Bécard and Fortin, 1988)
    20. MSR plate with colonized carrot root organ culture (Bécard and Fortin, 1988)
    21. Sucrose
    22. Gelrite (Duchefa Biochemie, catalog number: G1101 )
    23. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
    24. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291 )
    25. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    26. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
    27. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (Sigma-Aldrich, catalog number: C2786 )
    28. Calcium pantothenate (B5) (Sigma-Aldrich, catalog number: 705837 )
    29. Biotin (B7) (Sigma-Aldrich, catalog number: B4639 )
    30. Nicotinic acid (B3) (Sigma Aldrich; catalog number: N0761 )
    31. Pyridoxine (B6) (Sigma-Aldrich, catalog number: P5669 )
    32. Thiamine (B1) (Sigma-Aldrich, catalog number: T1270 )
    33. Cyanocobalamine (B12) (Sigma-Aldrich, catalog number: V6629 )
    34. NaFeEDTA (Sigma-Aldrich, catalog number: E6760 )
    35. Manganese(II) sulfate tetrahydrate (MnSO4·4H2O) (Merck, catalog number: 102786 )
    36. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z1001 )
    37. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
    38. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C8027 )
    39. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1651 )
    40. Ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O) (Sigma-Aldrich, catalog number: M1019 )
    41. Sodium citrate dihydrate (Sigma-Aldrich, catalog number: W302600 )
    42. Citric acid monohydrate (Sigma-Aldrich, catalog number: C1909 )
    43. MSR-medium (w/3% gelrite, w/10% sucrose) (see Recipes)
      1. Solution 1: Macroelements
      2. Solution 2: Calcium nitrate
      3. Solution 3: Vitamins
      4. Solution 4: NaFeEDTA
      5. Solution 5: Microelements
    44. MSR medium (w/ 3% gelrite, w/o 10% sucrose) (see Recipes and adjust accordingly)
      Note: Refer to MSR-medium (w/ 3% gelrite, w/10% sucrose) in Recipe section for preparation of this medium.
    45. MSR medium (w/o 3% gelrite, w/o sucrose) (see Recipes and adjust accordingly)
      Note: Refer to MSR-medium (w/ 3% gelrite, w/ 10% sucrose) in Recipe section for preparation of this medium.
    46. 10 mM citrate buffer (see Recipes)
      1. 0.1 M sodium citrate
      2. 0.1 M citric acid

  2. Isotopolog profiling
    1. 1.5 ml glass vials and screw caps for GC/MS autosampler (VWR, catalog numbers: 548-0018 and 548-0814 )
    2. MeOH containing 3 M HCl (Sigma-Aldrich, catalog number: 33050-U )
    3. Hexane, anhydrous (Sigma-Aldrich, catalog number: 296090 )

Equipment

  1. Growth system setup
    1. Mortar
    2. Rotator
    3. Incubator at 25 °C without lights
    4. Plant growth chamber, set at 24 °C, 16/8 h day/night cycle
    5. Clean bench
    6. Bunsen burner
    7. Microwave
    8. 25 ml flasks
    9. Balance
    10. Magnetic stirrer
    11. Stir bars
    12. Forceps
    13. Pipettes
    14. Microcentrifuge
    15. Benchtop N2 container
    16. -80 °C freezer

  2. Isotopolog profiling
    1. Freeze dryer (Christ, Osterode, Germany)
    2. Drying oven
    3. GCMS-QP 2010 plus with autosampler AOC20i and LabSolution software (Shimadzu, model: GCMS-QP 2010 Plus )
    4. Silica capillary column (equity TM-5; 30 m by 0.25 mm, 0.25-µm film thickness; Sigma-Aldrich, Supelco, catalog number: 28089-U )

Software

  1. LabSolution software: https://www.shimadzu.com/an/gc/advflowtech/sw-dl.html
  2. Isotopo: http://www.tr34.uni-wuerzburg.de/software_developments/isotopo/
  3. R (https://www.r-project.org/)

Procedure

  1. Growth system setup
    1. Cultivation of Lotus japonicus seedlings
      1. Lotus japonicus seeds are carefully scarified with sandpaper inside a mortar until the seeds turn slightly grey (Figures 1A-1D). They are then surface sterilized with 1% NaClO for 6 min, washed three times with sterile water and subsequently incubated in sterile water on a rotator for 180 min at room temperature. Imbibed seeds are placed in four rows, each row with 20 seeds, on square Petri dishes (Figure 1E) containing 0.8% Bacto agar and incubated in the dark at 24 °C for 3 days.
      2. Subsequently, the germinated seedlings on square Petri dishes are cultivated for additional 10 days in the light (16 h/8 h day-night cycle) at 24 °C. Then they are used for the two-compartment setup (Figure 1G). The plates are placed into dark-colored flat boxes (Figure 1F) to provide a dark-stimulus to the roots from the bottom and encourage straight growth. 


        Figure 1. Cultivation of Lotus japonicus seedlings. A and C. Lotus japonicus seeds are carefully scarified with sandpaper inside a mortar until the seeds turn slightly grey. Seeds before (B) and after (D) scarification. E. Imbibed seeds on 0.8% Bacto agar plates. F. Cultivation of seedlings in dark-coloured flat boxes (16 h light/8 h dark for 10 days). G. Seedlings ready for transfer to 2-compartement setups.

    2. Cultivation of carrot root organ culture on 2-compartment test Petri dishes
      1. All steps need to be performed in a clean bench to avoid contamination of root organ culture.
      2. Both compartments (tester & nurse compartment, Figure 2A) of the spilt Petri dish are filled with MSR medium. However, only the MSR medium of the nurse compartment contains 10% sucrose, whereas the MSR medium of the tester compartment does not. Both compartments need to be filled with medium up to the top of the dish in order to assure the availability of sufficient substrate during the entire course of cultivation.
      3. A fresh root piece (about 2.5 cm) of a carrot root organ culture is placed onto the medium in the nurse compartment. The Petri dish is closed, sealed with Parafilm and incubated for 1 week upside down in darkness at 25 °C.
      4. 7 days later, the carrot root culture in the nurse compartment is inoculated with R. irregularis. To this end, MSR-agar blocks (about 0.125 cm3) from another root organ culture containing fungal spores and mycelium are placed on two different spots on the growing carrot root culture in the nurse compartment. The plate is sealed and again incubated upside down in total darkness at 25 °C to allow the fungus to colonize the whole plate including the tester compartment.
      5. Incubate for 4 to 6 weeks. The plates need to be checked regularly (about once per week) in order to assure that only the fungus crosses the compartment barrier but the carrot roots do not. If carrot roots grow across the barrier to the tester compartment they need to be pruned using sterile scissors.
      6. When the fungal extraradical mycelium has spread over both compartments (both compartments are covered with fungal hyphae) you can proceed to the next stage.
    3. Placement of two Lotus seedlings into the tester compartment
      1. Clean the lid of the Petri dish with EtOH using a tissue paper to ensure maximum sterility.
      2. Heat forceps in the flame of a Bunsen burner and use the hot forceps to melt three holes (approx. 0.5 cm diameter) into the lid of the Petri dish on the tester compartment side. Two of the holes are needed for Lotus growth and the third is later used for labelled substrate application.
      3. 13 days old Lotus seedlings are placed through the prepared lid holes in the tester compartment. Take care that the root is well-attached to the medium and is not hanging loose in the air.
      4. Seal the Petri dish with Parafilm.
      5. Carefully seal the two seedling holes as well as the application hole with Parafilm to avoid contamination.
      6. Cover seedlings immediately with a 2 ml Eppendorf tube (like a mini-greenhouse) to prevent them from drying out. Keep seedlings covered for the first 5 days of incubation.
      7. Build black envelopes yourself from commercially available cardboard sheets to cover the Petri dish such that the roots and fungus are kept in the dark and the Lotus shoots are in the light (Figures 2B-2D).
      8. Incubate for 3 weeks at 24 °C, 16/8 h day/night cycle until the fungus has colonized the root.
    4. Application of [U13C6]-glucose
      1.  Labelled glucose is applied 3 weeks after Lotus seedling placement.
      2. 100 mg of [U13C6]-glucose are dissolved in 2 ml MSR-medium (w/o sugar, w/o gelrite).
      3. The 2 ml [U13C6]-glucose/MSR solution is carefully pipetted into the tester compartment via the application hole in the lid. Strictly avoid that the labelled glucose solution also drips in the nurse compartment and wait until the liquid has soaked into the agar before moving the plate.
      4. After application of the labelled substrate, the application hole of the Petri dish is sealed again and the setup is incubated in the growth chamber for another week.
        Note: One week after the stable isotope-labelled substrate has been added, the tester plants as well as the extraradical mycelium of the fungus are harvested.
    5. Harvest of the tester plants
      1. Both plants are carefully isolated from the plate. By slowly lifting the lid of the Petri dish the attached Lotus plants will be extricated from the medium. Make sure that no gelrite pieces remain on the roots.
      2. Use one of the plants for quantification of root length colonization. Place the root of this plant in 10% KOH. Later stain the root with ink & vinegar (Vierheilig et al., 1998) and quantify root length colonization using a modified gridline intersect method (McGonigle et al., 1990).
      3. The other root system is used for isotopolog profiling. Place it into a 2 ml Eppendorf tube and immediately shock freeze it in liquid nitrogen.
    6. Extraction of the fungal extraradical mycelium
      1. The MSR/gelrite medium of the tester compartment is cut into small pieces (about 0.125 cm3) and transferred into a 25 ml flask filled with citrate buffer.
      2. The buffer containing gelrite pieces is stirred for 15 min using a magnetic stirrer (750 rpm). Within this time the gelrite is dissolved and the fungal mycelium knots together.
      3. When knotted together, transfer the mycelium into a 1.5 ml Eppendorf tube by fishing it from the buffer with forceps.
      4. Centrifuge the tube for 1 min at 9,391 x g and remove the residual liquid.
      5. Subsequently shock freeze the sample in liquid nitrogen and keep it in a -80 °C freezer until mass spectrometry. 


        Figure 2. Growth system setup. A. Cultivated Lotus japonicus plants in the split Petri dish growth system at 4 weeks post placement. B. Example of a black carton envelope to keep the plate with roots and fungus in the dark. The red adhesive tape serves to close the opening of the paper envelope. C-D. Completed setup ready for incubation without (C) or with (D) Eppendorf tubes to prevent the seedlings from drying out.

  2. Isotopolog profiling
    1. Freeze dry the samples.
    2. Transfer samples to 1.5 ml glass vials.
    3. Derivatize with 500 µl MeOH containing 3 M HCl (Sigma-Aldrich) at 80 °C for 20 h.
    4. Dry the sample using a gentle stream of nitrogen gas.
    5. Add 100 µl dry hexane to dissolve methyl esters of the fatty acids.
    6. Analyze the samples by GC/MS: use a quadrupole GC/MS machine equipped with an autosampler and heated injection port (GCMS-QP 2010 plus; Shimadzu).
    7. GC/MS setup.
    8. Inject an aliquot of the solution in split mode (1:5) at an injector and interface temperature of 260 °C.
    9. Hold the column at 170 °C for 3 min, then increase temperature by a gradient of 2 °C/min to a temperature of 192 °C, afterwards by a temperature gradient of 30 °C/min to a final temperature of 300 °C.
    10. Perform one scan run for each sample, detecting a mass range from m/z 45-600, for unambiguous identification of the fatty acid and for confirmation of the retention times; this is especially important after a longer period, without this special analysis, and after a column change.
    11. Analyse samples in SIM (single ion monitoring) mode (m/z values 267 to 288) at least three times. Retention times for fatty acids 16:1 ω5 (unlabeled m/z 268) and 16:0 (unlabeled m/z 270) are 12.87 min and 13.20 min, respectively. For the analysis of other fatty acids, determine retention times and m/z values in scan runs and implement the values into the SIM method.
    12. Collect data with LabSolution software or the software connected to your GC/MS system.
    13. Calculate overall 13C enrichment and isotopolog composition by comparison with an unlabeled sample according to Ahmed et al. (2014). The software package is open source and can be downloaded by the following link: http://www.tr34.uni-wuerzburg.de/software_developments/isotopo/.
    14. Compare overall 13C excess (average value of 13C atoms incorporated into 16:0/16:1 ω5 fatty acids) as well as isotopomer distribution (M + 1, M + 2, M + 3, … M + 16). For detailed explanation of nomenclature see Eisenreich et al. (2013).
    15. Perform at least three independent labeling experiments.
    16. Data can be displayed as stacked columns as shown in Figure 3.


      Figure 3. Schematic representation of the istotopolog profiling pipeline. Analysis of samples via GC/MS SIM results in chromatograms of derivatized fatty acids. The individual mass spectra of 16:0 and 16:1 ω5 are extracted and the isotopomer distribution as well as the 13C overall excess (o.e) of these fatty acids is calculated. The isotopomer patterns can be displayed as stacked bars.

Data analysis

Statistical differences of overall 13C excess values for the different tested plant genotypes are analyzed via ANOVA followed by posthoc Tukey test in R, using at least 3 biological replicates per genotype.

Notes

Differences in root system development, distribution of labelled substrate on the Petri dish and the resulting differences in uptake of labelled substrate by the plant can lead to divergent isotopolog patterns among samples (see Keymer et al., 2017). However, when a metabolite (i.e., lipid) is transferred from the plant to the fungus, the isotopolog pattern among plant root and associated extraradical fungal mycelium is equivalent, notwithstanding the inter-sample variation.

Recipes

  1. MSR-medium (w/ 3% gelrite, w/ 10% sucrose)
    Medium preparation for 1 L:
    Solution 1
    10 ml
    Solution 2
    10 ml
    Solution 4
    5 ml
    Solution 5
    1 ml
    Sucrose
    10 g
    Adjust to pH 5.5

    Autoclave

    Solution 3
    5 ml
    1. Solution 1: Macroelements
      39.6 g MgSO4·7H2O
      3.8 g KNO3
      3.3 g KCl
      0.21 g KH2PO4 in 500 ml H2O
    2. Solution 2: Calcium nitrate
      17.95 g Ca(NO3)2·4H2O in 500 ml H2O
    3. Solution 3: Vitamins
      90 mg calcium panthotenate (B5)
      0.1 mg biotin (B7)
      100 mg nicotinic acid (B3)
      90 mg pyridoxine (B6)
      100 mg thiamine (B1)
      40 mg cyanocobalamine (B12) in 500 ml H2O
    4. Solution 4: NaFeEDTA
      0.4 g NaFeEDTA in 500 ml H2O
    5. Solution 5: Microelements
      (1) 1.225 g MnSO4·4H2O in 50 ml adjust to 100 ml
      (2) 0.14 g ZnSO4·7H2O in 50 ml adjust to 100 ml
      (3) 0.925 g H3BO3 in 50 ml adjust to 100 ml
      (4) 1.1 g CuSO4·5H2O in 30 ml adjust to 50 ml
      (5) 0.12 g Na2MoO4·2H2O in 50 ml adjust to 100 ml
      (6) 1.7 g (NH4)6Mo7O24·4H2O in 50 ml adjust to 100 ml
      Mix 100 ml (1) + 100 ml (2) + 100 ml (3) +5ml (4) + 1 ml (5) + 1 ml (6) and adjust to 500 ml to obtain Solution 5
  2. 10 mM citrate buffer
    180 ml (0.1 M sodium citrate) + 820 ml (0.1 M citric acid) (you need 25 ml per compartment)
    For 1.0 L stock solutions:
    1. 0.1 M sodium citrate: 29.41 g sodium citrate dehydrate (FW = 294.10 g/mol)
    2. 0.1 M citric acid: 21.01 g citric acid monohydrate (FW = 210.14 g/mol)

Acknowledgments

This protocol was developed for the work published in Keymer et al. (2017) financed by the Hans Fischer Gesellschaft e. V. to WE and by the Collaborative Research Center 924 (SFB924) of the Deutsche Forschungsgemeinschaft (DFG) ‘Molecular Mechanisms of Yield and Yield Stability in Plants’ (project B03) to CG. The authors have not conflicts of interest or competing interests.

References

  1. Ahmed, Z., Zeeshan, S., Huber, C., Hensel, M., Schomburg, D., Münch, R., Eylert, E., Eisenreich, W., and Dandekar, T. (2014). ‘Isotopo’ a database application for facile analysis and management of mass isotopomer data. Database (Oxford): bau077.
  2. Bécard, G. and Fortin, J. A. (1988). Early events of vesicular–arbuscular mycorrhiza formation on Ri T-DNA transformed roots. New Phytologist 108: 211-218.
  3. Eisenreich, W., Huber, C., Kutzner, E., Knispel, N. and Schramek, N. (2013). Isotopologue profiling: towards a better understanding of metabolic pathways. In: Weckwerth, W. and Kahl, G. (Eds). The Handbook of Plant Metabolomics. Wiley-Blackwell 26-56.
  4. Jiang, Y., Wang, W., Xie, Q., Liu, N., Liu, L., Wang, D., Zhang, X., Yang, C., Chen, X., Tang, D. and Wang, E. (2017). Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi. Science 356: 1172-1175.
  5. Keymer, A., Pimprikar, P., Wewer, V., Huber, C., Brands, M., Bucerius, S. L., Delaux, P. M., Klingl, V., Ropenack-Lahaye, E. V., Wang, T. L., Eisenreich, W., Dormann, P., Parniske, M. and Gutjahr, C. (2017). Lipid transfer from plants to arbuscular mycorrhiza fungi. Elife 6: e29107.
  6. Kuhn, H., Kuster, H. and Requena, N. (2010). Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula. New Phytol 185(3): 716-733.
  7. Luginbuehl, L. H., Menard, G. N., Kurup, S., Van Erp, H., Radhakrishnan, G. V., Breakspear, A., Oldroyd, G. E. D. and Eastmond, P. J. (2017). Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant. Science 356: 1175-1178.
  8. McGonigle, T. P., Miller, M. H., Evans, D. G., Fairchild, G. L. and Swan, J. A. (1990). A new method which gives an objective measure of colonization of roots by vesicular—arbuscular mycorrhizal fungi. New Phytol 115: 495-501.
  9. Mosse, B. and Hepper, C. (1975). Vesicular-arbuscule mycorrhizial infections in root organ cultures. Phys Plant Pathol 5: 215-223.
  10. Pfeffer, P. E., Douds Jr, D. D., Becard, G. and Shachar-Hill, Y. (1999). Carbon uptake and the metabolism and transport of lipids in an arbuscular mycorrhiza. Plant Physiol 120(2): 587-598.
  11. Rich, M. K., Nouri, E., Courty, P. E. and Reinhardt, D. (2017). Diet of arbuscular mycorrhizal fungi: bread and butter? Trends Plant Sci 22(8): 652-660.
  12. Trépanier, M., Becard, G., Moutoglis, P., Willemot, C., Gagne, S., Avis, T. J. and Rioux, J. A. (2005). Dependence of arbuscular-mycorrhizal fungi on their plant host for palmitic acid synthesis. Appl Environ Microbiol 71(9): 5341-5347.
  13. Vierheilig, H., Coughlan, A. P., Wyss, U. and Piche, Y. (1998). Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microbiol 64: 5004-5007.
  14. Wewer, V., Brands, M. and Dormann, P. (2014). Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus Rhizophagus irregularis during mycorrhization of Lotus japonicus. Plant J 79: 398-412.

简介

因为测序的丛枝菌根真菌基因组缺乏编码胞质脂肪酸合酶的基因(Wewer等人,2014; Rich等人,2014),因此假定脂质从宿主植物转移到丛枝菌根真菌数年。 / em>,2017)。最终通过两种独立的实验方法(Jiang等人,2017; Keymer等人,2017; Luginbuehl等人, ), 2017年)。一种方法使用称为同位素体谱分析的技术(Keymer等人,2017)。同位素体是分子,它们的同位素组成不同。对于同位素生物学分析,生物体被喂以重同位素标记的前体代谢物。随后,通过质谱分析代谢产物的标记同位素组成。检测到的目标代谢物的同位素体模式产生关于代谢途径和通量的信息(Ahmed et al。,2014)。以下协议描述了一个实验装置,该装置能够在由丛枝菌根真菌及其相关真菌胞外菌丝体定植的植物根中分离出脂肪酸的单独同位素分布图,以阐明两种共生生物之间的通量。我们预测,如果两种相互作用的生物体可以物理分离,则该策略还可以用于研究其他生物体之间的代谢物通量。

【背景】丛枝菌根真菌是生物营养生物。因此,它们不能独立栽培,而是依靠与寄主植物的相互作用来维持生命并完成它们的生命周期。这一特征使得研究两种共生生物,特别是单独的真菌具有挑战性。

为了培养,处理和收获与宿主根分开的真菌,开发了2室培养皿系统,并将其用于以前工作中的标记研究(Bécard和Fortin,1988; Pfeffer等人, 1999;Trépanieret al。,2005)。该系统由两部分组成;一个含有Ri(根诱导)T-DNA转化的胡萝卜根(Mosse和Hepper,1975),其含有真菌('胡萝卜隔室')和另一个仅含有真菌(真菌隔室),因为根外菌丝已经越过边界,分隔了两个隔间。库恩等人(2010)已经推进了这种设置以在真菌隔离区中定殖Medic藜苜蓿tester试验植物。

在这个协议中,我们使用了此前的知识,并进一步修改了系统以满足我们的需求。将这种生长系统与稳定的同位素标记和分析相结合(Eisenreich et al。,2013)可以分别分离植物和真菌代谢物,以获得关于两个共生体之间代谢物流量的信息。

关键字:丛枝菌根, 百脉根, 同位素标记谱分析, 稳定同位素标记, 异形根孢囊霉, MSR 培养基, 机体间脂质转运, 根器官培养, 保护植物系统

材料和试剂

  1. 增长系统设置
    1. 细砂纸(60微米)
    2. 2-Compartmented培养皿(直径9.4厘米)(Greiner Bio One International,目录号:635161)
    3. 方形培养皿(12厘米)(Greiner Bio One International,目录号:688161)
    4. 移液器吸头
    5. 手术刀
    6. Parafilm
    7. 2.0 ml Eppendorf管
    8. 1.5毫升Eppendorf管
    9. 黑色卡片纸(50×70厘米; 150克/米2 )
    10. 胶带
    11. 不育(Rhizophagus irregularis)孢子(Agronutrition,Carbonne,France; LOT:000300195)
    12. 1%NaClO溶液
    13. 细菌琼脂(BD,Bacto TM,产品目录号:214010)
    14. EtOH
    15. [U 13 C 16] - 葡萄糖(Sigma-Aldrich,目录号:389374)
    16. 10%KOH
    17. 墨水&醋染色溶液(Vierheilig et al。,1998)
    18. 液氮
    19. MSR板与胡萝卜根器官培养(Bécard和Fortin,1988)
    20. 具有殖民胡萝卜根器官培养物的MSR板(Bécard和Fortin,1988)
    21. 蔗糖
    22. Gelrite(Duchefa Biochemie,产品目录号:G1101)
    23. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M2773)
    24. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291)
    25. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    26. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
    27. 硝酸钙四水合物(Ca(NO 3)2·4H 2 O)(Sigma-Aldrich,目录号:C2786)
    28. 泛酸钙(B5)(Sigma-Aldrich,目录号:705837)
    29. 生物素(B7)(Sigma-Aldrich,目录号:B4639)
    30. 烟酸(B3)(Sigma Aldrich;目录号:N0761)
    31. 吡哆醇(B6)(Sigma-Aldrich,目录号:P5669)
    32. 硫胺素(B1)(Sigma-Aldrich,目录号:T1270)
    33. Cyanocobalamine(B12)(Sigma-Aldrich,目录号:V6629)
    34. NaFeEDTA(Sigma-Aldrich,目录号:E6760)
    35. 硫酸锰(II)四水合物(MnSO 4·4H 2 O)(Merck,目录号:102786)
    36. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z1001)
    37. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B6768)
    38. 五水合硫酸铜(II)(CuSO 4·5H 2 O)(Sigma-Aldrich,目录号:C8027)
    39. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(Sigma-Aldrich,目录号:M1651)
    40. 钼酸铵四水合物((NH4)6Mo7O24·4H2 / 2 > O)(Sigma-Aldrich,目录号:M1019)
    41. 柠檬酸钠二水合物(Sigma-Aldrich,目录号:W302600)
    42. 柠檬酸一水合物(Sigma-Aldrich,目录号:C1909)
    43. MSR培养基(w / 3%gelrite,w / 10%蔗糖)(见食谱)
      1. 解决方案1:宏元素
      2. 解决方案2:硝酸钙
      3. 解决方案3:维生素
      4. 解决方案4:NaFeEDTA
      5. 解决方案5:微元素
    44. MSR培养基(w / 3%gelrite,w / o 10%蔗糖)(见配方并相应调整)
      注:参考配方章节中的MSR培养基(w / 3%gelrite,w / 10%蔗糖)制备该培养基。
    45. MSR培养基(不含3%gelrite,不含蔗糖)(见配方并据此调整)
      注意:请参阅配方章节中的MSR培养基(w / 3%gelrite,w / 10%蔗糖)来制备此培养基。
    46. 10 mM柠檬酸盐缓冲液(见食谱)
      1. 0.1 M柠檬酸钠
      2. 0.1 M柠檬酸

  2. 同位素分析
    1. 用于GC / MS自动进样器的1.5 ml玻璃瓶和螺帽(VWR,产品目录号:548-0018和548-0814)
    2. 含3M HCl的MeOH(Sigma-Aldrich,目录号:33050-U)
    3. 己烷,无水(Sigma-Aldrich,目录号:296090)

设备

  1. 增长系统设置
    1. 砂浆
    2. 转子
    3. 在25°C没有灯的孵化器
    4. 植物生长室,设置在24°C,16/8小时昼夜周期
    5. 洁净工作台
    6. 本生燃烧器
    7. 微波炉

    8. 25毫升烧瓶
    9. 平衡
    10. 磁力搅拌器
    11. 搅拌棒
    12. 镊子
    13. 移液器
    14. 微量离心机
    15. 台式N 2 容器
    16. -80°C冷冻机

  2. 同位素分析

    1. 冷冻干燥机(Christ,Osterode,德国)
    2. 干燥箱
    3. GCMS-QP 2010 plus带自动进样器AOC20i和LabSolution软件(岛津,型号:GCMS-QP 2010 Plus)
    4. 二氧化硅毛细管柱(产品TM-5; 30m×0.25mm,0.25μm膜厚; Sigma-Aldrich,Supelco,目录号:28089-U)

软件

  1. LabSolution软件: https://www.shimadzu.com/an/gc/ advflowtech / sw-dl.html
  2. Isotopo: http://www.tr34.uni-wuerzburg.de/software_developments/isotopo/
  3. R( https://www.r-project.org/

程序

  1. 增长系统设置
    1. 莲藕幼苗的栽培
      1. 用研钵内的砂纸小心地将莲花种子种子弄湿,直到种子变成浅灰色(图1A-1D)。然后用1%NaClO将其表面灭菌6分钟,用无菌水洗涤三次,随后在室温下在旋转器中在无菌水中孵育180分钟。在含有0.8%Bacto琼脂的正方形培养皿(图1E)上放置四行,每行20粒种子,并在24℃黑暗中培养3天。
      2. 随后,将方培养皿上发芽的幼苗在24℃下光照(16小时/ 8小时昼夜循环)再培养10天。然后将它们用于双室设置(图1G)。将平板置于深色平坦的盒子中(图1F),以从底部向根部提供黑暗刺激并促进直线生长。 


        图1.栽培莲子幼苗 A和C.
    2. 在2室试验培养皿上培养胡萝卜根器官培养物
      1. 所有的步骤都需要在干净的工作台上进行,以避免污染根器官培养。
      2. 溢出的培养皿的两个隔室(检测器和护士隔室,图2A)都填充有MSR培养基。然而,只有护士隔室的MSR培养基含有10%蔗糖,而试验室隔室的MSR培养基则不含。两个隔间都需要填充培养基直到培养皿的顶部,以确保在整个培养过程中获得足够的底物。
      3. 将新鲜的根部碎片(约2.5厘米)的胡萝卜根器官培养物放置在护理室中的培养基上。将培养皿关闭,用Parafilm密封并在25℃黑暗中颠倒培养1周。
      4. 7天后,在护士隔室中的胡萝卜根培养物接种了R。 irregularis 。为此,将来自另一含有真菌孢子和菌丝体的根器官培养物的MSR-琼脂块(约0.125cm 3)置于护士隔室中生长的胡萝卜根培养物上的两个不同点上。将板密封并在25℃下在黑暗中再次颠倒孵育,以使真菌在包括测试器室在内的整个板上定殖。
      5. 孵育4至6周。需要定期检查平板(每周大约一次),以确保只有真菌穿过隔间屏障,但胡萝卜根不能。如果胡萝卜根横跨屏障进入测试室,则需要使用无菌剪刀进行修剪。
      6. 当真菌菌根菌丝体扩散到两个隔间(两个隔间都覆盖有真菌菌丝)时,您可以进入下一个阶段。
    3. 将两个莲花幼苗放入测试器室中

      1. 使用薄纸清洁EtOH培养皿的盖子以确保最大的无菌性。
      2. 在本生灯火焰中加热镊子,并使用热镊子将三个孔(约0.5cm直径)融化到测试器室侧的培养皿盖上。其中两个孔用于 Lotus 生长,第三个用于标记底物应用。
      3. 将13日龄荷花幼苗放置在测试器室中准备好的盖孔中。请注意,根部与媒体连接良好,不会悬空在空中。

      4. 用Parafilm密封培养皿。

      5. 。仔细密封两个幼苗孔和石蜡膜的应用孔以避免污染。
      6. 立即用2毫升Eppendorf管(如小温室)覆盖幼苗,以防止它们变干。孵化前5天保持幼苗。
      7. 使用市售的纸板自己制作黑色信封以覆盖陪替氏培养皿,使根和真菌保持在黑暗中,而莲藕芽在光照下(图2B-2D)。
      8. 在24°C,16/8小时昼夜循环下孵育3周,直至真菌已经侵染根部。
    4. 应用[U136C6] - 葡萄糖
      1.  标记的葡萄糖在莲花放置后3周施用。
      2. 将100mg [U 13 C 6] - 葡萄糖溶于2ml MSR培养基(无糖,无胶凝物)中。
      3. 通过盖上的应用孔小心地将2ml [U136C6] - 葡萄糖/ MSR溶液移液到测试室中。严格避免标记的葡萄糖溶液滴落在护士隔间中,等待液体浸入琼脂中,然后移动平板。
      4. 施加标记的底物后,再次密封培养皿的应用孔,并将设置在生长室中培养一周。
        注意:加入稳定同位素标记的底物一周后,收获测试植物以及真菌的根外菌丝体。
    5. 收获测试植物
      1. 这两种植物都是从平板上小心分离的。通过缓慢提起培养皿的盖子,附着的莲花植物将从培养基中排出。确保没有凝胶块残留在根部。
      2. 使用其中一种植物定量定殖根长。将此植物的根放入10%KOH中。稍后用油墨和水沾染根部。醋(Vierheilig等人,1998),并使用修改的网格线相交方法(McGonigle等人,1990)量化根长定植。
      3. 另一个根系用于同位素体谱分析。将其放入2 ml Eppendorf管中,立即用液氮将其冷冻。
    6. 提取真菌外根菌丝体
      1. 将试验室的MSR / gelrite培养基切成小块(约0.125cm 3)并转移到装满柠檬酸盐缓冲液的25ml烧瓶中。
      2. 使用磁力搅拌器(750rpm)将含有凝胶块的缓冲液搅拌15分钟。在这段时间内,凝胶溶解并且真菌菌丝结合在一起。
      3. 当打结在一起时,通过用镊子从缓冲液中捞出将菌丝体转移到1.5ml Eppendorf管中。

      4. 在9,391 em x g的条件下离心管1分钟,除去残留的液体。
      5. 随后在液氮中震荡冷冻样品并将其保存在-80°C冷冻箱中直至质谱分析。 


        图2.增长系统设置。 :一种。在放置后4周,在分裂的培养皿生长系统中栽培的 Lotus japonicus 植物。 B.黑色纸箱信封在黑暗中保持盘根和真菌的例子。红色胶带用于关闭纸质信封的开口。光盘。无需(C)或使用(D)Eppendorf管完成孵化的准备就绪,以防止幼苗变干。

  2. 同位素分析
    1. 冻结样品。
    2. 将样品转移至1.5 ml玻璃瓶。

    3. 在80℃下用含有3M HCl(Sigma-Aldrich)的500μlMeOH衍生20小时。

    4. 使用温和的氮气流干燥样品
    5. 加入100μl干燥己烷溶解脂肪酸甲酯。
    6. 通过GC / MS分析样品:使用配备自动进样器和加热进样口的四极杆GC / MS机器(GCMS-QP 2010 plus; Shimadzu)。
    7. GC / MS设置。
    8. 在分流模式下(1:5)注入等份的溶液,注射器和界面温度为260°C。
    9. 在170°C保持3分钟,然后以2°C / min的梯度升温至192°C,然后以30°C / min的温度梯度升温至300°C的最终温度。
    10. 对每个样品进行一次扫描,检测质量范围从
    11. 以SIM(单离子监测)模式( m / z <值> 267至288)分析样品至少三次。脂肪酸16:1ω5(未标记的m / z 268)和16:0(未标记的m / z 270)的保留时间分别为12.87分钟和13.20分钟。为了分析其他脂肪酸,确定扫描运行中的保留时间和em / m / z值,并将这些值应用到SIM方法中。
    12. 使用LabSolution软件或连接到GC / MS系统的软件收集数据。
    13. 根据Ahmed等人的方法,通过与未标记的样品比较来计算总体13 C富集度和同位素组成。 (2014)。该软件包是开源的,可以通过以下链接下载: http:// www .tr34.uni-wuerzburg.de / software_developments / isotopo /
    14. 比较总体13 C过量(掺入16:0/16:1ω5脂肪酸中的13 C原子的平均值)以及同位素异位分布(M + 1,M + 2,M + 3,... M + 16)。有关命名法的详细说明,请参见Eisenreich et al。(2013)。
    15. 执行至少三次独立的标记实验。
    16. 数据可以显示为堆叠列,如图3所示。


      图3.等位基因谱分析流程的示意图。通过GC / MS SIM分析样品得到衍生脂肪酸的色谱图。提取16:0和16:1ω5的单个质谱并计算这些脂肪酸的同位素体分布以及13 C总体过量(o.e)。同位素体模式可以显示为堆积条。

数据分析

使用每种基因型的至少3个生物学重复,通过ANOVA随后在R中进行posthoc Tukey测试来分析不同测试植物基因型的总体13 C过量值的统计差异。

笔记

根系发育的不同,标记底物在培养皿上的分布以及植物对标记底物的吸收差异可能导致样品之间不同的同位素体模式(参见Keymer等人,2017) 。然而,当代谢物(即,脂质)从植物转移到真菌时,尽管样品间变化,植物根和相关的外生真菌菌丝体之间的同位素模式是相等的。

食谱

  1. MSR培养基(w / 3%gelrite,w / 10%蔗糖)
    中等准备1升:
    解决方案1
    10毫升
    解决方案2
    10毫升
    解决方案4
    5毫升
    解决方案5
    1毫升
    蔗糖
    10克
    调整到pH 5.5

    高压灭菌器

    解决方案3
    5毫升
    1. 解决方案1:39.6克MgSO 4·7H 2 O0.21克KH 2 PO 4在500毫升H 2 O中的溶液
    2. 解决方案2:硝酸钙 在500毫升H 2 O中加入17.95克Ca(NO 3)2·4H 2 O, 
    3. 解决方案3:维生素90毫克泛酸钙(B5) 0.1毫克生物素(B7)100毫克烟酸(B3) 90毫克吡哆醇(B6)100毫克硫胺素(B1) 40毫克氰钴胺(B12)在500毫升H 2 O中
    4. 解决方案4:NaFeEDTA在500ml H 2 O中的0.4g NaFeEDTA
    5. 解决方案5:Microelements (1)将1.225克MnSO 4·4H 2 O在50毫升中调节至100毫升(2)将0.14g ZnSO 4·7H 2 O在50ml中调节至100ml(3)将0.925克H 3 BO 3在50毫升中调节至100毫升(4)将1.1g CuSO 4·5H 2 O在30ml中调节至50ml(5)将0.12克Na 2 MoO 4·2H 2 O在50毫升中调节至100毫升(6)1.7克(NH 4)6 Mo 7 O 24·4H 2 2在50毫升的O> O调整到100毫升 将100ml(1)+ 100ml(2)+ 100ml(3)+ 5ml(4)+ 1ml(5)+ 1ml(6)混合并调整至500ml以获得溶液5。
  2. 10 mM柠檬酸缓冲液
    180 ml(0.1 M柠檬酸钠)+ 820 ml(0.1 M柠檬酸)(您需要每隔25 ml)
    对于1.0 L库存解决方案:
    1. 0.1M柠檬酸钠:29.41g脱水柠檬酸钠(FW = 294.10g / mol)
    2. 0.1M柠檬酸:21.01g柠檬酸一水合物(FW = 210.14g / mol)
  3. 致谢

    该协议是为在Keymer等人发表的作品而开发的。 (2017)由Hans Fischer Gesellschaft e。提供资助。 V.到WE以及德国联合研究中心(DFG)的合作研究中心924(SFB924)的“植物产量和产量稳定性的分子机制”(项目B03)。作者没有利益冲突或利益冲突。

    参考

    1. Ahmed,Z.,Zeeshan,S.,Huber,C.,Hensel,M.,Schomburg,D.,Münch,R.,Eylert,E.,Eisenreich,W.和Dandekar,T.(2014)。 'Isotopo'是一种用于轻松分析和管理质量同位素异构体数据的数据库应用程序。 a> Database(Oxford):bau077。
    2. Bécard,G。和Fortin,J.A。(1988)。 Ri T-DNA上水泡 - 丛枝菌根形成的早期事件转化的根。新植物学家 108:211-218。
    3. Eisenreich,W.,Huber,C.,Kutzner,E.,Knispel,N.和Schramek,N.(2013)。 同位素分布图:为了更好地理解代谢途径。 在:Weckwerth,W.和Kahl,G。(Eds)。植物代谢组学手册。 Wiley-Blackwell 26-56。
    4. Jiang,Y.,Wang,W.,Xie,Q.,Liu,N.,Liu,L.,Wang,D.,Zhang,X.,Yang,C.,Chen,X.,Tang,D.and Wang,E。(2017)。 植物转移脂质以维持互生菌根和寄生真菌的定植。 科学 356:1172-1175。
    5. Keymer,A.,Pimprikar,P.,Wewer,V.,Huber,C.,Brands,M.,Bucerius,SL,Delaux,PM,Klingl,V.,Ropenack-Lahaye,EV,Wang,TL,Eisenreich, W.,Dormann,P.,Parniske,M.和Gutjahr,C。(2017)。 从植物到植物丛枝菌根真菌的脂质转移 Elife 6:e29107。
    6. Kuhn,H.,Kuster,H。和Requena,N。(2010)。 由扩散性真菌信号诱导的膜类固醇结合蛋白1对于苜蓿中的菌根化是至关重要的truncatula 。 New Phytol 185(3):716-733。
    7. Luginbuehl,L. H.,Menard,G. N.,Kurup,S.,Van Erp,H.,Radhakrishnan,G. V.,Breakspear,A.,Oldroyd,G. E. D.和Eastmond,P. J.(2017)。 丛枝菌根真菌中的脂肪酸是由寄主植物合成的。 科学 356:1175-1178。
    8. McGonigle,T.P.,Miller,M.H.,Evans,D.G.,Fairchild,G.L.和Swan,J.A。(1990)。 一种新的方法,可以客观衡量根源的定殖情况水泡 - 丛枝菌根真菌。 <新Phytol 115:495-501。
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Copyright Keymer et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Keymer, A., Huber, C., Eisenreich, W. and Gutjahr, C. (2018). Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi. Bio-protocol 8(7): e2786. DOI: 10.21769/BioProtoc.2786.
  2. Keymer, A., Pimprikar, P., Wewer, V., Huber, C., Brands, M., Bucerius, S. L., Delaux, P. M., Klingl, V., Ropenack-Lahaye, E. V., Wang, T. L., Eisenreich, W., Dormann, P., Parniske, M. and Gutjahr, C. (2017). Lipid transfer from plants to arbuscular mycorrhiza fungi. Elife 6: e29107.
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