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Feb 2019
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Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues
非水体系分离(NAF)用于拟南芥叶组织亚细胞组分的代谢研究   

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Abstract

The accurate determination of metabolite distribution in subcellular compartments is still challenging in plant science. Various methodologies, such as fluorescence resonance energy transfer-based technology, nuclear magnetic resonance spectroscopy and protoplast fractionation allow the study of metabolite compartmentation. However, large changes in metabolite levels occur during such procedures. Therefore, the non-aqueous fractionation (NAF) technique is currently the best method for the study of in-vivo metabolite distribution. Our protocol presents a detailed workflow including the NAF procedure and quantification of compartment-specific markers for three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker, and nitrate and acid invertase as vacuolar markers.

Keywords: In-vivo metabolite distribution (体内代谢物分布), Non-aqueous fractionation (NAF) (非水体系分离), Compartment-specific markers (微区特异性标记物)

Background

Metabolic activity and functionality require precise compartmentation of metabolism in eukaryotic cells. Despite knowing the spatial organization of metabolism within the plant cells, deciphering localization of the metabolites is still challenging due to metabolite redundancy, leakage, rapid turnover, futile cycling, and sophisticated transport systems and storage (Lunn, 2007; Klie et al., 2011; Sweetlove and Fernie, 2013). Several methods have been established to investigate metabolite compartmentation, among them non-aqueous fractionation (NAF) has been widely used for decades to determine the in-vivo subcellular metabolite distributions in eukaryotic cells. Firstly developed in animal sciences (Elbers et al., 1974), NAF was subsequently applied to plant leaf material from spinach (Gerhardt and Heldt, 1984 and 1987), bean (Sharkey and Vanderveer, 1989), maize (Weiner and Heldt, 1992) or barley (Winter et al., 1993). NAF is also successfully applied to rose petals (Yamada et al., 2009) and potato tubers (Farre et al., 2001). More recently, NAF has also been optimized to study metabolite compartmentation in the model plant Arabidopsis thaliana (Fettke et al., 2005; Krueger et al., 2009 and 2011; Klie et al., 2011; Arrivault et al., 2014; Shapiguzov et al., 2019). The NAF procedure prevents modification of metabolite levels (for example due to enzymatic activity) by having all steps performed at extremely low temperatures or under anhydrous conditions (Gerhardt and Heldt, 1984). After quenching plant material in liquid nitrogen, the frozen material is dehydrated by freeze drying, homogenized, and then applied to a gradient made of anhydrous organic solvents prior to centrifugation and subsequent collection of fractions. Subcellular compartments are separated on the basis of density in the centrifugation step and the fractions collected are enriched in organelles (such as chloroplasts, cytosol or vacuole; Gerhardt and Heldt, 1984; Lunn, 2006). By correlating the compartment-specific marker distributions with metabolite distributions over the gradient, it is possible to calculate compartment-specific metabolite distributions (Gerhardt and Heldt, 1984; Klie et al., 2011; Krueger et al., 2011). The software tool BestFit enables such calculations (Krueger et al., 2011 and 2014; Klie et al., 2011). Recently, a benchtop NAF method has been established and is adapted for a relatively small amount of tissue material (Fürtauer et al., 2016). This benchtop NAF method can be used as an alternative to the conventional NAF procedure. However, we still believe that the conventional NAF procedure is the method of choice for the determination of in vivo metabolite distribution among cellular compartments as (i) the plastidic and vacuolar compartments are more clearly separated on the density gradient used, and (ii) the higher amount of starting material leads to more material in each obtained fraction, giving the advantage to increase the number of sub-aliquots per fraction (thus increasing the range of measurements which can be done) and also to apply less sensitive quantification methods than mass spectrometry-based methods. A good example of the use of the conventional NAF procedure is a study performed by Arrivault et al. (2014) in which the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in Arabidopsis thaliana rosette leaves were analyzed, using the conventional NAF combined with MS-based approaches.

The protocol presented here is based on the method described by Krueger et al. (2014) with minor modifications. In our method, we provide a complete workflow for the NAF procedure in Arabidopsis thaliana and detailed information for the analysis of compartment–specific marker enzymes and metabolites which can be assigned to three subcellular compartments: ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker and nitrate and acid invertase as vacuolar markers.

Materials and Reagents

  1. NAF
    1. Plastic lids (Zinsser Analytic, catalog number: 3071400)
    2. 20 ml Polyvial–one per sample (Zinsser Analytic, catalog number: 3071400) 
    3. Steel balls:
      3 mm (Askubal, catalog number: 3 mm G100 1.4034)
      5 mm (Askubal, catalog number: 5 mm G100 1.4034)
      7 mm (Altmann, catalog number: KU.7G80-1.3541)
      Note: We recommend using steel balls of three different diameters 3, 5, and 7 mm (two of each size in each 20 ml Polyvial).
    4. Steel caps (custom made, please check Figure 1)
    5. 50 ml Falcon tubes–10 per sample (SARSTEDT, catalog number: 62.554.502)
    6. Paper tissue (KimWipes)
    7. pH-indicator paper (Merck, catalog number: 1.10962)
    8. Polyallomer centrifuge tubes (Beckman Coulter, catalog number: 326823) 
    9. Nylon mesh filter 20 µm pore size (SEFAR Nitex®, catalog number: 03-20/14)
    10. 1.5 ml screw cap tubes–11 per sample (SARSTEDT, catalog number: 72.692)
    11. Activated molecular sieve 4 Å (Carl Roth, catalog number: 1318-02-1)
    12. Plastic Pasteur pipette (NeoLab, catalog number: 2600111)
    13. Glass pipettes (10 ml, 20 ml)
    14. Glass funnel
    15. Metal rack
    16. Arabidopsis thaliana leaf material
    17. Liquid nitrogen
    18. n-Heptane (Carl Roth, catalog number: 142-82-5)
    19. Tetrachloroethylene (Carl Roth, catalog number: 127-18-4)
    20. Liquid nitrogen (~5 L per sample)
      Note: the required liquid nitrogen for harvesting (1 L), grinding (2 L), and aliquoting (2 L) samples suggested here is a rough estimate, since the amount of nitrogen needed will depend on several factors. Please consider that while planning your experiments.

  2. Measurement of metabolite markers
    1. 96 Deep-well plate (Sarstedt, catalog number: 82.1970.002)
    2. Steel balls (5 mm)
    3. Tubes holder for the Retsch mill (Retsch, catalog number: 22.008.0008)
    4. Ethanol (VMR Chemicals, catalog number: 20821.330)
    5. HEPES (CAS: 7365-45-9)
    6. NaOH (Carl Roth, catalog number: 6771.1)
    7. Potassium phosphate buffer 1 M, pH 7.5
      1. K2HPO4 (Sigma-Aldrich, CAS: 7758-11-4)
      2. KH2PO4 (Sigma-Aldrich, CAS: 7778-77-0)
    8. NADPH (Roche, CAS: 2646-71-1)
    9. Nitrate reductase (Roche, catalog number: 10981249001)
    10. Phenazine ethosulfate (PES) (Sigma-Aldrich, CAS: 10510-77-7)
    11. Sulfanilamide (Sigma-Aldrich, CAS: 63-74-1)
    12. N-(1-Naphthyl)ethylenediamine dihydrochloride (NNEDA) (Sigma-Aldrich, CAS: 1465-25-4)
    13. KNO3 (Sigma-Aldrich, CAS: 7757-79-1)
    14. Nitrate Standards (see Recipes)
      0, 0.2, 0.4, 0.8, and 1.6 mM in 70% ETOH with 10 mM MES pH 5.9

  3. Measurement of enzyme markers
    For the full description and order details of the reagents used in the measurements of enzyme activities, please see Supplemental Table 1.
    1. PVPP
    2. Deionized water
    3. MgCl2
    4. EDTA
    5. EGTA
    6. Benzamidine
    7. ε-aminocapronic acid
    8. BSA (protease free)
    9. Leupeptin 
    10. DTT 
    11. PMSF 
    12. Triton X-100
    13. Glycerol
    14. NaF
    15. 3-phosphoglycerate
    16. AGPase assay mix
    17. PPi
    18. Glycerokinase
    19. ADP-glucose
    20. GPOX
    21. GDH
    22. NaHCO3
    23. PEP
    24. Malate dehydrogenase
    25. MTT
    26. Alcohol Dehydrogenase
    27. Acid invertase assay Buffer
    28. Glucose oxidase
    29. Horse Radish Peroxidase
    30. Amplex Red
    31. DMSO
    32. Nitrate standards (see Recipes)
    33. Extraction buffers (see Recipes)
      1. Extraction Buffer 10x
      2. Extraction Buffer 1x
    34. AGPase activity (see Recipes)
      1. AGPase assay buffer
      2. AGPase assay mix
      3. AGPase determination mix
    35. PEPC activity (see Recipes)
      1. PEPC assay Buffer
      2. PEPC assay Mix
      3. PEPC determination mix
    36. Acid Invertase activity (see Recipes)
      1. Acid invertase assay Buffer
      2. Acid invertase Assay Mix
      3. Acid invertase Determination mix

Equipment

  1. NAF
    1. Capper/decapper machine (custom made, see Figure 1)
    2. Retsch mill (Retsch®, model: MM400)
    3. Freeze dryer (Christ, model: Alpha 2-4)
    4. Sonicator (BANDELIN SONOPULS HD 2070)
    5. Peristaltic gradient pump (Bio-Rad Econo Gradient Pump, catalog number: 731-9002)
    6. Gradient mixer (Bio-Rad Gradient mixer, catalog number: 731-8323)
    7. Centrifuge (Beckman Coulter, model: Allegra X-15R)
    8. Rotor (Beckman Coulter, model: SX4750A)
    9. Ultracentrifuge (Beckman Coulter, model: OptimaTM L80 XP)
    10. Swinging bucket rotor (Beckman Coulter, model: SW32Ti)
    11. Desiccator
    12. Vacuum pump

  2. Measurement of metabolite markers
    1. Thermomixer (Eppendorf, model: Compact 5350)
    2. Centrifuge (Eppendorf, model: 5430)
    3. Microplate Spectrophotometer (BioTeK®, Epoch 2)

  3. Measurement of enzyme markers
    1. Microplate Spectrophotometer (BioTeK®, ELx808)
    2. Microplate Fluorescence reader (BioTeK®, Synergy HT)

Software

  1. BestFit (Klie et al., 2011; http://www.csbdb.de/csbdb/bestfit/bestfit.html)

Procedure

  1. Harvesting, grinding and preparation of leaf material
    1. Harvest Arabidopsis thaliana leaf material and place into a 20 ml Polyvial (containing steel balls of three different diameters 3, 5, and 7 mm, two of each size in each 20 ml Polyvial) pre-cooled in liquid N2 and snap-freeze in liquid N2.
      Note: If the metabolites of interest have extremely fast turn-over time, plant material should be harvested by cutting rosettes and quenching them instantaneously in a bath of liquid N2 under the prevailing irradiance. The quenched material is then transferred into a 20 ml Polyvial (containing steel balls) pre-cooled in liquid N2. For an optimal grinding process, fill the vial to a maximum of 2/3 of its volume with the plant material. For NAF, 4 g of material is necessary, but it is recommended to harvest slightly more in order to have extra material to perform additional analysis in this material (such as quantification of metabolites, see section Data analysis–4).
    2. For homogenization, replace the plastic lids of the Polyvials by steel caps and place the Polyvials containing the leaf material into a pre-cooled steel grinding adapter for the Retsch mill (Figures 1A-1D).
      Note: Our grinding adapters and steel caps are custom-made for the 20 ml Polyvials. Alternatively, the grinding of the leaf material can be performed by using a pre-cooled mortar and pestle. Make sure that the leaf material is kept frozen during the whole procedure.
    3. Homogenize the tissue for 1 min at 25 Hz (two times) in the Retsch mill (Figures 1E and 1F).
      Note: Make sure that the ground tissue is very well homogenized (thin powder). We suggest grinding the leaf material twice for 1 min each at 25 Hz. Cool down the vials and adapters in between with liquid N2 to avoid thawing of the material.


      Figure 1. Grinding procedure. A. Cool down the Polyvials, steel grinding adapter, and steel caps in liquid nitrogen. B. Remove the plastic lid of the Polyvials and place the steel cap frozen. C. Use a capper/decapper machine (custom-made) to close the vial with the steel cap. D. Closed vial. E. Place the Polyvial inside steel grinding adapter. F. Place the grinding adapter in the Retsch mill and proceed as in Step A3. After grinding use the same capper/decapper machine for replacing the steel caps for the plastic lids. The samples can be stored at -80 °C or proceed to Step A4. Note that to avoid overheating of the vial, steel caps and grinding adapter deep them in liquid nitrogen or make use of dried ice while preparing a second sample.

    4. Weigh ~4 g of frozen tissue into a 50 ml Falcon tube pre-cooled in liquid N2.The samples can be stored at -80 °C for up to three months. Prepare as many Falcon tubes as necessary (each one will be used for one NAF).
      Note: Label the Falcon tubes and their corresponding lids. Record the weights of the empty Falcon tubes (with lids) and the precise amount of plant material weighed. 
    5. Freeze dry the homogenate. To avoid losing leaf material during this process, remove the lid and cover the Falcon tubes with paper tissue (KimWipes) fixed with an elastic band. Place the tubes in a metal rack into the freeze drier at 0.02 bar and -80 °C for 5 days. Close the tubes with their corresponding lids immediately after removing them from the freeze drier. The dried material can be stored under vacuum in a desiccator protected from light and humidity up to 6 months.
      Note: In order to avoid thawing, place the Falcon tubes in a polystyrene box containing dry ice and replace the lids by the paper tissue while the Falcon tubes are in the box. 
    6. Determine the plant material dry weight by weighing the closed Falcon tubes after the freeze drying process.

  2. Preparation of solutions and filters 
    1. Prepare the solutions for the NAF three days before starting the fractionation.
    2. Add the activated molecular sieve 4 Å to the Heptane (C7H16) and Tetrachloroethylene (C2Cl4) solutions to ensure that no residual water is present.
    3. Prepare a mixture of Tetrachloroethylene/Heptane 66:34 (v/v); density = 1.3 g cm-3. Add the activated molecular sieve 4 Å and store the mixture protected from light in a brown glass bottle.
      Note: We usually use the ~100 ml of activated molecular sieve in a 1 L bottle. For 10 samples it is needed: a) 1 L of the Tetrachloroethylene/Heptane mixture 66:34 (v/v): 660 ml of Tetrachloroethylene + 340 ml of Heptane, b) 2 L of Heptane, c) 500 ml of Tetrachloroethylene.
    4. Before use always check the pH of the solutions with a pH paper, especially Tetrachloroethylene. pH should be neutral. If Tetrachloroethylene solution turns acidic, the dried leaf material will become brown during gradient centrifugation and the NAF will fail.
    5. Cut 15 cm x 15 cm the nylon filters (one per NAF), weigh them and store them in clean 50 ml Falcon tubes.

  3. Fractionation procedure
    Note: The whole procedure should be performed under a fume hood. Only dried aliquots (after Step C17) can be processed on regular bench. For pouring solutions, use glass pipettes.
    1. Resuspend the dried leaf material in 20 ml of the Tetrachloroethylene/Heptane = 66:34 (v/v) mixture.
    2. Ultrasonicate the suspension for 2 min, with 6 x 10 cycles, 65% of power (Figure 2). To avoid overheating the suspension during the sonication process, keep the Falcon tube in an ice bath.
      Note: Place the sonicator tip inside the solution, but avoid touching the tube wall. Make sure the sonication is successful. This is a critical step in the fractionation, since insufficient sonication can produce aggregates that do not pass through nylon mesh.


      Figure 2. Setup for the ultrasonication procedure. Note that to avoid overheating the suspension during the process, the Falcon tube should be kept in an ice bath.

    3. Fold a nylon mesh into a glass funnel placed on top of a 50 ml Falcon tube and pour the suspension through the nylon mesh. Wash the nylon mesh 3 x with 10 ml of heptane.
      Note: The first two washing steps are performed by slowly pouring heptane on the nylon mesh and collecting the flow through in the Falcon tube below. For the third washing step, remove all the residual solution in the nylon mesh by squeezing it (while wearing adequate gloves). Place the nylon mesh back in a 50 ml Falcon tube and continue at Step C16. Be careful that all liquid go through the nylon mesh. If it´s the case, filter the whole obtained mixture again through a fresh nylon mesh. 
    4. Centrifuge for 10 min at 3,200 x g and 4 °C, using a swing-out rotor. Discard the supernatant (by pouring it into a waste glass bottle) and resuspend the pellet in 5 ml of Tetrachloroethylene/Heptane = 66:34 (v/v) mixture.
    5. Transfer 100 µl of the well homogenized resuspended pellet into a 1.5 ml screw cap tube. This aliquot is named fraction 0 (F0) and corresponds to the unfractionated material. Prepare three aliquots, add 900 µl of C7H16 to each, and keep protected from the light and continue at Step C15.
      Note: Use a cut 200 µl pipette tip to collect F0. Homogenize well the mixture before taking each aliquot.
    6. Prepare the NAF gradient (Figure 3A): Linear gradient (30 ml, density from 1.43 to 1.62 g cm-3)
      1. With a Pasteur pipette fill the 50 ml Falcon tube A with Tetrachloroethylene/Heptane mixture and the 50 ml Falcon tube B with Tetrachloroethylene.
      2. The dispensing needle is placed above a waste container.
        Note: Ensure that there are no air bubbles in any tubing by purging thoroughly the whole system.
      3. Set the peristaltic pump at a flow rate of 1.15 ml min-1 and the following program:
        Step
        ml
        Solvent A
        Solvent B
        1*
        0 70%
        30%
        2**
        3 70%
        30%
        3 28 0% 100%
        4 33 0% 100%
        Notes:
        1. *After 3 ml, pause the program. This step is used to ensure that the first mixture reaches the end of the dispensing needle before starting the actual gradient.
        2. **Remove the waste container and place the Polyallomer tube at this step. The needle should be placed slightly over the bottom of the Polyallomer tube and slightly touching the side of the tube (Figure 3B). Re-start the program.


          Figure 3. Setup of the gradient pump and the mixer. A. Overview of the setup for preparing the NAF gradient. Solution A (Tetrachloroethylene/Heptane mixture) and solution B (Tetrachloroethylene). B. Detail of the bottom-up preparation of the gradient. Tube holder is a 50 ml falcon tube with paper support on the bottom to help holding the gradient tube.

    7. Take the needle carefully out from the gradient and place the Polyallomer tube in a rack. Apply the suspension from Step C4 to the gradient, very carefully with a Pasteur pipette. Touch the wall of the tube with the pipette slightly above the gradient and release carefully to avoid disturbing the gradient.
    8. Place the gradients in the appropriate tube buckets from the ultracentrifuge. Verify their weights and to counterbalance, adjust the weight by adding 100% C2Cl4.
    9. Centrifuge for 50 min at 5,000 x g, and 4 °C using a swinging bucket rotor.
      Note: We always use the Ultracentrifuge Beckman Coulter OptimaTM L80 XP and Swinging bucket rotor Beckman Coulter SW32Ti. Set the acceleration/deceleration on three.
    10. Place the gradient tubes in a rack and mark eight fractions. Figure 4 illustrates how we usually separate the fractions based on the color.


      Figure 4. NAF gradient after centrifugation. Usually, we separate eight fractions based on their color. The dark green layer is enriched in chloroplast and usually divided into two fractions (F2 and F3) to avoid metabolite overloading. Note that fraction 8 is a pellet, which is the vacuolar-enriched fraction.

    11. Transfer carefully each fraction into clearly labeled clean 50 ml Falcon tube by using Pasteur pipette. The fractions are collected starting from the upper part (F1) to the bottom (F8). After F7 is removed, the pellet is resuspended in 20 ml heptane and the mixture transferred to a clean 50 ml Falcon tube. Use a new pipette for each fraction.
      Note: Transferring the fractions is a critical point for the method, since at this step cross contamination of the fractions should be avoided as much as possible. Therefore, always transfer the fractions from the very top of each layer.
    12. Add heptane up to 20 ml to the Falcon tubes containing F1 to F8 and mix carefully. Centrifuge all Falcon tubes for 10 min at 3,200 x g and at room temperature. 
    13. Discard the supernatant (by carefully pouring it into a waste glass bottle) and resuspend the pellet with 7 ml of Heptane.
      Note: The volume depends on how many aliquots you will need for your analysis. We usually prepare seven sub-aliquots per fraction. This number is enough for various metabolite analysis and determination of the compartment-specific markers.
    14. Transfer 6 x 1 ml from each suspension into 1.5 ml screw caps tubes. The pellet can easily re-aggregate at this step. Keep mixing by hand the solution while transferring into the tubes in order to obtain homogeneous aliquots from the same fraction.
      Note: Use a cut 1000 µl pipette tip to collect sub-aliquots from each fraction. As adding 7 ml generally does not allow to obtain seven aliquots of 1 ml, we generally take 0.8 ml for the seventh aliquot and clearly label the corresponding tube.
    15. Centrifuge for 10 min at 14,000 x g and at room temperature. Discard excess of supernatant (c.a. 900 µl) carefully, using a 1000 µl pipette tip. Process the F0 aliquots obtained in Step C5 in the same manner. 
    16. Place the screw caps tubes containing the sub-aliquots open in racks inside of a desiccator connected to a vacuum pump. Also, place the open Falcon tubes containing the filters from Step C3. Keep the aliquots under vacuum and protected from light overnight.
    17. Close the tubes and store them at -80 °C until further process. 
    18. Weigh the dried filters.

  4. Measurements of metabolite markers
    1. Extraction
      1. Add to the dried aliquots a steel ball and 250 µl 80% ethanol/10 mM HEPES pH 7 to the samples.
      2. Place the tubes in a cooled (4 °C) holder for the ball mill and disrupt the pellet for 1 min at 25 Hz.
      3. Incubate in a thermomixer for 30 min at 95 °C and 1000 rpm.
      4. Centrifuge for 10 min at 20,800 x g and 4 °C.
      5. Transfer the supernatant to a 96-deep well plate and keep it protected from light and on ice.
      6. Add to the Pellet 150 µl 80% ethanol/10 mM HEPES pH 7.
      7. Incubate in a thermomixer for 30 min at 95 °C 1000 rpm.
      8. Centrifuge for 10 min at 20,800 x g and 4 °C. 
      9. Transfer and combine with the previous supernatant. 
      10. Add to the Pellet 250 µl 50% ethanol/10 mM HEPES pH 7.
      11. Incubate in a thermomixer for 30 min at 95 °C and 1,000 rpm.
      12. Centrifuge for 10 min at 20,800 x g and 4 °C. 
      13. Transfer and combine with the previous two supernatants. It can be stored at -20 °C until further use.
    2. Nitrate (vacuolar marker)
      Nitrate content is determined following Cross et al. (2006). Nitrate can be used as a vacuolar marker in addition or alternatively to Acid Invertase.
      Note: Nitrite content must be determined and subtracted from the Nitrate values. For measuring nitrite content, nitrate reductase is replaced by water in the assay mix.
      Solutions: For storage recommendations, the solutions for nitrate measurement can be stored as follow:
      Solution:
      Storage temperature
      Potassium phosphate buffer 1 M pH 7.5
      -20 °C
      NADPH 50 mM in NaOH 20 mM
      -80 °C
      Nitrate reductase (5 U ml-1 in Potassium phosphate buffer 0.1 M)
      -80 °C
      PES 0.25 mM -80 °C
      Sulfanilamide 1% (w/v) in phosphoric acid 5%
      4 °C
      NNEDA 0.02% (w/v)
      4 °C
      Nitrate standards
      -80 °C
      Nitrate Standards: 0, 0.2, 0.4, 0.8, and 1.6 mM in 70% ETOH with 10 mM MES pH 5.9
      Protocol:
      1. For one reaction, dispense in a 96-well plate 95 µl of the assay mix:
        10.0 µl phosphate buffer
        0.5 µl NADPH
        2.0 µl Nitrate reductase
        82.5 µl deionized water
      2. Add 5 µl of standard or extract (1 to 10 diluted extract).
      3. Mix and incubate for 30 min at 25 °C, protected from light.
      4. Add 15 µl of PES.
      5. Mix and incubate for 20 min at 25 °C, protected from light.
      6. Add 50 µl of Sulphanilamide and 50 µl of NNEDA.
      7. Mix and incubate for 10 min at 25 °C, protected from light.
      8. Read the plate at 540 nm in a microplate reader.

  5. Measurements of enzyme markers
    1. Enzyme extraction
      1. Add to the dried extract ~2 mg of PVPP (one little spoon), one steel ball (5 mm diameter), and 500 µl of buffer 1x (see Recipe 1b). Keep the samples on ice.
      2. Place the tubes in a cooled (4 °C) holder for the ball mill and disrupt the pellet for 1 min at 25 Hz. Make sure that the entire pellet is dissolved. If not, repeat the pellet disruption procedure.
      3. Centrifuge for 10 min at 20,800 x g and 4 °C. Transfer the enzyme extract (supernatant) to a 96 deep-well plate. Keep on ice and proceed to the enzyme assays. If required, the supernatant can be stored at -80 °C.
    2. Enzyme assays
      All the three enzyme markers are measured according to Gibon et al. (2004).
      1. AGPase activity (plastid marker)
        1. Dispense in a 96-well plate placed on ice:
          14 µl of AGPase assay mix (see Recipe B2b)
          6 µl of standard or extract.
        2. Mix and incubate for 20 min at 25 °C.
        3. Add 20 µl of 0.5 M HCl in 100 mM Tricine/KOH pH 8.
        4. Mix and incubate for 10 min at room temperature.
        5. Add 20 µl of 0.5 M NaOH.
        6. Mix, add 50 µl of AGPase Determination mix (see Recipe B2c).
        7. Mix, spin down, and read at wavelength of 340 nm and 30 °C until the maximum rate of reaction stabilizes.
      2. PEPC (Cytosol marker)
        1. Dispense in a 96-well plate placed on ice.
          18 µl PEPC Assay Mix (see Recipe B3b)
          2 µl of standard or extract
        2. Mix and incubate for 20 min at 25 °C.
        3. Add 20 µl of 0.5 M HCl in 100 mM Tricine/KOH pH 9.
        4. Mix, spin down, and incubate at 95 °C for 5 min. Cool and spin down. Keep the plate on ice.
        5. Add 20 µl 0.5 M NaOH.
        6. Mix, add 45 µl of PEPC Determination mix (see Recipe 3c, protect from light).
        7. Add 5 µl of 4 mM PES (protect from light).
        8. Mix and read at wavelength of 570 nm and 30 °C until the maximum rate of reaction stabilizes.
      3. Acid Invertase (Vacuole marker)
        1. Dispense in a black 96-well microplate:
          10 µl Acid invertase Assay mix (see Recipe B4b)
          5 µl of standard or enzyme extract
        2. Mix and incubate at 25 °C for 5 and 40 min.
        3. Add 10 µl 0.5 M NaOH.
        4. Mix and incubate at R.T. for 10 min.
        5. Add 10 µl 0.5 M HCl in 100 mM Tricine/KOH pH 8.
        6. Mix and add 50 µl of Acid invertase Determination mix (see Recipe B4c).
        7. Mix and read the fluorescence for 10-15 min using the following setting:
          Excitation at 530 nm, emission at 590 nm, temperature: 30 °C, sensitivity at 25. Use the rate of reaction for calculations. We use the Synergy fluorescence meter.

Data analysis

  1. For evaluation of subcellular metabolite distributions, we recommend the statistical software BestFit. The BestFit is a C-language command line tool that allows calculation and evaluation of subcellular distributions from NAF data. The software requires; (i) the distribution of specific markers for each subcellular compartment analyzed throughout the different fractions (Figure 5) and (ii) the distribution of the metabolites of interest throughout the different fractions.


    Figure 5. Distribution in percentage of subcellular compartment-specific markers throughout the gradient showed in Figure 4. ADP glucose pyrophosphorylase (AGPase) as plastidic marker, phosphoenolpyruvate carboxylase (PEPC) as cytosolic marker and nitrate and acid invertase as vacuolar markers.

  2. For the values of either markers and metabolites of interest in the input file, we recommend using percentage, although absolute measurements can also be used (note that in this case values must be ≥ 0). 
  3. For more details on how to use BestFit, we suggest reading the documentation pdf file within the BestFit Folder (http://www.csbdb.de/csbdb/bestfit/bestfit.html). Alternatively, you can also find details regarding data input and use of BestFit in Krueger et al. (2014).
  4. BestFit outputs the subcellular-distribution of the metabolites of interest in percentage. In order to calculate which amounts this represents, metabolites are also measured in ground material not processed through NAF (see Note in A1). Various recovery calculations can be done by using the following information: (i) material fresh weight used for NAF (determined in step A4), (ii) corresponding dry weight (determined in step A6), (iii) how much material was applied to the gradient (determined in step C18 by weighing the residual material in the filter), (iv) F0 obtained step C6. We recommend always recording these data and collecting F0 for each NAF.

Recipes

  1. Measurement of metabolite markers
    1. Nitrate standards
      0, 0.2, 0.4, 0.8, and 1.6 mM in 70% ETOH with 10 mM MES pH 5.9

  2. Measurement of enzyme markers
    1. Extraction buffers
      1. Extraction Buffer 10x
        500 mM HEPES
        100 mM MgCl2
        10 mM EDTA
        10 mM EGTA
        10 mM Benzamidine
        10 mM ε-aminocapronic acid
        2.5% w/v BSA (protease-free)
        Bring volume to c.a. 40 ml, adjust pH to 7.5 with KOH 10 M, and then adjust the volume to 50 ml
        The extraction buffer 10x can be stored at -20 °C
        Prepare the following solutions and store accordingly:
        Leupeptin 2 mM (100x), freeze in liquid nitrogen and then store at -80 °C
        DTT 500 mM (1,000x), freeze in liquid nitrogen and then store at -80 °C
        PMSF 100 mM in isopropanol (100x), store at 4 °C, protect from light, stable for 1 month
        Triton X-100 10% v/v (10x), store at RT
        Glycerol (87% v/v), store at RT
      2. Extraction Buffer 1x
        Prepare only prior the extraction combining as the following:
        [Final]       
        Reagent
        [stock]
        For 1,000 µl
        1x
        Buffer 10x (see Recipe 1a)
        10x
        100 µl
        10 µM
        Leupeptin
        2 mM
        5 µl
        1 mM
        DTT
        500 mM
        2 µl
        1 mM
        PMSF
        100 mM in isopropanol
        10 µl
        1%
        Triton X-100
        10% v/v
        100 µl
        20%
        Glycerol
        87% (v/v)
        230 µl

        H2O

        553 µl
    2. AGPase activity
      1. AGPase assay buffer (can be stored at -20 °C)
        0.25 M HEPES/KOH pH 7.5
        7.5 mM NaF
        25 mM MgCl2
        5 mM 3-phosphoglycerate
        0.25% (v/v) Triton X-100
      2. AGPase assay mix
        7.6 µl water
        4 µl AGPase assay buffer (see Recipes B2a)
        2 µl 20 mM PPi
        0.2 µl Glycerokinase (200 U ml-1 in 200 mM Tricine/KOH pH 8, 10 mM MgCl2)
        0.2 µl ADP-glucose 0 mM (Blank) or 100 mM (maximum)
      3. AGPase determination mix
        37.8 µl water
        10 µl 1 M Tricine/KOH pH 8
        0.2 µl 1 M MgCl2
        0.5 µl GDH (200 U ml-1 in 200 mM Tricine/KOH pH 8, 10 mM MgCl2)
        0.5 µl GPOX (500 U ml-1 in 200 mM Tricine/KOH pH 8, 10 mM MgCl2)
        1 µl 66 mM NADH
    3. PEPC activity
      1. PEPC assay Buffer (can be stored at -20 °C)
        0.5 M Tricine/KOH pH 8
        100 mM MgCl2
        50 mM NaHCO3
        0.25% Triton X-100 
      2. PEPC assay Mix
        11.4 µl H2O
        4 µl PEPC assay Buffer (see Recipe B3a)
        2 µl 0 (Blank) or 20 mM PEP (maximum)
        0.4 µl 5 mM NADH
        0.2 µl Malate dehydrogenase (100 U ml-1 in 200 mM Tricine/KOH pH 8, 10 mM MgCl2)
      3. PEPC determination mix (protect from light)
        18 µl water
        10 µl 1M Tricine/KOH pH 9.0
        10 µl 10 mM MTT
        4 µl 200 mM EDTA
        2 µl 50% Ethanol
        1 µl Alcohol Dehydrogenase (2,000 U ml-1 in 200 mM Tricine/KOH pH 9, 10 mM MgCl2)
    4. Acid Invertase activity
      1. Acid invertase assay Buffer (can be stored at -20 °C)
        0.25 M Acetate/KOH pH 5 
      2. Acid invertase Assay Mix: for 1 reaction
        10 µl Acid invertase assay buffer (see Recipe B4a)
        35 µl water
      3. Acid invertase Determination mix (protect from light): for 1 reaction
        42 µl water
        5 µl 1 M HEPES/KOH pH 7
        1 µl Glucose oxidase (200 U ml-1 in 200 mM HEPES/KOH pH 7.0)
        1 µl Horse Radish Peroxidase (0.2 U ml-1 in 200 mM HEPES/KOH pH 7.0)
        1 µl 20 mM Amplex Red in DMSO

Acknowledgments

We thank Deutsche Forschungsgemeinschaft (DFG TRR 175 The Green Hub – Central Coordinator of Acclimation in Plants; FA and ARF) for funding.
  We also would like to thank the authors from Krueger et al. (2014), the previous work describing the NAF method, which we made the modification presented in the present protocol.

Competing interests

The authors declare no conflict of interest.

References

  1. Arrivault, S., Guenther, M., Florian, A., Encke, B., Feil, R., Vosloh, D., Lunn, J. E., Sulpice, R., Fernie, A. R., Stitt, M. and Schulze, W. X. (2014). Dissecting the subcellular compartmentation of proteins and metabolites in Arabidopsis leaves using non-aqueous fractionation. Mol Cell Proteomics 13(9): 2246-2259.
  2. Cross, J. M., von Korff, M., Altmann, T., Bartzetko, L., Sulpice, R., Gibon, Y., Palacios, N. and Stitt, M. (2006). Variation of enzyme activities and metabolite levels in 24 Arabidopsis accessions growing in carbon-limited conditions. Plant Physiol 142(4): 1574-1588.
  3. Elbers, R., Heldt, H. W., Schmucker, P., Soboll, S. and Wiese, H. (1974). Measurement of the ATP/ADP ratio in mitochondria and in the extramitochondrial compartment by fractionation of freeze-stopped liver tissue in non-aqueous media. Hoppe Seylers Z Physiol Chem 355(3): 378-393.
  4. Farre, E. M., Tiessen, A., Roessner, U., Geigenberger, P., Trethewey, R. N. and Willmitzer, L. (2001). Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiol 127(2): 685-700. 
  5. Fettke, J., Eckermann, N., Tiessen, A., Geigenberger, P. and Steup, M. (2005). Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L.: distinct SHG reside in the cytosol and in the apoplast. Plant J 43(4): 568-585.
  6. Furtauer, L., Weckwerth, W. and Nagele, T. (2016). A benchtop fractionation procedure for subcellular analysis of the plant metabolome. Front Plant Sci 7: 1912.
  7. Gerhardt, R. and Heldt, H. W. (1984). Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in nonaqueous media. Plant Physiol 75(3): 542-547.
  8. Gerhardt, R., Stitt, M. and Heldt, H. W. (1987). Subcellular metabolite levels in spinach leaves: regulation of sucrose synthesis during diurnal alterations in photosynthetic partitioning. Plant Physiol 83(2): 399-407.
  9. Gibon, Y., et al. (2004). A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. The Plant Cell 16(12): 3304-3325.
  10. Klie, S., Krueger, S., Krall, L., Giavalisco, P., Flugge, U. I., Willmitzer, L. and Steinhauser, D. (2011). Analysis of the compartmentalized metabolome - a validation of the non-aqueous fractionation technique. Front Plant Sci 2: 55.
  11. Krueger, S., Giavalisco, P., Krall, L., Steinhauser, M. C., Bussis, D., Usadel, B., Flugge, U. I., Fernie, A. R., Willmitzer, L. and Steinhauser, D. (2011). A topological map of the compartmentalized Arabidopsis thaliana leaf metabolome. PLoS One 6(3): e17806.
  12. Krueger, S., Niehl, A., Lopez Martin, M. C., Steinhauser, D., Donath, A., Hildebrandt, T., Romero, L. C., Hoefgen, R., Gotor, C. and Hesse, H. (2009). Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis. Plant Cell Environ 32(4): 349-367.
  13. Krueger, S., Steinhauser, D., Lisec, J. and Giavalisco, P. (2014). Analysis of subcellular metabolite distributions within Arabidopsis thaliana leaf tissue: a primer for subcellular metabolomics. Methods Mol Biol 1062: 575-596.
  14. Lunn, J. E. (2007). Compartmentation in plant metabolism. J Exp Bot 58(1): 35-47.
  15. Shapiguzov, A., Vainonen, J. P., Hunter, K., Tossavainen, H., Tiwari, A., Jarvi, S., Hellman, M., Aarabi, F., Alseekh, S., Wybouw, B., Van Der Kelen, K., Nikkanen, L., Krasensky-Wrzaczek, J., Sipari, N., Keinanen, M., Tyystjarvi, E., Rintamaki, E., De Rybel, B., Salojarvi, J., Van Breusegem, F., Fernie, A. R., Brosche, M., Permi, P., Aro, E. M., Wrzaczek, M. and Kangasjarvi, J. (2019). Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors. Elife 8. pii:e43284.
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  20. Yamada, K., Norikoshi, R., Suzuki, K., Imanishi, H. and Ichimura, K. (2009). Determination of subcellular concentrations of soluble carbohydrates in rose petals during opening by nonaqueous fractionation method combined with infiltration-centrifugation method. Planta 230(6): 1115-1127.

简介

在植物科学中,准确确定亚细胞区室中代谢物的分布仍然具有挑战性。各种方法,例如基于荧光共振能量转移的技术,核磁共振波谱和原生质体分级分离,都可以研究代谢物的区室化。但是,在此类操作过程中会发生代谢物水平的巨大变化。因此,非水分离技术是目前研究体内代谢产物分布的最佳方法。我们的协议提出了详细的工作流程,包括NAF程序和三个亚细胞区室的区室特异性标记物的定量:ADP葡萄糖焦磷酸化酶(AGPase)作为质体标记物,磷酸烯醇丙酮酸羧化酶(PEPC)作为胞质标记物,硝酸盐和酸转化酶作为液泡标记物。
【背景】 代谢活性和功能需要真核细胞中新陈代谢的精确分隔。尽管知道植物细胞内新陈代谢的空间组织,但由于代谢物的冗余,泄漏,快速周转,徒劳的循环以及复杂的运输系统和存储,对代谢物的定位仍具有挑战性(Lunn,2007; Klie等人)。 ,2011; Sweetlove和Fernie,2013)。已经建立了几种研究代谢物分隔的方法,其中非水分离法(NAF)几十年来已广泛用于确定真核细胞中体内亚细胞代谢物的分布。 NAF最初是在动物科学中发展的(Elbers等,1974),随后被应用于菠菜(Gerhardt和Heldt,1984和1987),豆类(Sharkey和Vanderveer,1989)中的植物叶片材料,玉米(Weiner和Heldt,1992)或大麦(Winter et al。,1993)。 NAF还成功地应用于玫瑰花瓣(Yamada等,2009)和马铃薯块茎(Farre等,2001)。最近,还对NAF进行了优化,以研究模型植物拟南芥(Erabidopsis thaliana)中的代谢物区室(Fettke et al。,2005年; Krueger et al。,2009年和2011年; Klie 等人,2011年; Arrivault 等人,2014年; Shapiguzov 等人,,2019年)。通过在极低温度或无水条件下进行所有步骤,NAF程序可防止代谢物水平发生变化(例如,由于酶促活性)(Gerhardt和Heldt,1984)。在液氮中淬灭植物物料后,将冷冻的物料通过冷冻干燥进行脱水,均质化,然后在离心和随后收集馏分之前,先后用无水有机溶剂制成的梯度液。在离心步骤中,根据密度将亚细胞区室分开,收集的级分富含细胞器(例如叶绿体,细胞质或液泡; Gerhardt和Heldt,1984; Lunn,2006)。通过将特定于隔室的标记物分布与梯度上的代谢物分布相关联,可以计算特定于隔室的代谢物分布(Gerhardt和Heldt,1984; Klie 等人。,2011; Krueger 等,2011)。软件工具BestFit支持此类计算(Krueger等,2011和2014; Klie等,2011)。最近,已经建立了台式NAF方法,并适用于相对少量的组织材料(Fürtauer等人,2016)。该台式NAF方法可以用作常规NAF程序的替代方法。
但是,我们仍然认为,常规的NAF程序是确定细胞腔室中体内代谢物分布的首选方法,因为(i)质体和液泡腔室在密度梯度上更清晰地分开(ii)较高的起始物料量会导致每个所得馏分中的物料更多,从而有利于增加每个馏分的等分试样数量(从而增加了可进行的测量范围)并适用灵敏度低于基于质谱的定量方法。使用常规NAF程序的一个很好的例子是Arrivault等人(2014)进行的一项研究,其中约有1,000种蛋白质和70种代谢物的分布,其中包括22种磷酸化的中间体。使用常规NAF结合基于MS的方法分析了拟南芥玫瑰花瓣叶。

此处提供的协议基于Krueger et al。(2014)所描述的方法,并进行了少量修改。在我们的方法中,我们为拟南芥(Arabidopsis thaliana)中的NAF程序提供了完整的工作流程,并提供了可区分为三个亚细胞区室的区室特异性标记酶和代谢物分析的详细信息:ADP葡萄糖焦磷酸化酶( AGPase)作为质体标记,磷酸烯醇丙酮酸羧化酶(PEPC)作为胞质标记,硝酸盐和酸转化酶作为液泡标记。

关键字:体内代谢物分布, 非水体系分离, 微区特异性标记物

材料和试剂

  1. 资产净值
    1. 塑料盖(Zinsser Analytic,目录号:3071400)
    2. 20 ml Polyvial-每个样品一个(Zinsser Analytic,目录号:3071400)
    3. 钢球:
      3毫米(Askubal,货号:3毫米G100 1.4034)
      5毫米(Askubal,目录号:5毫米G100 1.4034)
      7毫米(Altmann,目录号:KU.7G80-1.3541)
      注意:我们建议您使用直径分别为3、5和7毫米的三种直径的钢球(每20毫升Polyvial中每种尺寸两个)。
    4. 钢盖(定制,请检查图1)
    5. 50 ml猎鹰管–每个样品10个(SARSTEDT,目录号:62.554.502)
    6. 纸巾(KimWipes)
    7. pH指示纸(默克(Merck),目录号:1.10962)
    8. 聚异体离心管(Beckman Coulter,目录号:326823)
    9. 尼龙网状过滤器,孔径20 µm(SEFAR Nitex ®,目录号:03-20 / 14)
    10. 1.5 ml螺帽管–每个样品11个(SARSTEDT,目录号:72.692)
    11. 活化分子筛4Å(Carl Roth,目录号:1318-02-1)
    12. 巴斯德塑料移液器(NeoLab,目录号:2600111)
    13. 玻璃移液器(10 ml,20 ml)
    14. 玻璃漏斗
    15. 金属架
    16. 拟南芥叶片材料
    17. 液氮
    18. 正庚烷(卡尔·罗斯,目录号:142-82-5)
    19. 四氯乙烯(Carl Roth,目录号127-18-4)
    20. 液氮(每个样品约5 L)
      注意:此处建议的用于采集(1 L),研磨(2 L)和等分试样(2 L)样品所需的液氮是一个粗略的估计,因为所需的氮量将取决于几个因素。在计划实验时,请考虑这一点。

  2. 代谢物标记物的测定
    1. 96深孔板(Sarstedt,目录号:82.1970.002)
    2. 钢球(5毫米)
    3. Retsch轧机的管架(Retsch,目录号:22.008.0008)
    4. 乙醇(VMR Chemicals,目录号:20821.330)
    5. HEPES(CAS:7365-45-9)
    6. NaOH(Carl Roth,货号:6771.1)
    7. 磷酸钾缓冲液1 M,pH 7.5
      1. K 2 HPO 4 (Sigma-Aldrich,CAS:7758-11-4)
      2. KH 2 PO 4 (Sigma-Aldrich,CAS:7778-77-0)
    8. NADPH(罗氏(CAS):2646-71-1)
    9. 硝酸还原酶(罗氏(Roche),目录号:10981249001)
    10. 吩嗪乙硫酸盐(PES)(Sigma-Aldrich,CAS:10510-77-7)
    11. 磺胺(Sigma-Aldrich,CAS:63-74-1)
    12. N-(1-萘基)乙二胺二盐酸盐(NNEDA)(Sigma-Aldrich,CAS:1465-25-4)
    13. KNO 3 (Sigma-Aldrich,CAS:7757-79-1)
    14. 硝酸盐标准品(请参见食谱)
      在70%的ETOH中的0、0.2、0.4、0.8和1.6 mM以及10 mM MES pH 5.9

  3. 酶标记物的测定
    有关用于酶活性测量的试剂的完整描述和订购详细信息,请参见补充表1 。
    1. 聚氯乙烯
    2. 去离子水
    3. 氯化镁 2
    4. EDTA
    5. 埃格塔
    6. 苄am
    7. ε-氨基己酸
    8. BSA(无蛋白酶)
    9. 亮丙肽
    10. DTT
    11. PMSF 
    12. 海卫一X-100
    13. 甘油
    14. 氟化钠
    15. 3-磷酸甘油酸酯
    16. AGPase分析混合物
    17. 聚酰亚胺
    18. 甘油激酶
    19. ADP葡萄糖
    20. GPOX
    21. GDH
    22. NaHCO 3
    23. PEP
    24. 苹果酸脱氢酶
    25. MTT
    26. 酒精脱氢酶
    27. 酸性转化酶测定缓冲液
    28. 葡萄糖氧化酶
    29. 辣根过氧化物酶
    30. 浅红色
    31. 二甲基亚砜
    32. 硝酸盐标准(请参见食谱)
    33. 提取缓冲液(请参见食谱)
      1. 提取缓冲液10x
      2. 提取缓冲液1x
    34. AGPase活性(请参阅食谱)
      1. AGPase分析缓冲液
      2. AGPase分析混合物
      3. AGPase测定混合物
    35. PEPC活动(请参阅食谱)
      1. PEPC测定缓冲液
      2. PEPC分析混合物
      3. PEPC测定混合物
    36. 酸性转化酶活性(请参见食谱)
      1. 酸性转化酶测定缓冲液
      2. 酸性转化酶测定混合物
      3. 酸性转化酶测定混合物

设备

  1. 资产净值
    1. 封盖机/开盖机(定制,见图1)
    2. Retsch磨(Retsch ®,型号:MM400)
    3. 冷冻干燥机(基督,型号:Alpha 2-4)
    4. 超声波仪(BANDELIN SONOPULS HD 2070)
    5. 蠕动梯度泵(Bio-Rad Econo梯度泵,目录号:731-9002)
    6. 梯度混合器(Bio-Rad梯度混合器,目录号:731-8323)
    7. 离心机(贝克曼库尔特,型号:Allegra X-15R)
    8. 转子(贝克曼库尔特,型号:SX4750A)
    9. 超速离心机(贝克曼库尔特公司,型号:Optima TM L80 XP)
    10. 摆动铲斗转子(贝克曼库尔特,型号:SW32Ti)
    11. 干燥器
    12. 真空泵

  2. 代谢物标记物的测定
    1. Thermomixer(Eppendorf,型号:Compact 5350)
    2. 离心机(Eppendorf,型号:5430)
    3. 微孔板分光光度计(BioTeK ®,时代2)

  3. 酶标记物的测量
    1. 微孔板分光光度计(BioTeK ®,ELx808)
    2. 微孔板荧光读取器(BioTeK ®,Synergy HT)

软件

  1. BestFit(Klie et al。,2011; http:// www .csbdb.de / csbdb / bestfit / bestfit.html )

程序

  1. 收获,研磨和准备叶片材料
    1. 收获 注意:如果目标代谢物的周转时间非常快,则应通过切割玫瑰花结并在N 2 在主要辐照度下。然后将淬火的物料转移到预先在液态N 2 中预冷的20 ml聚乙烯瓶(含钢球)中。为了实现最佳研磨过程,请使用植物材料将小瓶装满最大容积的2/3。对于NAF,需要4 g的物质,但是建议多一点收获,以便有更多的物质对这种物质进行其他分析(例如代谢产物的定量,请参阅数据分析–4)。
    2. 为了均匀化,用钢盖代替Polyvials的塑料盖,然后将包含叶片材料的Polyvials放入用于Retsch轧机的预冷钢磨适配器(图1A-1D)。
      注意:我们的研磨适配器和钢盖是为20毫升Polyvials量身定制的。或者,可以通过使用预冷的研钵和研杵来研磨叶片材料。在整个过程中,确保叶片材料保持冷冻状态。
    3. 在Retsch磨机中以25 Hz(两次)将组织匀浆1分钟(图1E和1F)。
      注意:确保地面组织非常均匀(稀粉末)。我们建议将叶片材料以25 Hz的频率研磨两次,每次1分钟。用液态N 2 冷却小瓶和适配器之间的温度,以免材料融化。


      图1.研磨步骤。 A.在液氮中冷却Polyvials,钢研磨适配器和钢盖。 B.取下Polyvials的塑料盖,然后将钢盖冷冻。 C.使用封口机/开盖机(定制)用钢盖封闭小瓶。 D.封闭的小瓶。 E.将Polyvial放入钢制研磨适配器内。 F.将研磨适配器放入Retsch磨机中,然后按照步骤A3进行操作。研磨后,使用相同的封盖机/开盖器机器替换塑料盖的钢盖。样品可以保存在-80°C或进入步骤A4。请注意,为避免样品瓶过热,请在准备第二个样品时,用钢帽和研磨适配器将其深浸在液氮中或使用干冰。

    4. 称重约4 g冷冻组织到50 ml在N 2 液体中预冷的Falcon管中。样品可以在-80°C下保存三个月。准备所需数量的Falcon管(每根将用于一根NAF)。
      注意:标记猎鹰管及其相应的盖子。记录空Falcon管(带盖)的重量以及精确称量的植物材料。 
    5. 冷冻干燥匀浆。为避免在此过程中丢失叶片材料,请取下盖子,并用固定有松紧带的纸巾(KimWipes)覆盖猎鹰管。将试管放入金属架中,放入0.02 bar和-80°C的冷冻干燥器中5天。从冷冻干燥机中取出后,立即关闭具有相应盖子的试管。干燥后的材料可以在真空中保存在干燥器中,避免受到光和潮的影响,最长可保存6个月。
      注意:为避免解冻,请将Falcon管放在装有干冰的聚苯乙烯盒中,并在Falcon管处于盒子中的情况下用纸巾盖上盖子。
    6. 冷冻干燥后,通过称重密闭的Falcon管确定植物材料的干重。

  2. 准备溶液和过滤器
    1. 开始分馏前三天,为NAF准备溶液。
    2. 将活化的分子筛4Å添加到庚烷(C 7 H 16 )和四氯乙烯(C 2 C 14 >)解决方案,以确保不残留水。
    3. 制备四氯乙烯/庚烷的混合物66:34(v / v);密度= 1.3 g cm -3 。添加活化分子筛4Å,并将混合物避光保存在棕色玻璃瓶中。
      注意:我们通常在1 L的瓶子中使用约100 ml的活化分子筛。对于10个样品,需要:a)1 L四氯乙烯/庚烷混合物66:34(v / v):660 ml四氯乙烯+ 340 ml庚烷,b)2 L庚烷,c)500 ml四氯乙烯。
    4. 使用前,请始终用pH纸检查溶液的pH值,尤其是四氯乙烯。 pH应该是中性的。如果四氯乙烯溶液变成酸性,则干燥的叶片将在梯度离心过程中变成褐色,而NAF则失效。
    5. 切开15厘米x 15厘米的尼龙过滤器(每个NAF一个),称重,然后将其存储在干净的50毫升Falcon管中。

  3. 分馏程序
    注意:整个过程应在通风橱中进行。只能在常规工作台上处理干燥的等分试样(在步骤C17之后)。对于倾倒溶液,请使用玻璃移液器。
    1. 将干燥的叶片材料重悬于20 ml的四氯乙烯/庚烷= 66:34(v / v)混合物中。
    2. 用6 x 10个周期将悬浮液超声处理2分钟,功率的65%(图2)。为避免超声处理期间悬浮液过热,请将Falcon管放在冰浴中。
      注意:将超声仪的尖端放在溶液中,但避免接触管壁。确保超声处理成功。这是分馏的关键步骤,因为超声处理不足会产生无法通过尼龙网的聚集体。


      图2.超声处理程序的设置。请注意,为避免在处理过程中悬浮液过热,猎鹰管应置于冰浴中。

    3. 将尼龙网折叠到置于50 ml Falcon管顶部的玻璃漏斗中,然后将悬浮液倒入尼龙网中。用10毫升庚烷洗涤尼龙网3次。
      注意:前两个洗涤步骤是通过将庚烷缓慢倒入尼龙网并在下面的Falcon管中收集流来进行的。对于第三步洗涤,通过挤压(戴上足够的手套)除去尼龙网中的所有残留溶液。将尼龙网放回50 ml Falcon管中,并继续执行步骤C16。注意所有液体都流经尼龙网。如果是这种情况,请通过新的尼龙网再次过滤所有获得的混合物。
    4. 使用外摆式转子在3200 x g 和4°C下离心10分钟。弃去上清液(将其倒入废玻璃瓶中),然后将沉淀重悬于5 ml四氯乙烯/庚烷= 66:34(v / v)的混合物中。
    5. 将100 µl匀浆均匀的重悬沉淀物转移到1.5 ml螺帽管中。该等分试样称为分数0(F0),对应于未分级的物质。准备三个等分试样,向每个试样中加入900 µl C 7 H 16 ,并保持避光,然后继续执行步骤C15。
      注意:使用200 µl切割好的移液器吸头收集F0。均匀混合均匀后再取等分试样。
    6. 准备NAF梯度(图3A):线性梯度(30 ml,密度从1.43到1.62 g cm -3 )
      1. 用巴斯德移液器在50 ml的Falcon管A中填充四氯乙烯/庚烷混合物,在50 ml的Falcon管B中填充四氯乙烯。
      2. 分配针置于废物容器上方。
        注意:彻底清洗整个系统,确保所有管路中都没有气泡。
      3. 将蠕动泵的流量设置为1.15 ml min -1 ,并执行以下程序:
        class =“ ke-zeroborder” bordercolor =“#000000” style =“ width:500px;” border =“ 0” cellspacing =“ 0” cellpadding =“ 2”> <身体>步骤
        ml
        溶剂A
        溶剂B
        1 *
        0 70%
        30%
        2 **
        3 70%
        30%
        3 28 0% 100% 4 33 0% 100% 注释:
        1. * 3毫升后,暂停程序。此步骤用于确保在开始实际梯度之前,第一种混合物到达分配针的末端。
        2. **取出废物容器,然后在此步骤中放置聚异丁烯管。针头应稍微放在聚异丁烯管的底部上方,并轻轻接触管的侧面(图3B)。重新启动程序。


          图3.梯度泵和混合器的设置。 A.准备NAF梯度的设置概述。溶液A(四氯乙烯/庚烷混合物)和溶液B(四氯乙烯)。 B.渐变的自下而上准备的细节。试管架是50毫升的猎鹰管,底部装有纸支撑,可帮助固定梯度管。

    7. 小心地从梯度中取出针头,然后将 Polyallomer 管放在架子上。使用巴斯德移液器非常小心地将步骤C4的悬浮液应用于梯度。用移液管在梯度上方稍微接触管壁,然后小心释放,以免干扰梯度。
    8. 将梯度放入超速离心机的相应试管桶中。验证其重量并平衡,通过添加100%C 2 Cl 4 来调整重量。
    9. 使用摆动斗式转子在5,000 x g 和4°C下离心50分钟。
      注意:我们始终使用L80 XP超速离心贝克曼库尔特Optima TM L80 XP和摆桶式转子贝克曼库尔特SW32Ti。将加/减速设置为三个。
    10. 将梯度管放在架子上并标记八个分数。图4说明了我们通常如何根据颜色分离分数。


      图4.离心后的NAF梯度。通常,我们根据其颜色分离出八个馏分。深绿色层富含叶绿体,通常分为两个部分(F2和F3),以避免代谢物超载。请注意,级分8是沉淀,是富含液泡的级分。

    11. 使用巴斯德移液器将每个部分小心地转移到清晰标记的干净的50 ml Falcon管中。从上部(F1)到底部(F8)收集馏分。除去F7后,将沉淀重悬于20 ml庚烷中,并将混合物转移至干净的50 ml Falcon管中。对每个馏分使用新的移液器。
      注意:转移馏分是该方法的关键,因为在此步骤中应尽可能避免馏分的交叉污染。因此,请始终从每一层的最顶部转移分数。
    12. 向装有F1至F8的Falcon管中加入多达20 ml的庚烷,并仔细混合。将所有Falcon管在3,200 x g 和室温下离心10分钟。
    13. 弃去上清液(通过将其小心地倒入玻璃废液瓶中),然后用7 ml庚烷重悬沉淀。
      注意:数量取决于您需要进行分析的等分试样数。我们通常每个分数准备七个等分试样。此数字足以用于各种代谢物分析和确定隔室特异性标志物。
    14. 从每个悬浮液中将6 x 1 ml转移到1.5 ml螺帽管中。在此步骤中,沉淀物很容易重新聚集。转移到试管中时,用手继续混合溶液,以便从相同馏分中获得均质的等分试样。
      注意:用切好的1000 µl移液器吸头从每个馏分收集亚等分试样。由于添加7 ml通常不允许获得1 ml的7等分试样,因此我们通常取0.8 ml作为7等分试样,并清楚标记相应的试管。
    15. 在室温下以14,000 x g 离心10分钟。使用1000 µl移液器吸头小心丢弃过量的上清液( c.a。 900 µl)。以相同的方式处理在步骤C5中获得的F0等分试样。
    16. 将装有分等份的螺帽管放在连接到真空泵的干燥器内部的机架中。另外,放置装有步骤C3过滤器的开放式Falcon管。将等分试样置于真空中并避光过夜。
    17. 封闭试管,将其保存在-80°C直至进一步处理。
    18. 称重干燥的过滤器。

  4. 代谢物标记物的测定
    1. 萃取
      1. 向干燥的等分试样中加入钢球和250 µl 80%乙醇/ 10 mM HEPES pH 7。
      2. 将试管放入球磨机的冷却(4°C)支架中,以25 Hz的频率将沉淀破碎1分钟。
      3. 在热混合器中于95°C和1000 rpm孵育30分钟。
      4. 在20,800 x g 和4°C下离心10分钟。
      5. 将上清液转移到96孔深的孔板上,并保持避光和冰冻。
      6. 将150 µl 80%乙醇/ 10 mM HEPES pH 7加到颗粒中。
      7. 在95°C 1000 rpm的温度混合器中孵育30分钟。
      8. 在20,800 x g 和4°C下离心10分钟。
      9. 转移并与之前的上清液合并。
      10. 加入250微升50%乙醇/ 10 mM HEPES pH 7。
      11. 在95°C和1,000 rpm的温度混合器中孵育30分钟。
      12. 在20,800 x g 和4°C下离心10分钟。
      13. 转移并与前两个上清液合并。可以在-20°C下保存,直至进一步使用。
    2. 硝酸盐(真空标记)
      硝酸盐含量根据Cross et al。(2006)确定。除酸性转化酶外,硝酸盐还可用作液泡标记物。
      注意:亚硝酸盐含量必须确定并从硝酸盐值中减去。为了测量亚硝酸盐含量,将硝酸盐还原酶替换为分析混合物中的水。
      解决方案:对于存储建议,硝酸盐测量解决方案可以按以下方式存储:
      class =“ ke-zeroborder” bordercolor =“#000000” style =“ width:600px;” border =“ 0” cellspacing =“ 0” cellpadding =“ 2”> <身体>解决方案:
      存储温度
      磷酸钾缓冲液1 M pH 7.5
      -20°C
      NaOH中的NADPH 50 mM 20 mM
      -80°C
      硝酸还原酶(磷酸钾缓冲液0.1 M中的5 U ml -1 )
      -80°C
      PES 0.25毫米 -80°摄氏度 磺胺1%(w / v)的5%磷酸溶液
      4°C
      NNEDA 0.02%(w / v)
      4°C
      硝酸盐标准
      -80°C
      硝酸盐标准品:70%ETOH中的0、0.2、0.4、0.8和1.6 mM,MES pH 5.9为10 mM
      协议:
      1. 对于一个反应,将95 µl分析混合液分配到96孔板中:
        10.0 µl磷酸盐缓冲液
        0.5 µl NADPH
        2.0 µl硝酸盐还原酶
        82.5 µl去离子水
      2. 加入5 µl标准品或提取物(1至10稀释的提取物)。
      3. 混合并在避光下于25°C孵育30分钟。
      4. 加入15 µl PES。
      5. 混合并在避光条件下于25°C孵育20分钟。
      6. 加入50 µl磺胺甲酰胺和50 µl NNEDA。
      7. 混合并在避光下于25°C孵育10分钟。
      8. 在酶标仪中读取540 nm处的板。

  5. 酶标记物的测量
    1. 酶提取
      1. 向干燥的提取物中加入约2 mg PVPP(一把小汤匙),一个钢球(直径5 mm)和500μl1x缓冲液(请参见配方1b)。将样品放在冰上。
      2. 将试管放入球磨机的冷却(4°C)支架中,以25 Hz的频率将沉淀破碎1分钟。确保整个沉淀溶解。如果没有,请重复沉淀操作。
      3. 在20,800 x g 和4°C下离心10分钟。将酶提取物(上清液)转移到96深孔板中。保持冰上并进行酶测定。如果需要,可以将上清液保存在-80°C。
    2. 酶测定
      三种酶标记均根据Gibon等人(2004)进行测量。
      1. AGPase活性(质体标记)
        1. 分配在置于冰上的96孔板上:
          14 µl AGPase分析混合物(请参见配方B2b)
          6 µl标准品或提取物。
        2. 混合并在25°C下孵育20分钟。
        3. 加入100 µM Tricine / KOH pH 8中的20 µl 0.5 M HCl。
        4. 混合并在室温下孵育10分钟。
        5. 加入20 µl 0.5 M NaOH。
        6. 混合,加入50 µl AGPase定量混合物(请参见配方B2c)。
        7. 混合,旋转并在340 nm和30°C的波长下读取,直到最大反应速率稳定为止。
      2. PEPC(细胞溶胶标记物)
        1. 分配在置于冰上的96孔板上。
          18 µl PEPC分析混合物(请参见配方B3b)
          2 µl标准品或提取物
        2. 混合并在25°C下孵育20分钟。
        3. 加入100 µM Tricine / KOH pH 9中的20 µl 0.5 M HCl。
        4. 混合,旋转并在95°C下孵育5分钟。冷静下来。将盘子放在冰上。
        5. 加入20 µl 0.5 M NaOH。
        6. 混合,加入45 µl PEPC测定混合物(请参见配方3c,避光)。
        7. 加入5 µl 4 mM PES(避光)。
        8. 混合并在570 nm和30°C的波长下读取,直到最大反应速率稳定为止。
      3. 酸性转化酶(液泡标记)
        1. 在黑色的96孔微孔板上分配:
          10 µl酸性转化酶测定混合物(请参见配方B4b)
          5 µl标准品或酶提取物
        2. 混合并在25°C下孵育5和40分钟。
        3. 加入10 µl 0.5 M NaOH。
        4. 混合并在室温下孵育10分钟
        5. 加入100 µM Tricine / KOH pH 8中的10 µl 0.5 M HCl。
        6. 混合并加入50 µl的酸性转化酶测定混合物(请参见配方B4c)。
        7. 使用以下设置混合并读取荧光10-15分钟:
          在530 nm处激发,在590 nm处发射,温度:30°C,灵敏度为25。使用反应速率进行计算。我们使用Synergy荧光计。

数据分析

  1. 为了评估亚细胞代谢物的分布,我们建议使用统计软件BestFit。 BestFit是一种C语言命令行工具,允许从NAF数据计算和评估亚细胞分布。该软件要求; (i)在不同馏分中分析的每个亚细胞区室的特异性标志物的分布(图5)和(ii)在不同馏分中感兴趣的代谢物的分布。


    图5.图4中显示了整个梯度中亚细胞区室特异性标志物的百分比分布。 ADP葡萄糖焦磷酸化酶(AGPase)作为质体标志物,磷酸烯醇丙酮酸羧化酶(PEPC)作为胞质标志物以及硝酸盐和酸转化酶作为液泡标记物。

  2. 对于输入文件中感兴趣的标记物和代谢物的值,尽管也可以使用绝对测量(尽管在这种情况下值必须≥0),但我们建议使用百分比。
  3. 有关如何使用BestFit的更多详细信息,建议您阅读BestFit文件夹中的文档pdf文件( http://www.csbdb.de/csbdb/bestfit/bestfit.html )。另外,您也可以在Krueger等人(2014)中找到有关数据输入和BestFit使用的详细信息。
  4. BestFit以百分比形式输出目标代谢物的亚细胞分布。为了计算其代表的量,还对未通过NAF处理的研磨物料中的代谢物进行了测量(请参阅A1中的注释)。可以使用以下信息进行各种回收率计算:(i)用于NAF的物料新鲜重量(在步骤A4中确定),(ii)相应的干重(在步骤A6中确定),(iii)施加了多少物料梯度(在步骤C18中通过称量过滤器中的残留物确定),(iv)在步骤C6中获得F0。我们建议始终记录这些数据并为每个NAF收集F0。

菜谱

  1. 代谢物标记物的测定
    1. 硝酸盐标准
      在70%的ETOH中的0、0.2、0.4、0.8和1.6 mM以及10 mM MES pH 5.9

  2. 酶标记物的测量
    1. 提取缓冲液
      1. 提取缓冲液10x
        500 mM HEPES
        100 mM氯化镁 2
        10 mM EDTA
        10毫米EGTA
        10 mM苄am
        10 mMε-氨基己酸
        2.5%w / v BSA(不含蛋白酶)
        将音量调到c.a. 40 ml,用10 M KOH将pH调节至7.5,然后将体积调节至50 ml
        提取缓冲液10x可以在-20°C下保存

        准备以下解决方案并进行相应存储:
        Leupeptin 2 mM(100x),在液氮中冷冻,然后储存在-80°C
        DTT 500 mM(1,000x),在液氮中冷冻,然后在-80°C储存
        PMSF在异丙醇中的100 mM(100x),储存在4°C,避光,稳定1个月
        Triton X-100 10%v / v(10x),存放在RT
        甘油(87%v / v),储存在室温下
      2. 提取缓冲液1x
        仅在提取前准备,结合以下步骤:
        class =“ ke-zeroborder” bordercolor =“#000000” style =“ width:600px;” border =“ 0” cellspacing =“ 0” cellpadding =“ 2”> <身体>[最终]
        试剂
        [股票]
        用于1,000 µl
        1x
        缓冲液10x(请参见配方1a)
        10x
        100 µl
        10 µM
        Leupeptin
        2毫米
        5 µl
        1毫米
        DTT
        500毫米
        2 µl
        1毫米
        PMSF
        100 mM的异丙醇
        10 µl
        1%
        海卫一X-100
        10%v / v
        100 µl
        20%
        甘油
        87%(v / v)
        230微升

        H 2 O

        553微升
    2. AGPase活性
      1. AGPase分析缓冲液(可以在-20°C下保存)
        0.25 M HEPES / KOH pH 7.5
        7.5 mM NaF
        25 mM氯化镁 2
        5 mM 3-磷酸甘油酸酯
        0.25%(v / v)海卫X-100
      2. AGPase分析混合物
        7.6微升水
        4 µl AGPase分析缓冲液(请参见食谱B2a)
        2 µl 20 mM PPi
        0.2 µl甘油激酶(200 mM Tricine / KOH pH 8中的200 U ml-1,10 mM MgCl 2 )
        0.2 µl ADP葡萄糖0 mM(空白)或100 mM(最大)
      3. AGPase测定混合物
        37.8 µl水
        10 µl 1 M Tricine / KOH pH 8
        0.2 µl 1 M MgCl 2
        0.5 µl GDH(200 mM Tricine / KOH pH 8中的200 U ml-1,10 mM MgCl 2 )
        0.5 µl GPOX(200 mM Tricine / KOH pH 8中的500 U ml-1,10 mM MgCl 2 )
        1微升66 mM NADH
    3. PEPC活动
      1. PEPC分析缓冲液(可在-20°C下保存)
        0.5 M Tricine / KOH pH 8
        100 mM氯化镁 2
        50 mM NaHCO 3
        0.25%Triton X-100
      2. PEPC分析混合物
        11.4 µl H 2 O
        4 µl PEPC测定缓冲液(请参见配方B3a)
        2 µl 0(空白)或20 mM PEP(最大)
        0.4 µl 5 mM NADH
        0.2 µl苹果酸脱氢酶(200 mM Tricine / KOH pH 8中的100 U ml -1 ,10 mM MgCl 2 )
      3. PEPC测定混合物(避光)
        18 µl水
        10 µl 1M Tricine / KOH pH 9.0
        10 µl 10 mM MTT
        4 µl 200 mM EDTA
        2 µl 50%乙醇
        1 µl酒精脱氢酶(在200 mM Tricine / KOH pH 9中为2,000 U ml -1 ,10 mM MgCl 2 )
    4. 酸性转化酶活性
      1. 酸性转化酶测定缓冲液(可在-20°C下保存)
        0.25 M醋酸盐/ KOH pH 5
      2. 酸性转化酶测定混合物:用于1个反应
        10 µl酸性转化酶测定缓冲液(请参见配方B4a)
        35微升水
      3. 酸性转化酶测定混合物(避光):用于1个反应
        42 µl水
        5 µl 1 M HEPES / KOH pH 7
        1 µl葡萄糖氧化酶(在200 mM HEPES / KOH pH 7.0中为200 U ml -1 )
        1 µl辣根过氧化物酶(在200 mM HEPES / KOH pH 7.0中为0.2 U ml -1 )
        1 µl DMSO中的20 mM双联红色

致谢

我们感谢Deutsche Forschungsgemeinschaft(DFG TRR 175绿色枢纽–植物适应中心协调员; FA和ARF)提供的资金。
&nbsp;我们还要感谢Krueger et al。(2014)的作者,之前描述NAF方法的工作,我们对本协议进行了修改。

利益争夺

作者宣称没有利益冲突。

参考文献

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Copyright Medeiros 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. Medeiros, D. B., Arrivault, S., Alpers, J., Fernie, A. R. and Arabi, F. (2019). Non-aqueous Fractionation (NAF) for Metabolite Analysis in Subcellular Compartments of Arabidopsis Leaf Tissues. Bio-protocol 9(20): e3399. DOI: 10.21769/BioProtoc.3399.
  2. Shapiguzov, A., Vainonen, J. P., Hunter, K., Tossavainen, H., Tiwari, A., Jarvi, S., Hellman, M., Aarabi, F., Alseekh, S., Wybouw, B., Van Der Kelen, K., Nikkanen, L., Krasensky-Wrzaczek, J., Sipari, N., Keinanen, M., Tyystjarvi, E., Rintamaki, E., De Rybel, B., Salojarvi, J., Van Breusegem, F., Fernie, A. R., Brosche, M., Permi, P., Aro, E. M., Wrzaczek, M. and Kangasjarvi, J. (2019). Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors. Elife 8. pii:e43284.
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