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Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification
通过测量H+泵和定量H+-ATP酶分析测定拟南芥保卫细胞原生质体中H+-ATP酶的活性   

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Plant Physiology
Aug 2016

Abstract

The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of guard cell protoplasts and measure H+-pumping in the protoplasts. It is also necessary to determine the protein amount of H+-ATPase. In this protocol, we describe the procedures required for these preparations and measurements.

Keywords: Arabidopsis thaliana (拟南芥), Blue light (蓝光), Fusicoccin (壳梭孢素), H+-pumping (H+泵), Plasma membrane H+-ATPase (质膜H+-ATP酶)

Background

The opening of stomata in response to blue light is driven by membrane hyperpolarization mediated through H+-pumping across the plasma membrane in guard cells (Assmann et al., 1985; Shimazaki et al., 1986), and is brought about by the plasma membrane H+-ATPase (Kinoshita and Shimazaki, 1999). The H+-ATPase generates an electrochemical gradient across the membrane, and provides the energy required for numerous secondary transports in plant cells. However, it is not easy to measure the activity of H+-ATPase in vivo. Taking advantage of the blue light-sensitive properties of guard cells, our method makes it possible to measure H+-pumping as an in vivo H+-ATPase activity using Arabidopsis guard cell protoplasts (Ueno et al., 2005). Together with H+-ATPase quantification by Western blot (Yamauchi et al., 2016), this method allows comparing H+-ATPase activity under different conditions or mutant backgrounds.

Materials and Reagents

  1. Planter (640 x 230 x 185 mm) (Iris Oyama E-type, PR650EMK)
  2. Vermiculite (particle size 3-6 mm)
  3. Soil (potting soil containing peat moss)
  4. Cellophanes (Asahi KASEI)
  5. Nylon mesh (10 µm, 25 µm, 58 µm and 94 µm) (Kyoshin Riko)
  6. Micro slide glass (Matsunami Glass, catalog number: S011260 )
  7. Glass test tube (10 ml) (IWAKI, catalog number: TEST15-105NP )
  8. Gel loading tip (Thermo Fisher Scientific, Thermo Scientific, catalog number: 010-Q )
  9. Sample tube (1.5 ml) (INA•OPTIKA, catalog number: ST-0105F )
  10. Micro cuvette (100 µl) (Beckman Coulter, catalog number: 523270 )
    Note: This product has been discontinued. Any micro cuvette (100 µl) that can be fixed in the spectrophotometer can be substituted.
  11. Blender cup (100 ml) (Waring Lab, catalog number: MC2 )
  12. Glass pipette (10 ml) (Sansyo, catalog number: 73-0045 )
  13. Glass centrifuge tube (50 ml) (Sansyo, catalog number: 84-0182 )
  14. Orange-colored film (Kodak, Cinemoid 5A)
  15. Filter paper (3MM CHR CHROMATOGRAPHY PAPER, GE Healthcare, catalog number: 3030-928 )
  16. Arabidopsis thaliana ecotype Col-0
  17. Arabidopsis thaliana mutant aha1-10
  18. Bradford protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 5000006JA )
  19. Aprotinin (Merck, Calbiochem, catalog number: 616399 )
  20. Protease inhibitor cocktail set III (Merck, Calbiochem, catalog number: 539134 )
  21. MG132 (Sigma-Aldrich, catalog number: M8699-1MG )
  22. Trichloroacetic acid (TCA) (NACALAI TESQUE, catalog number: 06275-24 )
  23. Anti-rabbit IgG HRP (Bio-Rad Laboratories, catalog number: 1706515 )
  24. Anti H+-ATPase antibodies (Kinoshita and Shimazaki, 1999) or Agrisera anti H+-ATPase antibodies (Agrisera, catalog number: AS07 260 )
  25. Clarity Western ECL substrate (Bio-Rad Laboratories, catalog number: 1705061 )
  26. Cellulase R-10 (Yakult, Pharmaceutical Industry, Tokyo, Japan)
  27. Macerozyme R-10 (Yakult, Pharmaceutical Industry, Tokyo, Japan)
  28. Polyvinylpyrrolidone K 30 (PVP-K30) (NACALAI TESQUE, catalog number: 28314-95 )
  29. Bovine serum albumin (BSA) (Thermo Fisher Scientific, GibcoTM, catalog number: 30036727 )
  30. Mannitol (Wako Pure Chemical Industries, catalog number: 130-00855 )
  31. Calcium chloride dihydrate (CaCl2·2H2O) (Wako Pure Chemical Industries, catalog number: 031-00435 )
  32. 2-(N-morpholino) ethanesulfonic acid (MES) (NACALAI TESQUE, catalog number: 02442-44 )
  33. Cellulase RS (Yakult, Pharmaceutical Industry, Tokyo, Japan)
  34. Hydrochloric acid (HCl) (Wako Pure Chemical Industries, catalog number: 081-03475 )
  35. Potassium chloride (KCl) (NACALAI TESQUE, catalog number: 28513-85 )
  36. Fusicoccin (Fc) (Sigma-Aldrich, catalog number: F0537-1MG )
  37. DMSO (Wako Pure Chemical Industries, catalog number: 046-21981 )
  38. Tris (hydroxymethyl) aminomethane (Wako Pure Chemical Industries, catalog number: 514-98121 )
  39. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  40. Sodium dodecyl sulfate (SDS) (NACALAI TESQUE, catalog number: 31606-75 )
  41. Ammonium peroxodisulfate (Wako Pure Chemical Industries, catalog number: 012-20503 )
  42. 30% (w/v) Acrylamide/Bis mixed solution (37.5:1) (Wako Pure Chemical Industries, catalog number: 018-25625 )
  43. N,N,N’,N’-tetramethylethylenediamine (NACALAI TESQUE, catalog number: 33401-72 )
  44. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14112-94 )
  45. Nα-Tosyl-Lys chloromethyl ketone (Merck, Calbiochem, catalog number: 616382 )
  46. Ponceau S (NACALAI TESQUE, Catalog number: 28322-72 )
  47. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00251 )
  48. Tween 20 (MP Biomedicals, catalog number: 0210316890 )
  49. Skim milk (Snow Brand Milk Products, Ltd.)
  50. Glycine (Wako Pure Chemical Industries, catalog number: 077-00735 )
  51. Methanol (Wako Pure Chemical Industries, catalog number: 137-01823 )
  52. Ethylenediaminetetraacetate acid (EDTA) (Wako Pure Chemical Industries, catalog number: 343-01861 )
  53. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
  54. Coomassie Brilliant Blue G-250 (NACALAI TESQUE, catalog number: 09409-42 )
  55. 2-Mercaptoethanol (NACALAI TESQUE, catalog number: 21417-52 )
  56. 1st digestion solution (see Recipes)
  57. 2nd digestion solution (see Recipes)
  58. 2x H+-pumping buffer (see Recipes)
  59. Reaction mixture for H+ pumping (see Recipes)
  60. Fc stock solution (see Recipes)
  61. Reaction mixture for inhibitor treatment (see Recipes)
  62. HCl stock solution (see Recipes)
  63. 10x TBS (see Recipes)
  64. 4x separation buffer (see Recipes)
  65. 4x stacking buffer (see Recipes)
  66. Ammonium peroxodisulfate stock solution (see Recipes)
  67. 10% acrylamide gel (see Recipes)
  68. DTT stock solution (see Recipes)
  69. TLCK stock solution (see Recipes)
  70. Ponceau S (see Recipes)
  71. Tween 20 stock solution (see Recipes)
  72. Blocking buffer (see Recipes)
  73. T-TBS (see Recipes)
  74. Transfer buffer (see Recipes)
  75. 3x SDS buffer (see Recipes)
  76. 1x SDS sample buffer (see Recipes)

Equipment

  1. Metal shelf made by stainless steel (120 x 61 x 230cm) (Iris Oyama, MR-230P)
  2. Blender (Waring Lab, model: 51BL31 , catalog number: 7011BU)*
  3. Funnel (TGK, catalog number: 416-09-24-04 )*
  4. Erlenmeyer flask (500 ml) (IWAKI, catalog number: 82-0088 )
  5. Erlenmeyer flask (300 ml) (IWAKI, catalog number: 82-0087 )
  6. Refrigerated centrifuge (TOMY, model: AX-310 )*
  7. Refrigerated centrifuge (TOMY, model: MX-100 )*
  8. Incubator with shaker (TAITEC, model: Personal Lt10 )
  9. Unit thermostat bath (TAITEC, THERMO MINDER model: JR 80 )*
  10. Spectrophotometer (Beckman Coulter, model: DU800 )*
  11. pH glass electrode (Beckman Coulter, catalog number: 39532 )*
  12. pH meter (Beckman Coulter, model: Φ71 pH meter )*
  13. Chart recorder (YOKOGAWA ELECTRIC WORKS, model: 3066 )*
  14. Magnetic stirrer (RANK BROTHERS, model: Model 300 )
  15. Chiller bath circulator (Thermo Fisher Scientific, Thermo ScientificTM, model: RTE7 )
  16. LED panel (CCS, model: ISL 150X150 H4RRHB , Made by order)
    Note: LEDs for both red and blue are fixed on the same panel, and each LED can be turned on and off by the power supply.
  17. LED power supply (CCS, model: ISC-201-2 )
  18. Microscope (Nikon Instruments, model: Eclipse TS100 )
  19. Pipetman (P200)
  20. Power supply (Bio-Rad Laboratories, model: PowerPacTM HC High-Current Power Supply )
  21. Transfer Cell (Bio-Rad Laboratories, model: Trans-Blot® SD Semi-Dry Transfer Cell )
  22. Electrophoresis vessel (ATTO, model: AE6200 , catalog number: 2392385)*
  23. Chemical Luminescence analyzer (Bio-Rad Laboratories, model: ChemiDocTM Touch Imaging System )
  24. Gel cassette (ATTO, model: AE-6210 )

*Note: This product has been discontinued.

Software

  1. ImageJ

Procedure

  1. Preparation of guard cell protoplasts from Arabidopsis leaves
    1. Sow 200 Arabidopsis seeds per planter on soil and vermiculite mixture (1:1).
    2. Grow Arabidopsis plants for 4- to 6-weeks under white light (60 µmol m-2 sec-1) with14/10 h light/dark cycle at 24 °C (see Figure 1).


      Figure 1. Four weeks old Arabidopsis grown under white light. Arabidopsis plants were grown for 4-weeks under white light (60 µmol m-2 sec-1) with 14/10 h light/dark cycle at 24 °C. The bar represents 1 cm.

    3. Harvest the fully expanded rosette leaves from the 200 plants and remove the main petioles (~2 cm-length, see Figure 2) before bolting.


      Figure 2. Fully expanded leaves from 4 weeks old Arabidopsis. Fully expanded leaves were harvested from Arabidopsis and main petioles were removed before bolting. The bar represents 1 cm.

    4. Place the leaves (25 g) in ice-cold water (see Figure 3).


      Figure 3. Leaves in ice-cold water. Harvested leaves were put into a glass beaker filled with ice-cold water.

    5. Move the ice-cold leaves into the cup of Waring blender (see Figure 4A). Homogenize the leaves in the blender covered with two layers of cellophane (see Figure 4B) at full speed for 45 sec in 70 ml ice-cold distilled water. Pour the resulting homogenate onto the nylon mesh placed on a funnel (see Figure 5). Discard the filtrate (flow-through) throughout the protocol.


      Figure 4. Waring blender with blender cup. A. Picture of Waring blender with blender cup; B. Fully expanded rosette leaves were put into blender cup filled with ice-cold water. Blender cup was sealed with cellophane. Leaves were homogenized at maximum speed for 45 sec.

    6. Transfer the epidermal tissues retained on the 58 µm mesh (see Figure 5) into the cup of Waring blender by pouring ice-cold distilled water (see Video 1) and subject them to homogenization again for 1 min in 70 ml water at full speed, and collect them by filtering through the 58 µm nylon mesh as above. The nylon mesh can be reused after cleaning with distilled water.


      Figure 5. Epidermal tissues collected with layers of 58 µm nylon mesh. Homogenized epidermal tissues were collected with layers of 58 µm nylon mesh after 45 sec homogenization. Collected tissues were placed into the blender cup again and homogenized for 1 min (see Video 1). The epidermal tissues were collected again with the 58 µm nylon mesh and resuspended in the 1st digestion solution.

      Video 1. Transfer the epidermal fragments by pouring the solution onto the fragments on the mesh using a wash bottle (or 10 ml Komagome pipette, not shown here)

    7. Transfer 12-13 g of the wet epidermal fragments on the mesh (see Figure 7) into a 500 ml Erlenmeyer flask by pouring the 1st digestion solution (see Recipes) using a wash bottle or a 10 ml Komagome pipette (see Figure 6 and Video 1). Adjust the total volume of the digestion solution to 100 ml by adding the same solution


      Figure 6. Komagome pipette (10 ml) with silicone rubber bulb


      Figure 7. Mesophyll cell removal by 1st digestion solution. A. Epidermal fragment before 1st digestion solution treatment. Mesophyll cells adhered to epidermal tissue. B. Epidermal fragment after 1st digestion solution treatment. All mesophyll cells are detached from the epidermal cells. The bars represent 10 µm.

    8. Pass the epidermal fragments gently into 100 ml of 1st digestion solution in the Erlenmeyer flask through the same Komagome pipette several times up and down to thoroughly mix the suspension and to ensure homogeneous digestion (see Video 2).

      Video 2. Pipetting the partially digested epidermal fragments to remove the mesophyll cells using Komagome pipette
       
    9. Incubate the suspension in the Erlenmeyer flask at 24 °C for 30 min, shaking at 70 strokes min-1.
    10. Pippet the partially digested epidermal fragments in the 1st digestion solution 30 times up and down through Komagome pipette to remove the attached mesophyll cells (see Video 2). Inspect the mesophyll cells attached to the epidermal fragments on a micro slide glass using a microscope (150x).
    11. Repeat Step A9 until all mesophyll cells attached to the epidermis are removed (see Figure 7).
    12. Pass the suspension through a 58 µm nylon mesh, and keep the epidermal fragments retained on the mesh.
    13. Transfer the epidermal fragments into 0.3 M mannitol and 1 mM CaCl2 solution by pouring the solution onto the fragments on the mesh (see Video 1). Keep the fragments in the solution for 30 min to adapt them to the high osmotic pressure.
    14. Pass the osmotically adapted epidermis through the 58 µm nylon mesh. Transfer the epidermis retained on the mesh into a 300 ml Erlenmeyer flask by pouring the 2nd digestion solution (see Recipes) on the epidermis using Komagome pipette (see Video 1). Adjust the total volume to 50 ml by adding the same 2nd digestion solution.
      Note: Since the epidermal peels were partially digested and fragile, do not touch the epidermis in this step.
    15. Incubate the epidermis in the 2nd digestion solution with 50 strokes min-1 for 50-60 min at 27 °C.
    16. Using several epidermal fragments, inspect whether most of the guard cells have become spherical under a microscope (see Figure 8).


      Figure 8. Guard cell spheroidization by 2nd digestion solution. Epidermal fragments were incubated with 2nd digestion solution. The spherical nature of the guard cells was examined under the microscope (Black arrows). The bar represents 10 µm.

    17. Place the Erlenmeyer flask containing the digested epidermis on ice, and pippet the suspension gently up and down 60 times through a Komagome pipette to detach guard cell protoplasts from the epidermis (see Video 2).
    18. Pass the suspension through two layers of piled nylon meshes of 94 and 25 µm (the 94 µm mesh is on top of the 25 µm one) to separate guard cell protoplasts from the epidermal remnants.
    19. Pass the separated guard cell protoplasts through two layers of 10 µm nylon mesh to remove contamination by other small tissues.
    20. Centrifuge the suspension of guard cell protoplasts at 420 x g for 18 min at 4 °C to collect guard cell protoplasts as a pellet. Discard the supernatant.
    21. Resuspend the pellet in a 50 ml solution of 0.4 M mannitol and 1 mM CaCl2.
    22. Repeat Steps A19 and A20 twice (total three times) to remove the residual enzymes.
    23. Resuspend the pellet of guard cell protoplasts (see Figure 9) in 200 to 500 µl of 0.4 M mannitol and 1 mM CaCl2 in 10 ml test tube and keep them on ice.
      Note: It takes 6-8 h to prepare guard cell protoplasts from 25 g leaves. The protoplasts can be stored in the refrigerator overnight under darkness until measurement. Keep the protoplasts in the dark for at least 1 h on ice for immediate measurement. The time required for one measurement is 2-3 h because it takes 1-2 h until the pH of the protoplast suspension shows a constant value under red light (RL). The measurement time for Arabidopsis is longer than for Vicia faba (Shimazaki et al., 1986).
    24. Determine protein concentration by Bradford method (Bradford, 1976) using 10 µl of the protoplast suspension.


      Figure 9. Guard cell protoplasts in 0.4 M mannitol and 1 mM CaCl2. Guard cell protoplasts isolated from Arabidopsis. Guard cell protoplasts were resuspended in 200 to 500 µl 0.4 M mannitol and 1 mM CaCl2. The typical yield of guard cell protoplasts was 4.3 x 107 cells per 5,000 leaves with a purity of 98%. The bar represents 10 µm.

  2. Measurement of blue light- and fusicoccin-dependent H+-pumping
    1. Blue light-dependent H+-pumping
      1. Keep guard cell protoplasts in the dark at 4 °C for 2 h or overnight.
      2. Circulate water at 24 °C in a Plexiglas water jacket to maintain the temperature. Mix X ml of guard cell protoplasts suspension (50 μg proteins) with 0.5-X ml of 0.4 M mannitol and 1 mM CaCl2 in an open glass vessel (see Figure 10A) enclosed in the water jacket. Add 0.5 ml of 2x H+-pumping buffer (see Recipes) to the suspension to give a final volume of 1 ml reaction mixture in the vessel. Stir the protoplasts in the reaction mixture by a small magnetic stirrer inside the vessel.
        Note: Add the protoplasts in the absence of blue light. Remove blue portion of light by covering the fluorescent lamp with orange-colored film, which prevents blue light penetration.


        Figure 10. Open glass vessel surrounded by water jacket. Water was circulated around the vessel in the water jacket through a nylon (or silicone) tube. A. Empty glass vessel; B. pH glass electrode and guard cell protoplasts in the vessel.

      3. Place a pH glass electrode in the reaction mixture, and record the signal from the electrode on a recorder via a pH meter (see Figure 10B).
      4. Use the LED illuminator (see Figure 11) to apply red light (600 µmol m-2 sec-1) to protoplasts for 2 h by turning on the switch for the red photodiode until the pH shows an almost constant value (see Figure 13). The distance from the illuminator to the sample is about 10 cm. Regulate light intensity at the sample by the voltage controller.


        Figure 11. LED illuminator. Both red and blue LEDs were fixed on a plate. When the LEDs were turned off, all LED bulbs were seen as 242-243 dark-brown spots (A. Light off). When red LED was turned on, red-colored light could be seen (B. Red light). When both red and blue LEDS were turned on, the pink-colored light could be seen (C. Red and blue light).

      5. Use the LED illuminator (see Figure 11) to apply blue light pulse (100 µmol m-2 sec-1) to protoplasts for 30 sec by turning on the switch for the blue photodiode superimposed on red light. Incubate the protoplasts for about 20 min under red light to follow the pH change in the mixture (Figure 13).
      6. Add 3 or 5 µl of 1 mM HCl to the mixture to calculate the magnitude of H+-pumping in response to the blue light.
    2. Fusicoccin (Fc)-dependent H+-pumping
      1. Steps from B1a-B1f follow the same procedure as above. Use the same samples used above.
      2. Add 2.5 µl of 4 mM Fc in DMSO to the suspension (at 10 µM) through a gel loading tip instead of the usual tip and induce H+-pumping. Calculate the rate after the pumping becomes constant (5 min after the addition of Fc).

  3. Determination of total H+-ATPase amount
    1. Suspend 10 µg proteins of guard cell protoplasts in the reaction mixture (150 µl, see Recipes) in a 1.5 ml sampling tube. The concentration of guard cell proteins had been determined as per the measurement Steps A22-A23.
    2. Mix the protoplasts suspension gently by pipetting up and down.
    3. Add the inhibitors of protease or proteasome to the suspension and mix gently by pipetting up and down every 15 min.
      Inhibitors as indicated below were separately administered to the protoplasts:
      1. 10 µM aprotinin (inhibitor for serine protease) and 2 mM DTT.
      2. 1 mM Nα-Tosyl-Lys Chloromethyl Ketone (TLCK; inhibitor for serine protease).
      3. 1% (v/v) protease inhibitor cocktail set III (contains various protease inhibitors).
      4. 50 µM MG132 (inhibitor of proteasome).
    4. Incubate the protoplast suspension containing the inhibitors or without inhibitor as controls for 30 min at 24 °C with pipetting up and down every 15 min.
    5. Add 10% trichloroacetic acid (TCA) to the suspension to cease the reaction and pipette the suspension up and down gently through PIPETMAN (P200) 10 times to denature the proteins.
    6. Place the suspension on ice for 10 min to avoid excessive denaturalization.
    7. Centrifuge the suspension at 17,800 x g for 10 min at 4 °C to collect the proteins.
    8. Remove the supernatant and suspend the pellet with 200 µl of 50 mM Tris-HCl (pH 8.0). Repeat Steps C7-C8 twice to remove TCA.
    9. Add 30 µl 1x SDS sample buffer (see Recipes) to the pellet and solubilize it.
    10. Load 5 µg of the solubilized protein of guard cell protoplasts on SDS-PAGE.
      Separate the proteins by SDS-PAGE using a 10% acrylamide gel (see Recipes).
    11. Transfer the proteins onto a nitrocellulose membrane.
      1. Immerse the nitrocellulose membrane, the acrylamide gel that contains separated proteins, and 6 filter papers of the same size as that of the gel in 200 ml transfer buffer for 15 min.
      2. Stack them by placing 3 layers of filter papers on the bottom, the nitrocellulose membrane, the gel, and 3 layers of filter papers on the top in the Transfer Cell.
      3. Transfer the proteins from the gel onto the nitrocellulose membrane electrically using the Trans blot transfer cell.
    12. Stain the membrane by 0.5 % Ponceau S in 1 % acetic acid for 5 sEC (see Recipes) and use as the loading control of proteins by taking a picture (see Figure 12, lower panel).


      Figure 12. Determination of H+-ATPase by the immunological method. Immunodetection of H+-ATPase of guard cell after treatment with MG132 (Yamauchi et al., 2016). Guard cell protoplasts from wild-type and aha1-10 mutant were incubated with 50 µM MG132 for 30 min at 24 °C. Upper panel shows immunoblot using H+-ATPase antibodies. Lower panel shows Ponceau S staining as loading control. Each lane contains 5 µg guard cell protein.

    13. Wash the membrane by distilled water and incubate it in the blocking buffer (see Recipes) for 30 min.
    14. Incubate the membrane in the presence of anti-H+-ATPase antibodies (dilute 3,000x by blocking buffer) overnight at 4 °C without shaking.
    15. Wash the membrane in T-TBS (see Recipes) for 10 min by incubation.
      Note: Repeat this process three times.
    16. Incubate the membranes in the presence of anti-rabbit IgG HRP (Bio-Rad; dilute 3,000x by blocking buffer) for 2 h at room temperature.
    17. Wash the membrane by T-TBS for 10 min three times.
    18. Incubate the membranes with Clarity Western ECL substrate (Bio-Rad) for 3min.
    19. Detect the H+-ATPase by Chemical Luminescence analyzer (Bio-Rad)
    20. Determine the band density on the membrane by ImageJ, and calculate the amount of the H+-ATPase.

Data analysis

Calculation of the magnitude and the maximum rate of H+-pumping (see Figure 13).

  1. See Figure 13 that indicates H+-pumping (shown by decrease in pH over time) by guard cell protoplasts in response to a pulse of blue light (BL) under red light (RL).
  2. Determine the amounts of acid equivalents by pH decrease caused by the addition of 3 nmol H+ (3 µl of 1 mM HCl) to the suspension at the end of each experiment (see A). 3 nmol H+ corresponds to 3.2 cm on the chart recorder. Thus, 1 nmol H+ corresponds to 1.07 cm pH decrease on the chart.
  3. Calculate the magnitude of H+-pumping in response to the pulse. The pH decrease is 4.20 cm per pulse (see B). The magnitude is calculated to be 4.20/1.07 = 3.93 nmol H+ per pulse. Since the mixture contains 50 µg protein, the magnitude of H+-pumping was calculated to be 0.079 nmol H+ per pulse µg protein-1.
  4. Draw an approximate line at the steepest slope of H+-pumping, which occurs around 2.5 min after the pulse, to calculate the maximum rate of H+-pumping (see red line).
  5. Determine the pH decrease on a chart on the basis of this line. It exhibited 4.0 cm of pH decrease on the chart in 3 min (see C).
  6. Calculate the maximum rate of H+ pumping on the basis guard cell proteins used. Since the pH decrease per pulse is 1.33 cm min-1 (4.0/3) and 1 nmol H+ corresponds to 1.07 cm of pH decrease, the maximum rate is calculated to be 1.33 cm/1.07 cm x 60 nmol h-1/50 µg protein is 1.49 nmol H+ h-1 µg protein-1.


    Figure 13. Time course of H+-pumping by guard cell protoplasts in response to blue light (BL). Guard cell protoplasts in the reaction mixture containing 50 µg proteins were illuminated with a 30 sec pulse of BL (100 μmol m-2 sec-1) superimposed on RL (600 μmol m-2 sec-1). The vertical and horizontal axes indicate pH and time, respectively. The pumping initiated a pH decrease 30 sec after the pulse and reached the maximum rate around 2-3 min after the pulse. The pumping was recorded on a chart recorder at the speed of 20 cm h-1 (= 1 cm 3 min-1). A: Magnitude of pH decrease by the addition of 3 nmol H+. B: Magnitude of H+-pumping in response to a pulse of BL. C: Magnitude of H+-pumping in 3 min in response to BL.

Notes

The H+-pumping activities vary with the viability of guard cell protoplasts. An important criterion to obtain viable guard cell protoplasts is enzyme quality (Cellulase RS and R-10). Activity and toxicity, which often cause reduction in protoplast recovery, vary with enzyme lot numbers. We usually check the enzyme activity on a small scale through preparation of the protoplasts and H+-pumping measurement. If the lot produces viable protoplasts, we purchase it in larger amounts.

Recipes

Note: Enzyme solutions for 1st and 2nd digestion should be prepared fresh.

  1. 1st digestion solution
    0.5% (w/v) cellulase R-10
    0.05% (w/v) macerozyme R-10
    0.1% (w/v) polyvinyl pyrrolidone (PVP-K30)
    0.2% (w/v) BSA
    0.25 M mannitol
    1 mM CaCl2·2H2O
    10 mM MES-KOH (pH 5.4)
  2. 2nd digestion solution
    1.5% (w/v) cellulase RS
    0.5% (w/v) macerozyme R-10
    0.2% (w/v) BSA
    0.4 M mannitol
    1 mM CaCl2·2H2O
    10 mM MES-KOH (pH 5.4)
    Adjust pH to 5.4 with 2 N HCl
  3. 2x H+-pumping buffer
    0.25 mM MES-KOH (pH 6.0)
    20 mM KCl
    0.4 M mannitol
    1 mM CaCl2·2H2O
    Store at 4 °C
  4. Reaction mixture for H+-pumping
    500 µl 2x H+-pumping buffer
    X µl guard cell protoplasts (50-100 µg protein)
    500-X µl 0.4 M mannitol and 1 mM CaCl2
  5. Fc stock solution
    4 mM Fc in DMSO
    Store at -20 °C
  6. Reaction mixture for inhibitor treatment
    75 µl 2x H+-pumping buffer
    X µl guard cell protoplasts (10 µg protein)
    75-X µl 0.4 M mannitol and 1 mM CaCl2
  7. HCl stock solution
    1 mM HCl
    Store at room temperature
  8. 10x TBS
    200 mM Tris-HCl (pH 7.4)
    1400 mM NaCl
    Store at room temperature
  9. 4x separation buffer
    1.5 M Tris-HCl (pH 8.8)
    4% (w/v) SDS
    Store at room temperature
  10. 4x stacking buffer
    1.0 M Tris-HCl (pH 6.8)
    4% (w/v) SDS
    Store at room temperature
  11. Ammonium peroxodisulfate stock solution
    10% (w/v) ammonium peroxodisulfate in distilled water
    Store at 4 °C
  12. 10% acrylamide gel
    1. Separation gel
      6.7 ml Acrylamide/Bis mixed solution (30% [w/v], Wako Pure Chemical Industries)
      5.0 ml 4x separation buffer
      200 µl 1% (v/v) ammonium peroxodisulfate stock solution
      20 µl 0.1% (v/v) N,N,N’,N’-tetramethylethylenediamine
      Add distilled water to the solution until 20 ml
      Prepare as required
    2. Stacking gel
      1.67 ml acrylamide/Bis mixed solution (30% [w/v], Wako Pure Chemical Industries)
      2.5 ml 4x stack buffer
      100 µl ammonium peroxodisulfate stock solution
      10 µl N,N,N’,N’-tetramethylethylenediamine
      Add distilled water to the solution until 10 ml
      Prepare as required
  13. DTT stock solution
    1 M DTT in distilled water
    Store at -20 °C
  14. TLCK stock solution
    100 mM TLCK in DMSO
    Store at -20 °C
  15. Ponceau S
    0.5% (w/v) Ponceau S
    1% (v/v) acetic acid
    Store at room temperature
  16. Tween 20 stock solution
    25% (v/v) Tween 20 in 1x TBS buffer
    Store at 4 °C
  17. Blocking buffer
    5% (w/v) skim milk in T-TBS
    Prepare as required
  18. T-TBS
    0.5% (v/v) Tween 20 in 1x TBS
    Store at room temperature
  19. Transfer buffer
    39 mM glycine
    48 mM Tris
    20% (v/v) methanol
    Store at room temperature
  20. 3x SDS buffer
    30 mM Tris-HCl (pH 8.0)
    3 mM EDTA
    3% (w/v) SDS
    30% (w/v) sucrose
    Store at -20 °C
  21. 1x SDS sample buffer
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA
    10% (w/v) sucrose
    1% (w/v) SDS
    0.04% (v/v) 2-mercaptethanol
    0.003% (w/v) Coomassie Brilliant Blue G-250
    Prepare as required

Acknowledgments

This protocol is adapted from Shimazaki et al. (1986), Ueno et al. (2005), and Yamauchi et al. (2016). This work was supported by JSPS KAKENHI Grant Number 26251032 (to K.S.). There is no conflict of interest.

References

  1. Assmann, S. M., Simoncini, L., and Schroeder, J. I. (1985). Blue light activates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318: 285-287.
  2. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  3. Kinoshita, T. and Shimazaki, K. (1999). Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells. EMBO J 18(20): 5548-5558.
  4. Shimazaki, K., Iino, M. and Zeiger, E. (1986). Blue light-dependent proton extrusion by guard-cell protoplasts of Vicia faba. Nature 319, 324-326.
  5. Ueno, K., Kinoshita, T., Inoue, S., Emi, T. and Shimazaki, K. (2005). Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light. Plant Cell Physiol 46(6): 955-963.
  6. Yamauchi, S., Takemiya, A., Sakamoto, T., Kurata, T., Tsutsumi, T., Kinoshita, T. and Shimazaki, K. (2016). The plasma membrane H+-ATPase AHA1 plays a major role in stomatal opening in response to blue light. Plant Physiol 171(4): 2731-2743.

简介

响应蓝光的植物气孔的开放是由保卫细胞中的质膜H + -ATPase驱动的。 为了评价体内H + -ATP酶的激活,我们可以使用H + +保卫细胞对蓝光的响应,fusicoccin。 为此,需要制备大量的保卫细胞原生质体,并测量原生质体中的H + - 抽吸。 还需要确定H + -ATP酶的蛋白质量。 在这个协议中,我们描述了这些准备和测量所需的程序。

【背景】响应于蓝光的气孔的开放是由穿过保卫细胞质膜上的H +介导的膜超极化驱动的(Assmann等,1985; Shimazaki等人,1986),并且是由质膜H + -ATP酶引起的(Kinoshita和Shimazaki,1999)。 H + -ATP酶在膜上产生电化学梯度,并提供植物细胞中许多次级运输所需的能量。然而,测量体内H + -ATP酶活性并不容易。利用保卫细胞的蓝光敏感特性,我们的方法可以将体内H +泵送作为体内测量H + + 使用拟南芥保卫细胞原生质体的ATP酶活性(Ueno等人,2005)。与通过蛋白质印迹(Yamauchi等人,2016)的Hβ+ -ATPase定量一起,该方法允许比较Hβ+ -ATPase活性不同的条件或突变背景。

关键字:拟南芥, 蓝光, 壳梭孢素, H+泵, 质膜H+-ATP酶

材料和试剂

  1. 播种机(640 x 230 x 185毫米)(Iris Oyama E型,PR650EMK)
  2. 蛭石(粒度3-6毫米)
  3. 土壤(含泥炭土的盆栽土)
  4. 玻璃纸(Asahi KASEI)
  5. 尼龙网(10μm,25μm,58μm和94μm)(Kyoshin Riko)
  6. 微型载玻片(Matsunami Glass,目录编号:S011260)
  7. 玻璃试管(10毫升)(IWAKI,目录号:TEST15-105NP)
  8. 凝胶加样尖(Thermo Fisher Scientific,Thermo Scientific,目录号:010-Q)
  9. 样品管(1.5 ml)(INA•OPTIKA,目录号:ST-0105F)
  10. 微量杯(100μl)(Beckman Coulter,目录号:523270)
    注:本产品已停产。任何可以在分光光度计中固定的微量比色皿(100μl)都可以替代。
  11. 搅拌杯(100毫升)(Waring实验室,目录号:MC2)
  12. 玻璃吸管(10毫升)(Sansyo,目录号:73-0045)
  13. 玻璃离心管(50毫升)(Sansyo,目录号:84-0182)
  14. 橙色电影(柯达,Cinemoid 5A)
  15. 滤纸(3MM CHR CHROMATOGRAPHY PAPER,GE Healthcare,目录号:3030-928)
  16. 拟南芥生态型Col-0
  17. 拟南芥突变体 aha1-10
  18. Bradford蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006JA)
  19. 抑肽酶(Merck,Calbiochem,目录号:616399)
  20. 蛋白酶抑制剂混合物组III(Merck,Calbiochem,目录号:539134)
  21. MG132(Sigma-Aldrich,目录号:M8699-1MG)
  22. 三氯乙酸(TCA)(NACALAI TESQUE,目录号:06275-24)
  23. 抗兔IgG HRP(Bio-Rad Laboratories,目录号:1706515)
  24. 抗-H + -ATP酶抗体(Kinoshita和Shimazaki,1999)或Agrisera抗H + -ATP酶抗体(Agrisera,目录号:AS07 260)
  25. 清晰的Western ECL底物(Bio-Rad Laboratories,目录号:1705061)
  26. 纤维素酶R-10(Yakult,制药工业,日本东京)
  27. Macerozyme R-10(Yakult,制药工业,日本东京)
  28. 聚乙烯吡咯烷酮K30(PVP-K30)(NACALAI TESQUE,目录号:28314-95)
  29. 牛血清白蛋白(BSA)(Thermo Fisher Scientific,GibcoTM,目录号:30036727)
  30. 甘露醇(Wako Pure Chemical Industries,目录号:130-00855)
  31. 氯化钙二水合物(CaCl 2•2H 2 O)(Wako Pure Chemical Industries,目录号:031-00435)
  32. 2-(N-吗啉代)乙磺酸(MES)(NACALAI TESQUE,目录号:02442-44)
  33. 纤维素酶RS(Yakult,制药工业,日本东京)
  34. 盐酸(HCl)(Wako Pure Chemical Industries,目录号:081-03475)
  35. 氯化钾(KCl)(NACALAI TESQUE,目录号:28513-85)
  36. Fusicoccin(Fc)(Sigma-Aldrich,目录号:F0537-1MG)
  37. DMSO(和光纯药工业,目录编号:046-21981)
  38. 三(羟甲基)氨基甲烷(Wako Pure Chemical Industries,目录号:514-98121)
  39. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  40. 十二烷基硫酸钠(SDS)(NACALAI TESQUE,目录号:31606-75)
  41. 过氧二硫酸铵(和光纯药工业,目录编号:012-20503)
  42. 30%(w / v)丙烯酰胺/ Bis混合溶液(37.5:1)(Wako Pure Chemical Industries,目录号:018-25625)
  43. N',N'-四甲基乙二胺(NACALAI TESQUE,目录号:33401-72),N,N',N'-四甲基乙二胺
  44. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14112-94)
  45. Nα - 甲苯磺酰-Lys氯甲基酮(Merck,Calbiochem,目录号:616382)
  46. Ponceau S(NACALAI TESQUE,目录编号:28322-72)
  47. 乙酸(Wako Pure Chemical Industries,目录号:017-00251)
  48. 吐温20(MP Biomedicals,目录号:0210316890)
  49. 脱脂牛奶(雪牌奶制品有限公司)
  50. 甘氨酸(和光纯药工业,目录号:077-00735)
  51. 甲醇(Wako Pure Chemical Industries,目录号:137-01823)
  52. 乙二胺四乙酸(EDTA)(Wako Pure Chemical Industries,目录号:343-01861)
  53. 蔗糖(Wako Pure Chemical Industries,目录号:196-00015)
  54. 考马斯亮蓝G-250(NACALAI TESQUE,目录号:09409-42)
  55. 2-巯基乙醇(NACALAI TESQUE,目录号:21417-52)
  56. 1消化液(见食谱)
  57. 2消解液(见食谱)
  58. 2x H - - 抽取缓冲区(请参阅食谱)
  59. 用于H +泵送的反应混合物(见食谱)
  60. Fc储备液(见食谱)
  61. 反应混合物的抑制剂处理(见食谱)
  62. HCl储备液(见食谱)
  63. 10倍TBS(见食谱)
  64. 4倍分离缓冲液(见食谱)
  65. 4倍堆叠缓冲区(请参阅食谱)
  66. 过氧二硫酸铵储备液(见配方)
  67. 10%丙烯酰胺凝胶(见食谱)
  68. DTT原液(见食谱)
  69. TLCK储备液(见食谱)
  70. Ponceau S(见食谱)
  71. 吐温20储备液(见食谱)
  72. 阻塞缓冲区(见食谱)
  73. T-TBS(见食谱)
  74. 传输缓冲区(见食谱)
  75. 3倍SDS缓冲液(见食谱)
  76. 1x SDS样品缓冲液(见食谱)

设备


  1. 不锈钢制成的金属架(120 x 61 x 230厘米)(Iris Oyama,MR-230P)
  2. 搅拌器(Waring Lab,型号:51BL31,目录号:7011BU)*
  3. 漏斗(TGK,目录号:416-09-24-04)*
  4. 锥形瓶(500ml)(IWAKI,目录号:82-0088)
  5. 锥形瓶(300ml)(IWAKI,目录号:82-0087)
  6. 冷冻离心机(TOMY,型号:AX-310)*
  7. 冷冻离心机(TOMY,型号:MX-100)*
  8. 孵化器与振动筛(TAITEC,型号:个人Lt10)
  9. 单位恒温槽(TAITEC,THERMO MINDER型号:JR 80)*
  10. 分光光度计(Beckman Coulter,型号:DU800)*
  11. pH玻璃电极(Beckman Coulter,目录号:39532)*
  12. pH计(Beckman Coulter,型号:Φ71pH计)*
  13. 图表记录仪(横河电机工厂,型号:3066)*
  14. 磁力搅拌器(RANK BROTHERS,型号:300型)
  15. 冷水浴循环器(Thermo Fisher Scientific,Thermo Scientific TM,型号:RTE7)
  16. LED面板(CCS,型号:ISL 150X150 H4RRHB,按订单生产)
    注意:红色和蓝色的LED固定在同一个面板上,每个LED都可以通过电源打开和关闭。
  17. LED电源(CCS,型号:ISC-201-2)
  18. 显微镜(尼康仪器,型号:Eclipse TS100)
  19. 移液器(P200)
  20. 电源(Bio-Rad Laboratories,型号:PowerPac TM HC大电流电源)
  21. 转移细胞(Bio-Rad Laboratories,型号:Trans-Blot SD Semi-Dry Transfer Cell)
  22. 电泳容器(ATTO,型号:AE6200,目录号:2392385)*
  23. 化学发光分析仪(Bio-Rad Laboratories,型号:ChemiDoc TM Touch成像系统)
  24. 凝胶盒(ATTO,型号:AE-6210)

注:此产品已停产。

软件

  1. 图片J

程序

  1. 从拟南芥叶制备保卫细胞原生质体
    1. 在土壤和蛭石混合物(1:1)中播种200个拟南芥种子/

    2. 在白光(60μmolm -2 s -1)下,用14 / 10h光照/植物生长拟南芥植株4-6周,在24°C的黑暗周期(见图1)。


      图1.在白光下生长的四周龄的拟南芥(Arabidopsis)植物在白光下(60μmolm )生长4周> -2秒-1),在24℃下进行14/10小时光照/黑暗循环。酒吧代表1厘米。

    3. 从200株植物中收获完全展开的莲座叶,取下主叶柄(〜2厘米长,见图2),然后抽紧。


      图2:从4周龄的拟南芥完全展开的叶子从拟南芥中收获完全展开的叶子,并且在抽薹之前除去主叶柄。酒吧代表1厘米。

    4. 将叶子(25克)放入冰水中(见图3)。


      图3.在冰冷的水中的叶子将收获的叶子放入盛有冰水的玻璃烧杯中。

    5. 将冰冷的叶子移入Waring搅拌器杯中(参见图4A)。在70ml冰冷的蒸馏水中,将覆盖有两层玻璃纸的搅拌器中的叶子全速均匀化(参见图4B)45秒。将得到的匀浆倒在放在漏斗上的尼龙网上(见图5)。在整个协议中丢弃滤液(流通)。


      图4.带有搅拌杯的挥舞着搅拌器。 B.充分展开的莲座叶放入装有冰水的搅拌杯中。搅拌杯用玻璃纸密封。
      最大速度均化叶片45秒。

    6. 通过倒入冰冷的蒸馏水(参见视频1)将保留在58μm筛上的表皮组织(参见图5)转移到Waring搅拌器杯中,并使其在70ml水中全速再次均化1分钟,并通过如上所述通过58μm尼龙网过滤收集它们。
      尼龙网可以在用蒸馏水清洗后重新使用

      图5.用58μm尼龙网层收集的表皮组织在均化45秒后,用58μm尼龙网格层收集均质的表皮组织。将收集的组织再次放入搅拌器杯中并匀化1分钟(参见视频1)。用58μm尼龙网再次收集表皮组织,并重新悬浮在1消化液中。

    7. 将12-13g湿的表皮碎片(参见图7)转移到500ml的锥形烧瓶中,通过使用洗瓶或10ml的注射器倒入1ml消化溶液(参见食谱)驹pipe吸管(见图6和视频1)。
      加入相同的溶液,将消解液的总体积调整到100毫升

      图6.带有硅橡胶灯泡的驹pipe吸管(10 ml)


      图7.通过1 st消化液除去叶肉细胞A. A.表皮碎片在1 st消化液处理前。叶肉细胞粘附于表皮组织。 B. 1消化液处理后的表皮碎片。所有的叶肉细胞都从表皮细胞分离。条纹代表10微米。

    8. 将表皮碎片轻轻地通过同样的Komagome移液管上下几次,在100ml的锥形瓶中的1ml消化溶液中上下混合以彻底混合悬浮液并确保均匀消化(参见视频2)。

      视频2

    9. 在24°C的锥形瓶中孵育悬浮液30分钟,以70次/分钟的速度摇动。
    10. 通过Komagome移液管上下移动30次消化液中部分消化的表皮片段以除去附着的叶肉细胞(参见视频2)。
      使用显微镜(150x)检查微载玻片上附着于表皮碎片的叶肉细胞。
    11. 重复步骤A9,直到所有附着于表皮的叶肉细胞都被除去(见图7)。
    12. 将悬浮液通过58μm的尼龙网,并将表皮碎片保留在网片上。
    13. 将表皮碎片倒入0.3M甘露醇和1mM CaCl 2溶液中,将溶液倒入网片上(参见视频1)。保持碎片在解决方案30分钟,以适应他们的高渗透压。
    14. 通过渗透适应表皮穿过58微米尼龙网。用Komagome移液器(见视频1)将表面上的第二消化溶液(参见食谱)倒入300ml锥形瓶中,将保留在网上的表皮转移到300ml锥形瓶中。通过加入相同的2snd消解溶液将总体积调节至50ml。
      注意:由于表皮的部分消化和脆弱,在这一步不要触摸表皮。
    15. 在第二次消化溶液中用50分钟min -1 -1的表皮在27℃孵育表皮50-60分钟。
    16. 使用几个表皮碎片,检查大多数保卫细胞在显微镜下是否变成球形(见图8)。


      图8.通过第二次消化溶液进行的保卫细胞球化。将表皮片段与第二次消化溶液一起温育。在显微镜下观察保卫细胞的球形性质(黑色箭头)。该条代表10微米。

    17. 将含有消化表皮的锥形瓶放在冰上,用Komagome移液管轻轻上下60次,使保护细胞原生质体从表皮脱落(见视频2)。
    18. 将悬浮液通过94和25μm的两层堆叠的尼龙网(94μm网目位于25μm的网的顶部)以将保卫细胞原生质体与表皮残余物分开。
    19. 将分离的保卫细胞原生质体通过两层10μm尼龙网以除去其他小组织的污染。
    20. 在4℃下将保卫细胞原生质体的悬浮液在420gxg离心18分钟以收集保卫细胞原生质体作为沉淀。丢弃上清液。
    21. 在50ml 0.4M甘露醇和1mM CaCl 2溶液中重悬沉淀。
    22. 重复步骤A19和A20两次(总共三次)以除去残留的酶。
    23. 在10ml试管中,将保卫细胞原生质体的沉淀重悬于200-500μl0.4M甘露醇和1mM CaCl 2中,并保持在冰上。
      注意:需要6-8小时从25克叶片制备保卫细胞原生质体。原生质体可以在黑暗中在冰箱中储存过夜,直到测量。保持原生质体在冰上至少1小时的黑暗中立即测量。一次测量所需的时间为2-3小时,因为需要1-2小时直到在红光(RL)下原生质体悬浮液的pH显示恒定的值。拟南芥的测量时间比蚕豆(Vicima faba)长(Shimazaki等,1986)。
    24. 用Bradford法(Bradford,1976)用10μl原生质体悬浮液测定蛋白质浓度。


      图9. 0.4M甘露醇和1mM CaCl 2保卫细胞原生质体从拟南芥中分离的保卫细胞原生质体。将保卫细胞原生质体重悬于200-500μl0.4M甘露醇和1mM CaCl 2中。保卫细胞原生质体的典型产量为每5,000个叶片4.3×10 7个细胞,纯度为98%。该条代表10微米。

  2. 测量蓝光和依赖fococcin的H + +抽血
    1. 蓝光取决于H - - 抽水
      1. 保持细胞原生质体在黑暗中4°C 2小时或过夜。
      2. 在24℃的有机玻璃水套中循环水以保持温度。将Xml的保卫细胞原生质体悬浮液(50μg蛋白质)与0.5-Xml的0.4M甘露醇和1mM CaCl 2在密封在水套中的开放式玻璃容器(见图10A)中混合。向该悬浮液中加入0.5ml 2×H 2 +抽吸缓冲液(见配方),使容器中的最终体积为1ml反应混合物。通过容器内的小型磁力搅拌器搅拌反应混合物中的原生质体。
        注意:在没有蓝光的情况下添加原生质体。
        用橙色薄膜覆盖荧光灯,从而消除蓝光部分。


        图10.被水套环绕的敞开的玻璃容器水通过尼龙(或硅树脂)管在水套周围循环。 A.空的玻璃容器; B.容器中pH玻璃电极和保卫细胞原生质体。

      3. 将pH玻璃电极放入反应混合物中,并通过pH计将记录来自电极的信号记录在记录仪上(见图10B)。
      4. 使用LED照明器(参见图11),通过打开红色开关,将红色光(600μmolm -2 s -1 sup )施加到原生质体2小时光电二极管直到pH显示几乎恒定的值(见图13)。照明器到样品的距离约为10厘米。
        通过电压控制器调节样品的光强度

        图11. LED照明器。红色和蓝色LED固定在平板上。当LED关闭时,所有的LED灯泡被视为242-243个深褐色斑点(A.熄灭)。当红色LED亮起时,可以看到红色的光(B.红灯)。当红色和蓝色LED都打开时,可以看到粉红色的光(C.红色和蓝色光)。

      5. 使用LED照明器(见图11),将蓝光脉冲(100μmolm -2 sup-1 -1)施加到原生质体30秒,通过打开蓝色光电二极管叠加在红光。在红光下孵育原生质体约20分钟以跟踪混合物中的pH变化(图13)。
      6. 向混合物中加入3或5μl的1mM HCl,以计算响应于蓝光的H +泵浦的幅度。
    2. Fusicoccin(Fc) - 依赖性H + +抽取
      1. 从B1a-B1f开始,按照上述相同的步骤。使用上面使用的相同样品。
      2. 添加2.5μL的4mM的Fc在DMSO中的悬浮液(10μM)通过凝胶加载尖端,而不是通常的提示,并诱导H + +泵。计算泵送恒定后(加入Fc后5分钟)的速率。

  3. 测定总H + -ATPase量
    1. 在1.5ml取样管中,将10μg保卫细胞原生质体的蛋白质悬浮在反应混合物中(150μl,见食谱)。根据测量步骤A22-A23确定了保卫细胞蛋白质的浓度。
    2. 通过上下移液轻轻混合原生质体悬浮液。
    3. 将蛋白酶或蛋白酶体的抑制剂加入到悬浮液中,每15分钟上下吸取轻轻混合。
      如下所示的抑制剂分别施用于原生质体:
      1. 10μM抑肽酶(丝氨酸蛋白酶抑制剂)和2 mM DTT。
      2. 1mM Nα-Tosyl-Lys氯甲基酮(TLCK;丝氨酸蛋白酶抑制剂)。
      3. 1%(v / v)蛋白酶抑制剂混合物组III(含有各种蛋白酶抑制剂)。
      4. 50μMMG132(蛋白酶体抑制剂)。
    4. 将含有抑制剂或无抑制剂的原生质体悬浮液作为对照,在24℃下每15分钟上下移液30分钟。
    5. 向悬浮液中加入10%三氯乙酸(TCA)以停止反应,并通过PIPETMAN(P200)轻轻地上下吸取悬浮液10次以使蛋白质变性。
    6. 悬浮在冰上10分钟,以避免过度变性。
    7. 在4℃下将悬浮液在17,800×gg离心10分钟以收集蛋白质。
    8. 取出上清液并用200μl50mM Tris-HCl(pH8.0)悬浮沉淀。重复步骤C7-C8两次,以删除TCA。
      在脉搏后2.5分钟左右的H +泵抽速最陡的斜率处绘制一条近似直线,计算出H +泵的最大速率(见红色线)。
    9. 根据这条线确定图表上的pH值下降。在3分钟内显示出4.0cm的pH下降(参见C)。
    10. 根据所使用的保卫细胞蛋白质计算H +泵的最大速率。由于每脉冲的pH降低为1.33cm / min(4.0 / 3),而1nmol H +对应于1.07cm的pH降低,所以最大速率计算为是1.33cm / 1.07cm×60nmol h -1 /50μg蛋白质是1.49nmol H + -h -1蛋白质+ -1 。


      图13.响应于蓝光(BL)的保卫细胞原生质体的H + +抽吸的时间进程照射包含50μg蛋白质的反应混合物中的保卫细胞原生质体与RL(600μmolm -2 s -1 )。纵轴和横轴分别表示pH和时间。在脉冲之后,泵送开始30秒的pH降低,并在脉冲之后2-3分钟达到最大速率。在记录仪上以20cm h -1(= 1cm 3 min -1)的速度记录泵送。 A:通过加入3nmol H + +降低pH值的大小。 B:响应于BL的脉冲的H +泵的量值。 C:响应于BL,3分钟内H +泵的强度。

    笔记

    根据保卫细胞原生质体的生存能力,H +抽提活性不同。获得有活力的保卫细胞原生质体的重要标准是酶质量(纤维素酶RS和R-10)。经常引起原生质体回收减少的活性和毒性随酶批号而变化。我们通常通过制备原生质体和H +抽吸测量来检查酶活性。如果这批产生可行的原生质体,我们购买大量。

    食谱

    注意:第一次和第二次消化的酶溶液应该新鲜制备。

    1. 1 消解溶液
      0.5%(w / v)纤维素酶R-10
      0.05%(w / v)macerozyme R-10
      0.1%(w / v)聚乙烯吡咯烷酮(PVP-K30)
      0.2%(w / v)BSA
      0.25 M甘露醇
      1mM CaCl 2•2H 2 O 0 10 mM MES-KOH(pH 5.4)
    2. 2 消解溶液
      1.5%(w / v)纤维素酶RS
      0.5%(w / v)macerozyme R-10
      0.2%(w / v)BSA
      0.4 M甘露醇
      1mM CaCl 2•2H 2 O 0 10 mM MES-KOH(pH 5.4)
      用2N HCl将pH调节至5.4
    3. 2x H - - 抽取缓冲区
      0.25mM MES-KOH(pH 6.0)
      20 mM KCl
      0.4 M甘露醇
      1mM CaCl 2•2H 2 O 0 在4°C储存
    4. 反应混合物用于H +泵 - 泵 500μl2x H <+> - 泵缓冲液
      Xμl保卫细胞原生质体(50-100微克蛋白质)
      500-Xμl0.4M甘露醇和1mM CaCl 2
    5. Fc储备液
      4mM Fc在DMSO中
      在-20°C储存
    6. 用于抑制剂处理的反应混合物
      75μl2x H - 抽吸缓冲液
      Xμl保卫细胞原生质体(10微克蛋白质)
      75-μμl0.4M甘露醇和1mM CaCl 2
    7. HCl储备液
      1 mM HCl
      在室温下储存
    8. 10倍TBS
      200mM Tris-HCl(pH 7.4)
      1400 mM NaCl
      在室温下储存
    9. 4倍分离缓冲液
      1.5M Tris-HCl(pH8.8)
      4%(w / v)SDS
      在室温下储存
    10. 4倍堆栈缓冲区
      1.0M Tris-HCl(pH6.8)
      4%(w / v)SDS
      在室温下储存
    11. 过氧二硫酸铵储备液
      过氧二硫酸铵在蒸馏水中10%(w / v)
      在4°C储存
    12. 10%丙烯酰胺凝胶
      1. 分离凝胶
        6.7 ml丙烯酰胺/ Bis混合溶液(30%[w / v],Wako Pure Chemical Industries) 5.0毫升4X分离缓冲液
        200μl1%(v / v)过氧二硫酸铵储备液
        20μl0.1%(v / v)N,N,N',N'-四甲基乙二胺
        加入蒸馏水到溶液直到20毫升
        按要求准备
      2. 堆叠凝胶
        1.67ml丙烯酰胺/ Bis混合溶液(30%[w / v],Wako Pure Chemical Industries) 2.5毫升4×堆栈缓冲液
        100μl过氧二硫酸铵储备液
        10微升N,N,N',N' - 四甲基乙二胺
        加入蒸馏水到溶液中,直到10毫升
        按要求准备
    13. DTT库存解决方案
      1 M DTT在蒸馏水中
      在-20°C储存
    14. TLCK库存解决方案
      在DMSO中的100mM TLCK
      在-20°C储存
    15. Ponceau S
      0.5%(w / v)Ponceau S
      1%(v / v)乙酸
      在室温下储存
    16. 吐温20储备液

      25%(v / v)Tween 20在1x TBS缓冲液中 在4°C储存
    17. 阻塞缓冲区
      T-TBS中5%(w / v)脱脂牛奶
      按要求准备
    18. T-TBS
      1%TBS中的0.5%(v / v)吐温20 在室温下储存
    19. 传输缓冲区
      39 mM甘氨酸
      48 mM Tris
      20%(v / v)甲醇
      在室温下储存
    20. 3倍SDS缓冲液
      30 mM Tris-HCl(pH 8.0)
      3 mM EDTA
      3%(w / v)SDS
      30%(w / v)蔗糖
      在-20°C储存
    21. 1x SDS样品缓冲液
      10 mM Tris-HCl(pH 8.0)
      1 mM EDTA
      10%(w / v)蔗糖
      1%(w / v)SDS
      0.04%(v / v)2-巯基乙醇
      0.003%(w / v)考马斯亮蓝G-250
      按要求准备

    致谢

    该协议是从Shimazaki et al 改编的。 (1986),上野等人。 (2005)和Yamauchi等人。 (2016)。这项工作得到了JSPS KAKENHI资助号码26251032(K.S.)的支持。没有利益冲突。

    参考

    1. Assmann,S.M.,Simoncini,L。和Schroeder,J.I。(1985)。 蓝光激活Vicia faba保卫细胞原生质体中的电生成离子泵。 />自然 318:285-287。
    2. Bradford,M.M。(1976)。 利用蛋白质 - 染料结合的原理,快速而灵敏地定量蛋白质的微克数量。 Anal Biochem 72:248-254。
    3. Kinoshita,T。和Shimazaki,K。(1999)。 蓝光通过磷酸化激活质膜H + -ATPase在气孔保卫细胞中的C端。 EMBO J 18(20):5548-5558。
    4. Shimazaki,K.,Iino,M.和Zeiger,E。(1986)。 保卫细胞原生质体蓝光依赖质子挤压蚕豆(Vicia faba)。 Nature 319,324-326。
    5. 上野,K.,Kinoshita,T.,井上,S.,Emi,T.和Shimazaki,K.(2005)。 质膜H +的生化表征 - ATPase在拟南芥保卫细胞原生质体中对蓝光的响应。植物细胞生理学(Plant Cell Physiol)46(6):955-963。
    6. Yamauchi,S.,Takemiya,A.,Sakamoto,T.,Kurata,T.,Tsutsumi,T.,Kinoshita,T。和Shimazaki,K。(2016)。 质膜H + -ATPase AHA1在气孔中起主要作用开放以响应蓝光。植物生理学 171(4):2731-2743。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yamauchi, S. and Shimazaki, K. (2017). Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification. Bio-protocol 7(24): e2653. DOI: 10.21769/BioProtoc.2653.
  2. Yamauchi, S., Takemiya, A., Sakamoto, T., Kurata, T., Tsutsumi, T., Kinoshita, T. and Shimazaki, K. (2016). The plasma membrane H+-ATPase AHA1 plays a major role in stomatal opening in response to blue light. Plant Physiol 171(4): 2731-2743.
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