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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

SY Shota Yamauchi
KS Ken-ichiro Shimazaki

发布: 2017年12月20日第7卷第24期 DOI: 10.21769/BioProtoc.2653 浏览次数: 8787

评审: Marisa RosaAna Espinosa-RuizRenate WeizbauerAnonymous reviewer(s)

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Cover of Plant Physiology, featuring study using the protocol.

Aug 2016

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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) search Blue light (蓝光) search Fusicoccin (壳梭孢素) search H+-pumping (H+泵) search Plasma membrane H+-ATPase (质膜H+-ATP酶) search

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物细胞生物学 > 细胞染色

植物科学 > 植物生物化学 > 蛋白质

细胞生物学 > 细胞成像 > 活细胞成像

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