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Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
番茄果实有色体的分离和ATP含量的测定

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Abstract

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.

Keywords: Chromoplast(成色素细胞), Tomato(番茄), Fruit(水果), ATP levels(ATP水平)

Materials and Reagents

  1. Red tomato fruits (MicroTom or Ailsa Craig, harvested 5-7 and 7-9 days after breaker stage, respectively)
  2. Ultrapure water
  3. NaCl (Sigma-Aldrich, catalog number: S7653 )
  4. Trizma® base (Tris) (Sigma-Aldrich, catalog number: T6066 )
  5. HCl (Merck KGaA, catalog number: 100317 )
  6. Sorbitol (Sigma-Aldrich, catalog number: S1878 )
  7. MgCl2 (Sigma-Aldrich, catalog number: M2670 )
  8. KCl (Sigma-Aldrich, catalog number: P9541 )
  9. L-Ascorbic acid (Sigma-Aldrich, catalog number: 255564 )
  10. L-Cysteine (Sigma-Aldrich, catalog number: C7352 )
  11. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43819 )
  12. Sucrose (Sigma-Aldrich, catalog number: S8501 )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  14. MnCl2 (Sigma-Aldrich, catalog number: M3634 )
  15. KH2PO4 (Merck KGaA, catalog number: 104873 )
  16. β-Nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma-Aldrich, catalog number: N7505 )
  17. β-Nicotinamide adenine dinucleotide phosphate hydrate (NADP+) (Sigma-Aldrich, catalog number: N5755 )
  18. β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH) (Sigma-Aldrich, catalog number: N8129 )
  19. β-Nicotinamide adenine dinucleotide hydrate (NAD+) (Sigma-Aldrich, catalog number: N1636 )
  20. Flavin adenine dinucleotide disodium salt hydrate (FAD) (Sigma-Aldrich, catalog number: F6625 )
  21. Sodium pyruvate (Sigma-Aldrich, catalog number: P2256 )
  22. ATP Bioluminescence Assay Kit HS II (Roche Diagnostics, catalog number: 11699709001 )
  23. ENLITEN® rLuciferase/Luciferin Reagent (Promega Corporation, catalog number: FF2021 )
  24. RC DC Protein Assay kit (Bio-Rad Laboratories, catalog number: 500-0122 )
  25. Adenosine 5’-diphosphate sodium salt (ADP) (Sigma-Aldrich, catalog number: A2754 )
  26. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A2383 )
  27. P1, P5-di(adenosine-5’) pentaphosphate pentasodium salt (DAPP) (Sigma-Aldrich, catalog number: D4022 )
  28. 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) (Sigma-Aldrich, catalog number: T1375 )
  29. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H4034 )
  30. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  31. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  32. Miracloth (pore size of 22-25 µm) (Calbiochem®, catalog number: 475855 )
  33. 1 mM DAPP (stored at -20 °C)
  34. 10 mM FAD (stored at -80 °C)
  35. 100 mM NADH (stored at -80 °C)
  36. 100 mM NAD+ (stored at -80 °C)
  37. 100 mM NADPH (stored at -80 °C)
  38. 100 mM NADP+ (stored at -80 °C)
  39. 10 µM ATP standard solutions (stored at -80 °C)
  40. 0.1 µM ATP standard solutions (stored at -80 °C)
  41. 0.001 µM ATP standard solutions (stored at -80 °C)
  42. 1 M DTT (stored at -20 °C)
  43. 0.5 mM ADP
  44. Washing solution (see Recipes)
  45. 0.5 M Tris-HCl buffer (see Recipes)
  46. 15% sucrose solution (see Recipes)
  47. 30% sucrose solution (see Recipes)
  48. 40% sucrose solution (see Recipes)
  49. 50% sucrose (see Recipes)
  50. Buffer A (see Recipes)
  51. Buffer B (see Recipes)
  52. Buffer C (see Recipes)
  53. Buffer D (see Recipes)
  54. Buffer E (see Recipes)

Equipment

  1. Glomax® 96 Microplate Luminometer w/Dual Injectors (Promega Corporation, catalog number: E6521 )
  2. Centrifuge Avanti J-E (Beckman Coulter)
  3. Ultracentrifuge Optima L-100xPI (Beckman Coulter)
  4. SW 28 swinging-bucket rotor (Beckman Coulter) or equivalent
  5. JA-14 rotor (Beckman Coulter) or equivalent
  6. JA-20 rotor (Beckman Coulter) or equivalent
  7. Polypropylene centrifuge tubes (250 ml) (Beckman Coulter)
  8. Polypropylene centrifuge tubes (30 ml) (Beckman Coulter)
  9. Polyallomer centrifuge tubes for SW 28 rotor (Beckman Coulter)
  10. Waring blender 8011S (Dynamics Corporation of America)
  11. Support stand, clamp holder and extension clamp
  12. Glass beakers of different volume capacity
  13. 96-well microplates white (Porvair Sciences, catalog number: 204003 )
  14. Bench orbital shaker
  15. Micropipettes
  16. Heat block
  17. Benchtop microcentrifuge
  18. Gauze (1 m x 1 m)

Software

  1. Microsoft Excel

Procedure

  1. Chromoplast isolation
    1. Prepare in advance the buffers for chromoplast isolation (buffers A and B) and the sucrose solutions for ultracentrifugation in discontinuous gradients.
    2. Wash about 300 g tomato fruits with washing solution for 15 min.
    3. Cut fruits and remove seeds and the gelatinous material of the locular cavities. Store the pericarp tissue in a glass beaker placed on ice.
    4. Cut the pericarp tissue in small pieces (approximately 1 cm2) with a razor blade.
    5. Weigh the pericarp tissue and place in the cold Waring blender jar. 
    6. Add two volumes of ice-cold buffer A. 
    7. Homogenize with three pulses of 1 sec each at low speed. 
    8. Filter the homogenate through 8 layers of gauze and then through 2 layers of Miracloth. Transfer the flow through to two polypropylene centrifuge tubes of 250 ml. Keep samples on ice.
    9. Centrifuge at 200 x g for 2 min at 4 °C.
    10. Carefully transfer the supernatant to new polypropylene centrifuge tubes of 250 ml. 
    11. Centrifuge at 5,000 x g for 10 min at 4 °C.
    12. Discard the supernatant and gently resuspend the pellets in 25 ml of ice-cold buffer B.
    13. Centrifuge at 5,000 x g for 10 min at 4 °C.
    14. Gently resuspend the chromoplast pellet in 4 ml of ice-cold buffer B using a 1 ml micropipette (cut the tip to increase its diameter for not harming the chromoplasts).
    15. In advance, prepare the sucrose differential density blocks in two 35 ml SW28 ultracentrifuge tubes by carefully loading the corresponding sucrose solutions in the following order (from bottom to top): 8 ml of 50% sucrose solution, 8 ml of 40% sucrose solution, 8 ml of 30% sucrose solution and 8 ml of 15% sucrose solution. Keep at 4 °C. 
    16. Carefully load 2 ml of the resuspended chromoplast sample on top of the 15 % sucrose solution.
    17. Centrifuge at 50,000 x g for 60 min at 4 °C in a SW28 rotor (mild brake).
    18. Carefully remove the tubes from the buckets and hold them using an extension clamp.
    19. Carefully harvest the chromoplasts present in the 30-40% interface using a Pasteur pipette and transfer into a prechilled 30 ml centrifuge tube. 
    20. Add one volume of ice-cold buffer B and centrifuge at 5,000 x g for 10 min at 4 °C.
    21. Resuspend chromoplasts in the appropriate buffer according to the protocols used for ATP measurements described in steps B and C.

  2. Measurement of ATP levels (end point measurement)
    1. Resuspend chromoplasts (step A21) in 750 µl of ice-cold buffer C by gentle pipetting. Samples should be kept continuously on ice.
    2. Aliquot the resuspended chromoplasts into microcentrifuge tubes (30 µl per tube) and incubate samples at room temperature (23 °C) on a bench orbital shaker for as long as needed according to the experiment (usually 10 to 60 min). This step allows chromoplasts to perform the biochemical reactions needed for the de novo synthesis of ATP. At the end of the incubation, samples can be processed immediately or stored at -80 °C.
    3. Add 90 µl of the “Cell Lysis Reagent” (contained in the kit “ATP Bioluminescence Assay Kit HS II”) to each tube, mix and incubate the samples for 2 min at 95 °C in a heat block, then transfer the samples on ice.
    4. Centrifuge the samples for 3 min in a microcentrifuge at high speed and room temperature to pellet chromoplasts debris. Transfer the supernatants to fresh microcentrifuge tubes. Keep samples on ice. 
    5. Transfer 50 µl from each sample to a 96-well microplate. Keep samples on ice.
    6. Perform the ATP determination reactions using the Glomax 96 Microplate Luminometer. For these experiments only one injector is used, primed with the “Luciferase reagent” included in the kit “ATP Bioluminescence Assay Kit HS II” and prepared in advance following the instructions provided by the supplier. The instrument is set to add automatically 50 µl of the “Luciferase reagent” to each sample and to start the measurements after 1 sec delay. The final outcome values of the instrument show relative luminescence (light) of the samples, which expresses ATP content according to the reaction: D-luciferin + ATP + O2 → oxyluciferin + PPi + AMP + CO2 + light. The values of the blank samples (samples without chromoplasts, but only the buffer C) have to be subtracted from the sample values.
      Note: The above described method using the “ATP Bioluminescence Assay Kit HS II” kit allows the determination of relative ATP values and the comparison of the ATP content of different samples according to the luminescence observed values. Furthermore, absolute ATP values can be measured using the same kit and method. For this purpose, the ATP standard (contained in the kit) has to be prepared, according to the kit manual, in serial dilutions of known ATP concentration. Next, the luminescence of these serial dilutions has to be determined using the Glomax 96 Microplate Luminometer to generate an ATP standard curve. Comparison of the luminescence values of the samples with those of the ATP standard curve can help for the estimation of absolute ATP values of the samples of interest. The estimated ATP amount can be referred to chromoplast protein content (see step D).

  3. Measurement of ATP synthesis rates (kinetics measurements)
    1. Reconstitute the “ENLITEN® rLuciferase/Luciferin Reagent” following the instructions provided by the supplier. The reagent has to be stored at -20 °C in 2 ml aliquots for no more than two weeks.
    2. Resuspend chromoplasts (step A21) in 500 µl of buffer D by gentle pipetting. Keep chromoplast samples on ice and start the ATP measurements within the first 15 min after chromoplast isolation.
    3. Prime injector 1 of the luminometer with 0.5 mM ADP. Then select the “kinetic protocol” with injector 1 and adjust the settings as indicated: i) injector volume: 40 µl, ii) delay after injection: 0.4 sec, and iii) integration time: 60 sec.
    4. Allow the following reagents to equilibrate at room temperature for approximately 5 min. Then, add the reagents in this order into the wells of a 96-well microplate:
      rLuciferase/Luciferin Reagent
      80 µl
      Buffer E
      80 µl
      DAPP
      4 µl
      NADH 100 mM and NAD+ 100 mM
      (or NADPH 100 mM and NADP+ 100 mM)
      2 µl of each
      Chromoplasts resuspended in buffer D
      20 µl
      Note: DAPP is an inhibitor of adenylate kinase that does not affect the ATP synthase.
    5. Place the microplate in the luminometer and start the “kinetic protocol”. The injector should inject automatically 40 µl of 0.5 mM ADP and light emission data should be collected during 60 sec for each well.
    6. To prepare the ATP calibration curve, select the “non-injection kinetic protocol” in the luminometer and adjust the following settings: i) delay in the first measurement: 0 sec, and ii) integration time: 30 sec.
    7. Add the following components in the appropriate wells of the microplate (at room temperature):
      rLuciferase/luciferin Reagent 
      80 µl
      Buffer
      80 µl
      Ultrapure water
      20 µl
      DAPP
      4 µl
      Corresponding ATP standard (10 µM, 0.1 µM or 0.001 µM ATP)
      20 µl
      Chromoplasts resuspended in buffer D
      20 µl
      Note: Chromoplasts are added because they quench part of the light emitted in the luciferase reaction. 
    8. Place the plate in the luminometer and start the “non-injection kinetic protocol”.
    9. Data analysis: Relative luminescence data will appear in an Excel spreadsheet. Calculate the ATP standard curve equation using the Excel functions and apply this equation to obtain the ATP concentration of all samples at each measurement time (sec). Then, the ATP synthesis rate can be represented in a graph (Y-axis: nmol ATP; X-axis: sec) and can be expressed as nmol ATP x mg protein-1 x sec-1 performing the appropriate calculations. 
      Note: Data from the first 5 sec of kinetics measurements are discarded because they are usually erratic.

  4. Determination of protein content of chromoplast samples
    1. Aliquots of 50 µl of chromoplast samples resuspended in buffer C or D are supplemented with Tween-20 at 1% final concentration and incubated at 4 °C in an orbital shaker for 30 min.
    2. Process the samples using the RC DC Protein Assay kit following the indications provided by the supplier.


      Figure 1. Transversal section of a tomato fruit (var. MicroTom). (A). Preparation of fruit samples prior to homogenization (B, C and D).


      Figure 2. Schematic overview of the procedures included in this protocol showing the typical results obtained in each case

Recipes

  1. Washing solution
    Mix 12.5 g of NaCl in 500 ml of distilled water
  2. 0.5 M Tris-HCl (pH 7.4) buffer
    Stored at 4 °C
  3. 15% sucrose solution (50 ml) (prepare before use)
    Mix 7.5 g of sucrose with 5 ml 0.5 M Tris (pH 7.4) buffer and 50 µl of 1 M DTT
    Complete to 50 ml with ultrapure water
  4. 30% sucrose solution (50 ml) (prepare before use)
    Mix 15 g of sucrose with 5 ml 0.5 M Tris (pH 7.4) buffer and 50 µl of 1 M DTT
    Complete to 50 ml with ultrapure water
  5. 40% sucrose solution (50 ml) (prepare before use)
    Mix 20 g of sucrose with with 5 ml 0.5 M Tris (pH 7.4) buffer and 50 µl of 1 M DTT
    Complete to 50 ml with ultrapure water
  6. 50% sucrose (50 ml) (prepare before use)
    Mix 25 g of sucrose, 5 ml 0.5 M Tris (pH 7.4) buffer and 50 µl of 1 M DTT
    Complete to 50 ml with ultrapure water
  7. Buffer A (prepare fresh before use)
    100 mM Tris-HCl (pH 8.2)
    330 mM sorbitol
    2 mM MgCl2
    10 mM KCl
    8 mM EDTA
    10 mM L-ascorbic acid
    5 mM L-cysteine
    0.2% BSA (can be stored at 4 °C)
    Just before use add DTT (to 1 mM final concentration)
    Finally, add PVPP to 1% and mix.
  8. Buffer B (prepare fresh before use)
    Buffer A without PVPP
  9. Buffer C (prepare fresh prior to use)
    100 mM HEPES-HCl (pH 7.4)
    10 mM MgCl2
    2 mM MnCl2
    10 mM KH2PO4
    1 mM NADPH
    1 mM NADP+
    20 μM FAD
    2 mM pyruvate
    330 mM sorbitol
  10. Buffer D
    100 mM HEPES-HCl (pH 7.4)
    10 mM MgCl2
    2 mM MnCl2
    10 mM KH2PO4
    330 mM sorbitol
    Before use add FAD to 10 µM final concentration
    Stored at -20 °C
  11. Buffer E
    10 mM TES-HCl (pH 7.4)
    600 mM sorbitol
    2 mM MgCl2
    25 mM KH2PO4
    0.33 mM EDTA
    Stored at -20 °C
    Note: All buffers and solutions are prepared with ultrapure water.

Acknowledgments

This protocol has been adapted from methods previously described in Angaman et al. (2012) and Pateraki et al. (2013). This work has been supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2009-09523) and Ministerio de Economía y Competitividad (AGL2013-43522-R), both including FEDER Funds, the Spanish Consolider-Ingenio 2010 Program (CSD2007-00036 Centre for Research in Agrigenomics) and the Generalitat de Catalunya (2009SGR0026). Marta Renato is a recipient of a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte.

References

  1. Angaman, D. M., Petrizzo, R., Hernandez-Gras, F., Romero-Segura, C., Pateraki, I., Busquets, M. and Boronat, A. (2012). Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts. Plant Methods 8(1): 1.
  2. Pateraki, I., Renato, M., Azcón‐Bieto, J. and Boronat, A. (2013). An ATP synthase harboring an atypical γ–subunit is involved in ATP synthesis in tomato fruit chromoplasts. Plant J 74(1): 74-85.

简介

最近报道了番茄果实色素可以使用含有非典型γ亚基的ATP合酶复合物来合成ATP,所述亚基也存在于多种植物物种中。 然而,与这个过程基础的生化过程相关的许多方面仍然很大程度上未知。 在这里我们描述了隔离番茄果实色质体和ATP水平(终点测量)和ATP合成率(动力学测量)在这些细胞器中使用基于生物发光荧光素/荧光素酶的测定的确定详细协议。

关键字:成色素细胞, 番茄, 水果, ATP水平

材料和试剂

  1. 红番茄果实(MicroTom或Ailsa Craig,分别在破碎期后5-7和7-9天收获)
  2. 超纯水
  3. NaCl(Sigma-Aldrich,目录号:S7653)
  4. Trizma底物(Tris)(Sigma-Aldrich,目录号:T6066)
  5. HCl(Merck KGaA,目录号:100317)
  6. 山梨醇(Sigma-Aldrich,目录号:S1878)
  7. MgCl 2(Sigma-Aldrich,目录号:M2670)
  8. KCl(Sigma-Aldrich,目录号:P9541)
  9. L-抗坏血酸(Sigma-Aldrich,目录号:255564)
  10. L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
  11. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43819)
  12. 蔗糖(Sigma-Aldrich,目录号:S8501)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  14. MnCl 2(Sigma-Aldrich,目录号:M3634)

  15. (Merck KGaA,目录号:104873)
  16. β-烟酰胺腺嘌呤二核苷酸2'-磷酸盐还原四钠盐水合物(NADPH)(Sigma-Aldrich,目录号:N7505)
  17. β-烟酰胺腺嘌呤二核苷酸磷酸水合物(NADP )(Sigma-Aldrich,目录号:N5755)
  18. β-烟酰胺腺嘌呤二核苷酸还原二钠盐水合物(NADH)(Sigma-Aldrich,目录号:N8129)
  19. β-烟酰胺腺嘌呤二核苷酸水合物(NAD +)(Sigma-Aldrich,目录号:N1636)
  20. 黄素腺嘌呤二核苷酸二钠盐水合物(FAD)(Sigma-Aldrich,目录号:F6625)
  21. 丙酮酸钠(Sigma-Aldrich,目录号:P2256)
  22. ATP生物发光测定试剂盒HS II(Roche Diagnostics,目录号:11699709001)
  23. ENLITEN R荧光素酶/荧光素试剂(Promega Corporation,目录号:FF2021)
  24. RC DC蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0122)
  25. 腺苷5'-二磷酸钠盐(ADP)(Sigma-Aldrich,目录号:A2754)
  26. 腺苷5'-三磷酸二钠盐水合物(ATP)(Sigma-Aldrich,目录号:A2383)
  27. P1,P5-二(腺苷-5')五磷酸五钠盐(DAPP)(Sigma-Aldrich,目录号:D4022)
  28. 2 - [(2-羟基-1,1-双(羟甲基)乙基)氨基]乙磺酸,N- [三(羟甲基)甲基] -2-氨基乙磺酸(TES)(Sigma-Aldrich,目录号:T1375)
  29. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N-(2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H4034)
  30. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  31. 吐温20(Sigma-Aldrich,目录号:P1379)
  32. Miracloth(孔径为22-25μm)(Calbiochem ,目录号:475855)
  33. 1mM DAPP(储存于-20℃)
  34. 10mM FAD(储存于-80℃)
  35. 100mM NADH(储存于-80℃)
  36. 100mM NAD +(储存于-80℃)
  37. 100mM NADPH(储存于-80℃)
  38. 100mM NADP +(储存于-80℃)
  39. 10μMATP标准溶液(储存于-80℃)
  40. 0.1μMATP标准溶液(储存于-80℃)
  41. 0.001μMATP标准溶液(储存于-80℃)
  42. 1 M DTT(储存于-20°C)
  43. 0.5 mM ADP
  44. 洗涤液(见配方)
  45. 0.5 M Tris-HCl缓冲液(见配方)
  46. 15%蔗糖溶液(见配方)
  47. 30%蔗糖溶液(见配方)
  48. 40%蔗糖溶液(见配方)
  49. 50%蔗糖(见配方)
  50. 缓冲区A(参见配方)
  51. 缓冲液B(参见配方)
  52. 缓冲区C(参见配方)
  53. 缓冲区D(参见配方)
  54. 缓冲区E(参见配方)

设备

  1. Glomax 96双微注射器微孔板光度计(Promega公司,目录号:E6521)
  2. 离心机Avanti J-E(Beckman Coulter)
  3. 超速离心机Optima L-100xPI(Beckman Coulter)
  4. SW 28摆动叶片转子(Beckman Coulter)或等同物
  5. JA-14转子(Beckman Coulter)或等同物
  6. JA-20转子(Beckman Coulter)或等同物
  7. 聚丙烯离心管(250ml)(Beckman Coulter)
  8. 聚丙烯离心管(30ml)(Beckman Coulter)
  9. SW 28转子(Beckman Coulter)的聚合物离心管
  10. Waring搅拌机8011S(美国动力公司)
  11. 支撑架,夹具支架和延伸夹具
  12. 不同容量的玻璃烧杯
  13. 96孔微孔板(Porvair Sciences,目录号:204003)
  14. 台式轨道摇床
  15. 微量移液器
  16. 热块
  17. 台式微量离心机
  18. 纱布(1米x 1米)

软件

  1. Microsoft Excel

程序

  1. Chromoplast隔离
    1. 提前准备用于色素分离的缓冲液(缓冲液A和B)和用于在不连续梯度中超速离心的蔗糖溶液。
    2. 用洗液洗涤约300g番茄果实15分钟
    3. 切割水果,删除种子和局部腔的凝胶状材料。 将果皮组织储存在置于冰上的玻璃烧杯中。
    4. 用刀片切割小片(约1cm <2> )的果皮组织。
    5. 称取果皮组织并置于冷的Waring搅拌器罐中。
    6. 添加两卷冰冷缓冲液A. 
    7. 以每秒1秒的三个脉冲均匀化。
    8. 过滤匀浆通过8层纱布,然后通过2层Miracloth。 转移流量通过两个聚丙烯离心管250毫升。 将样品保存在冰上。
    9. 在4℃下以200×g离心2分钟。
    10. 小心地将上清液转移到250 ml的新聚丙烯离心管中。
    11. 在4℃下以5,000xg离心10分钟。
    12. 弃去上清液,轻轻地将沉淀重悬在25ml冰冷的缓冲液B中
    13. 在4℃下以5,000xg离心10分钟。
    14. 使用1ml微量移液管轻轻地将色原体沉淀物重悬在4ml冰冷的缓冲液B中(切割尖端以增加其直径,不伤害色质体)。
    15. 事先,通过小心地按照以下顺序(从底部到顶部)加载相应的蔗糖溶液,在两个35ml SW28超速离心管中制备蔗糖差异密度块:8 ml 50%蔗糖溶液,8ml 40%蔗糖溶液,8ml 30%蔗糖溶液和8ml 15%蔗糖溶液。 保持在4°C。
    16. 小心加载2ml重悬的色原体样品在15%蔗糖溶液的顶部
    17. 在SW28转子(轻度制动)中在4℃下以50,000×g离心60分钟。
    18. 小心地从水桶中取出管道,并使用延长夹具固定
    19. 使用巴斯德移液管小心地收获存在于30-40%界面中的色原体,并转移到预先冷却的30ml离心管中。 
    20. 加入一体积的冰冷缓冲液B,并在4℃下以5,000xg离心10分钟。
    21. 根据步骤B和C中所述的用于ATP测量的方案,将色原体重悬于合适的缓冲液中。

  2. 测量ATP水平(终点测量)
    1. 通过温和吸取重悬染色质(步骤A21)在750μl冰冷的缓冲液C. 样品应连续保存在冰上。
    2. 将重悬的色原体分装到微量离心管中(每管30μl),并根据实验(通常10至60分钟)在台式轨道摇床上在室温(23℃)孵育样品。 该步骤允许染色质进行ATP的新合成所需的生物化学反应。 在孵育结束时,样品可立即处理或储存在-80℃
    3. 向每个管中加入90μl"细胞裂解试剂"(包含在试剂盒"ATP生物发光测定试剂盒HS II"中),混合并在加热块中在95℃温育样品2分钟,然后将样品转移 冰。
    4. 在微量离心机中高速和室温离心样品3分钟,以沉淀色质体碎片。转移上清液到新鲜的微量离心管。将样品保存在冰上。
    5. 转移50微升从每个样品到96孔微板。将样品保存在冰上。
    6. 使用Glomax 96微孔板光度计进行ATP测定反应。对于这些实验,仅使用一个注射器,用包含在试剂盒"ATP生物发光测定试剂盒HS II"中的"荧光素酶试剂"引发,并且根据供应商提供的说明书预先制备。仪器设置为向每个样品自动添加50μl的"荧光素酶试剂",并在延迟1秒后开始测量。仪器的最终结果值显示样品的相对发光(光),其根据反应表达ATP含量:D-荧光素+ ATP + O 2→氧化荧光素+ PP + AMP + CO <2> +光。必须从样品值中减去空白样品(没有色原体的样品,但只有缓冲液C)的值。
      注意:使用"ATP生物发光测定试剂盒HS II"试剂盒的上述方法允许根据发光观察值测定不同样品的相对ATP值和ATP含量的比较。此外,可以使用相同的试剂盒和方法测量绝对ATP值。为此,必须根据试剂盒手册以已知ATP浓度的系列稀释液制备ATP标准品(包含在试剂盒中)。接下来,这些连续稀释的发光必须使用Glomax 96微孔板光度计测定以产生ATP标准曲线。样品的发光值与ATP标准曲线的发光值的比较可以帮助估计感兴趣样品的绝对ATP值。估计的ATP量可以参考染色质体蛋白质含量(参见步骤D)
  3. 测量ATP合成速率(动力学测量)
    1. 按照供应商提供的说明重组"ENLITEN ® rLuciferase/Luciferin Reagent"。试剂必须在-20℃下以2ml等分试样保存不超过两周
    2. 通过温和吸取重悬染色质(步骤A21)在500μl缓冲液D中。保持chromoplast样品在冰上,并在chromoplast隔离后的前15分钟开始ATP测量
    3. 具有0.5mM ADP的发光计的注射器1。然后使用进样器1选择"动力学方案",并如下所示调整设置:i)进样器体积:40μl,ii)注射后延迟:0.4秒,以及iii)积分时间:60秒。
    4. 让以下试剂在室温下平衡约5分钟。然后,按此顺序将试剂加入96孔微孔板的孔中:
      r萤光素酶/荧光素试剂
      80μl
      缓冲区E
      80μl
      DAPP
      4微升
      NADH 100mM和NAD + 100mM</sup> 100mM (或NADPH 100mM和NADP + 100mM) 每个2微升
      色素重悬于缓冲液D中
      20μl
      注意:DAPP是不影响ATP合酶的腺苷酸激酶抑制剂。
    5. 将微孔板置于光度计中,开始"动力学方案"。 注射器应自动注射40μl的0.5 mM ADP,并且每个孔应在60秒内收集光发射数据
    6. 要准备ATP校准曲线,请在光度计中选择"非注射动力学协议",并调整以下设置:i)第一次测量延迟:0秒,ii)积分时间:30秒。
    7. 将以下组分加入微孔板的适当孔中(室温):
      r荧光素酶/荧光素试剂
      80μl
      缓冲区
      80μl
      超纯水
      20微升
      DAPP
      4微升
      相应的ATP标准品(10μM,0.1μM或0.001μMATP)
      20微升
      色素重悬于缓冲液D中
      20微升
      注意:添加色原体是因为它们抑制了荧光素酶反应中发出的部分光。 
    8. 将板放在光度计中,开始"非注射动力学方案"
    9. 数据分析:相对发光数据将显示在Excel电子表格中。使用Excel函数计算ATP标准曲线方程,并应用此方程式以获得每个测量时间(秒)时所有样品的ATP浓度。然后,ATP合成速率可以以曲线图(Y轴:nmol ATP; X轴:秒)表示,并且可以表示为nmol ATP×mg蛋白 -1 x sec < -1 执行适当的计算。
      注意:动态学测量前5秒的数据被丢弃,因为它们通常不稳定。

  4. 测定色原体样品的蛋白质含量
    1. 将悬浮于缓冲液C或D中的50μl染色质试样的等分试样补充以1%终浓度的吐温-20,并在轨道振荡器中在4℃温育30分钟。
    2. 使用RC DC蛋白测定试剂盒根据供应商提供的指示处理样品

      图1.番茄果实(var.MicroTom)的横截面。(A)。在均化(B,C和D)之前制备水果样品

      图2.本协议中包含的程序示意图,显示了每种情况下获得的典型结果

食谱

  1. 洗涤溶液
    将12.5g NaCl混合在500ml蒸馏水中
  2. 0.5M Tris-HCl(pH7.4)缓冲液
    储存在4°C
  3. 15%蔗糖溶液(50ml)(使用前制备)
    将7.5g蔗糖与5ml 0.5M Tris(pH7.4)缓冲液和50μl1M DTT混合 用超纯水完成至50ml
  4. 30%蔗糖溶液(50ml)(使用前制备)
    将15g蔗糖与5ml 0.5M Tris(pH7.4)缓冲液和50μl1M DTT混合 用超纯水完成至50ml
  5. 40%蔗糖溶液(50ml)(使用前制备)
    将20g蔗糖与5ml 0.5M Tris(pH7.4)缓冲液和50μl1M DTT混合 用超纯水完成至50ml
  6. 50%蔗糖(50ml)(使用前制备)
    混合25g蔗糖,5ml 0.5M Tris(pH7.4)缓冲液和50μl1M DTT 用超纯水完成至50ml
  7. 缓冲液A(使用前准备新鲜)
    100mM Tris-HCl(pH8.2)
    330mM山梨醇 2mM MgCl 2/
    10 mM KCl
    8 mM EDTA
    10mM L-抗坏血酸 5mM L-半胱氨酸 0.2%BSA(可在4℃贮存) 在使用前加入DTT(最终浓度为1mM)
    最后,将PVPP加到1%并混合
  8. 缓冲液B(使用前准备新鲜)
    无PVPP的缓冲区
  9. 缓冲液C(使用前准备新鲜)
    100mM HEPES-HCl(pH7.4) 10mM MgCl 2/
    2mM MnCl 2
    10mM KH 2 PO 4 sub/
    1mM NADPH 1 mM NADP +
    20μMFAD
    2mM丙酮酸 330mM山梨醇
  10. 缓冲区
    100mM HEPES-HCl(pH7.4) 10mM MgCl 2/
    2mM MnCl 2
    10mM KH 2 PO 4 sub/
    330mM山梨醇 在使用前,将FAD加到10μM终浓度 储存于-20°C
  11. 缓冲区E
    10mM TES-HCl(pH7.4) 600mM山梨醇 2mM MgCl 2/
    25mM KH 2 PO 4 sub/
    0.33mM EDTA 储存于-20°C
    注意:所有缓冲液和溶液都用超纯水制备。

致谢

该方案已经改变自先前在Angaman等人(2012)和Pateraki等人(2013)中描述的方法。 这项工作得到了西班牙部长奖学金的支持 (BOD2009-09523)和经济部长部长(AGL2013-43522-R),包括FEDER基金,西班牙语Consolider-Ingenio 2010计划(CSD2007-00036农业研究中心)和加泰罗尼亚通用研究中心(2009SGR0026)。 Marta Renato是西班牙文化教育部长教授的受教育者。

参考文献

  1. Angaman,D.M.,Petrizzo,R.,Hernandez-Gras,F.,Romero-Segura,C.,Pateraki,I.,Busquets,M.and Boronat,A。(2012)。 分离的番茄果实色霉中的前体吸收测定和代谢分析植物方法 8(1):1.
  2. Pateraki,I.,Renato,M.,Azcón-Bieto,J.and Boronat,A。(2013)。 含有非典型γ亚基的ATP合酶参与番茄果实色素体中的ATP合成。 Plant J 74(1):74-85。
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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。
引用:Hernández-Gras, F., Petrizzo, R., Pateraki, I., Renato, M., Angaman, M., Azcón-Bieto, J. and Boronat, A. (2014). Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels. Bio-protocol 4(15): e1192. DOI: 10.21769/BioProtoc.1192.
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