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Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts
使用完整拟南芥叶绿体进行阳离子(Ca2+和Mn2+)的分布测定   

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Abstract

Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

Keywords: Arabidopsis(拟南芥), Chloroplast(叶绿体), Ion transport(离子传输), Thylakoid membrane(类囊体膜), Envelope membrane(叶绿体被膜), Photosynthesis(光合作用)

Background

Calcium and manganese are structural components of photosystem II and form the inorganic Mn4CaO5 cluster, where water oxidation takes place with the outcome of electrons, protons and molecular oxygen. Ca2+ and Mn2+ fluxes across the chloroplast envelope membrane and the thylakoid membrane are fundamental processes enabling the plant cell to meet the high demand of PSII for these cations. In a previous study, a Ca2+/H+ antiport activity was analyzed using isolated thylakoid membranes from pea plants (Ettinger et al., 1999). In the model plant Arabidopsis thaliana hardly any Ca2+/H+ antiport activity is detectable in isolated thylakoid membranes (Schneider et al., 2016), thus a protocol which allows the thylakoid membrane system to reside in its naturally physiological environment, namely the chloroplast is presented. Furthermore, this protocol permits the relative distribution of Ca2+ and Mn2+ in chloroplasts to be determined. The protocol given here has been tested with Arabidopsis as well as with pea plants.

Materials and Reagents

  1. Corex® centrifuge tubes (30 ml) (Corning, USA)
    Note: This product has been discontinued. Replaceable item: 30 ml glass tubes from Krackeler Scientific, catalog number: 6-45500-30 .
  2. Miracloth (pore size: 22-25 µm) (EMD Millipore, catalog number: 475855 )
  3. Pipette tips (10 µl, 200 µl, 1 ml) and ‘cut-off’ pipette tips (1 ml) with the top (~2 mm) cut with a scissors
  4. Falcon tubes (50 ml) (Greiner Bio One, catalog number: 227261 )
  5. 2 ml tubes
  6. Aluminum foil
  7. Scintillation vials (SARSTEDT, catalog number: 58.536 ) and push caps (SARSTEDT, catalog number: 65.816 )
  8. Arabidopsis plants, approximately 4 weeks old (see Notes)
  9. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  10. H2Obidest
  11. 80% (v/v) acetone (Carl Roth, catalog number: 9372.2 )
  12. 45Ca stock solution (1 mCi [37 MBq], CaCl2 > 10 Ci/g) (PerkinElmer, catalog number NEZ013001MC )
  13. 54Mn stock solution (200 µCi [7.4 MBq], MnCl2 in 0.5 N HCl > 20 Ci/g) (PerkinElmer, catalog number: NEZ040200UC )
  14. Calciumchlorid (CaCl2) (Carl Roth, catalog number: A119.1 )
  15. Mangan(II)-sulfat monohydrate (MnSO4·H2O) (Carl Roth, catalog number: 4487.1 )
  16. 0.1% (w/v) SDS (Carl Roth, catalog number: CN30.4 )
  17. Rotiszint® Eco Plus scintillation cocktail (Carl Roth, catalog number: 0016.3 )
  18. Sorbitol (Carl Roth, catalog number: 6213.2 )
  19. Tricine (Carl Roth, catalog number: 6977.5 )
  20. Natriumhydroxid (NaOH) (Carl Roth, catalog number: P031.2 )
  21. EDTA (Carl Roth, catalog number: 8040.2 )
  22. Magnesiumchlorid (MgCl2) (Carl Roth, catalog number: KK36.1 )
  23. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  24. BSA (Carl Roth, catalog number: 8076.3 )
  25. Manganese(II) chloride dehydrate (MnCl2) (EMD Millipore, catalog number: 105934 )
  26. EGTA (Carl Roth, catalog number: 3054.2 )
  27. HEPES
  28. 45Ca working solution (see Recipes)
  29. 54Mn working solution (see Recipes)
  30. 5x resuspension buffer, pH 8.4 (see Recipes)
  31. Homogenization buffer, pH 8.4 (see Recipes)
  32. Import buffer, pH 8.0 (see Recipes)
  33. Washing buffer, pH 8.0 (see Recipes)
  34. Lysis buffer, pH 7.6 (see Recipes)

Equipment

  1. Cold room equipped with green light (500-530 nm)
  2. Automatic pipette controller (BrandTech Scientific, model: Accu-Jet® Pro ) and a 10 ml glass pipette
  3. Waring blender (Waring Laboratory Science, model: 7011S) with a steel container and four razor blades
  4. Beckman-AvantiTM J-25 centrifuge with rotors JA-14 and JS-13.1 (Beckman Coulter, model: AvantiTM J-25 )
    Note: This product has been discontinued. Replaceable item: Beckman Avanti J-E series centrifuge (Beckman Coulter, model: Avanti J-E).
  5. Centrifuge Type 4K15C for Falcon tubes (Sigma Laborzentrifugen, model: 4K15C) or any other centrifuge, which is adequate for Falcon tubes
  6. UV/Vis spectrophotometer (Biochrom, model: UltrospecTM 2100 ) with quartz cuvettes
  7. Light source Faser-Illuminator (Heinz Walz, model: FL-460 ) set at 90 µmol photons m-2 sec-1
  8. Vortex shaker (Scientific Industries, model: Genie2 )
  9. Centrifuge 5418 R with rotor FA 45-18-11 for 2 ml tubes (Eppendorf, model: 5418 R)
  10. Liquid scintillation counter (PerkinElmer, model: Tri-Carb® 2910TR )
  11. Isotope lab with permission for 54Mn handling, lead coat and lead shields (approximately 5 cm)

Procedure

  1. Preparation of a Percoll gradient
    1. Place all materials, reagents and buffers in the cold room for temperature equilibration at 4 °C.
    2. Prepare 10 ml Percoll solution (40%) by mixing 4 ml Percoll, 2 ml 5x resuspension buffer, pH 8.4 and 4 ml H2Obidest.
    3. Prepare 10 ml Percoll solution (80%) by mixing 8 ml Percoll and 2 ml 5x resuspension buffer, pH 8.4.
    4. Fill into a Corex tube 9 ml of 40% Percoll solution and carefully underlay 9 ml of 80% Percoll solution using a 10 ml glass pipette and an automatic pipette controller, keep the gradient on ice until further use (Figure 1).


      Figure 1. Preparation of Percoll gradient and isolation of intact chloroplasts (Procedures A and B)

  2. Isolation of intact chloroplasts
    1. Harvest 12 g fresh leaf material of overnight dark adapted Arabidopsis plants (see Notes). Perform all steps in a cold room equipped with green light (500-530 nm).
    2. Add 180 ml of homogenization buffer, pH 8.4 to the leaf material and homogenize in a Warring blender for 4 x 3 sec at 18,000 rpm (corresponding to button ‘Hi’).
    3. Filter the homogenate through two layers of Miracloth and centrifuge at 1,500 x g for 5 min at 4 °C in a JA-14 rotor.
    4. Remove supernatant and resuspend the pellet in 1 ml of 1x resuspension buffer pH 8.4 by carefully inverting the tube.
    5. Transfer the resuspended pellet (= chloroplast suspension) by using a cut-off pipette tip carefully on top of the Percoll gradient (Figure 1).
    6. The density gradient centrifugation is carried out in a JS-13.1 rotor for 15 min at 4 °C and 7,000 x g, use a slow acceleration and turn deceleration off. After centrifugation broken chloroplasts reside in the top fraction, intact chloroplasts reside in the interface of the two Percoll layers (Figure 1).
    7. Remove broken chloroplasts and the upper solution and carefully transfer intact chloroplasts using a cut-off pipette tip into a Falcon tube.
    8. Wash intact chloroplasts with 25 ml of 1x resuspension buffer pH 8.4. This step removes residual Percoll. Centrifuge at 1,500 x g for 5 min at 4 °C.
    9. Resuspend pellet (= Intact chloroplasts) in 500 µl import buffer, pH 8.0 (Ettinger et al., 1999), transfer to a 2 ml tube, wrap in aluminum foil and keep on ice.

  3. Determination of chlorophyll concentration
    1. Dilute 10 µl of intact chloroplast solution with 990 µl 80% acetone and incubate for 10 min on ice and in the dark.
    2. Centrifuge at 16,000 x g (centrifuge 5418 R) for 2 min at room temperature and immediately transfer supernatant to a quartz cuvette for measurement at 750 nm (for baseline correction), 663 nm and 646 nm in a UV/Vis spectrophotometer. Use 80% acetone as a blank sample.
    3. Calculate the chlorophyll content with following formulae (Porra, 2002):
      Chla = 12.25 x A663 - 2.55 x A646
      Chlb = 20.31 x A646 - 4.91 x A663
      (Chla + Chlb) x 100 = xxx µg Chl/ml
      Adjust chlorophyll concentration to 0.3 mg/ml with import buffer, pH 8.0 and divide sample into 45 µl aliquots (= Intact chloroplasts), wrap in aluminum foil and keep on ice. The whole procedure requires 15 aliquots per isotope experiment.

  4. Preparation of 45Ca or 54Mn working solution
    1. Perform all steps in the isotope lab. For handling 54Mn it is necessary to wear a lead coat and to shield radioactive samples with a lead wall (5 cm). Perform 45Ca assays and 54Mn assays separately, e.g., on two following days, but use freshly prepared chloroplasts.
    2. Take 4 µl of 45Ca stock solution and dilute it (1:20) with 76 µl of CaCl2 (15 µM). Mix well, this mixture corresponds to the 45Ca working solution and it is suitable for 15 (+1) reactions.
    3. Take 4 µl of 54Mn stock solution and dilute it (1:20) with 76 µl of MnSO4 (1.5 µM). Mix well, this mixture corresponds to the 54Mn working solution and it is suitable for 15 (+1) reactions.

  5. Uptake of 45Ca or 54Mn into chloroplasts (Figure 2)


    Figure 2. Schema of experimental set-up. Incubation of intact chloroplasts with 45Ca (A and B) or 54Mn (C and D) is done for 20 min in 90 µmol photons m-2 sec-1. After light incubation samples are incubated in sorbitol, without vortexing to keep chloroplasts intact (A and C) or samples are incubated without sorbitol, but with vortexing to release thylakoids (B and D). EGTA is used as chelating agent to complex divalent cations, attached to the outside of the membranes.

    1. To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of 45Ca working solution (step D2) or with 54Mn working solution (step D3), remove aluminum foil and immediately expose samples for 20 min to 90 µmol photons m-2 sec-1.
      Set up the reaction with three aliquots corresponding to three replicates.
    2. Add 1 ml import buffer, pH 8.0 to each aliquot and incubate on ice, in the dark for 10 min (the total incubation time is 30 min). The import buffer contains sorbitol therefore the chloroplasts are kept intact during this incubation step.
    3. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 1 ml washing buffer, pH 8.0 and vortex for 2 sec. This step removes 45Ca or 54Mn bound at the surface of the membrane, because the washing buffer contains EGTA as chelating agent.
    4. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 50 µl of 0.1% SDS and immediately transfer to 4 ml scintillation cocktail. Mix well and keep samples at room temperature until counting.

  6. Uptake of 45Ca or 54Mn into chloroplasts and subsequent release of thylakoids (Figure 2)
    1. To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of 45Ca working solution (step D2) or with 54Mn working solution (step D3), remove aluminum foil and immediately expose samples for 20 min to 90 µmol photons m-2 sec-1.
      Set up the reaction with three aliquots corresponding to three replicates.
    2. Add 1 ml import buffer, pH 8.0 to each aliquot and incubate on ice, in the dark for 10 min (the total incubation time is 30 min). The import buffer contains sorbitol therefore the chloroplasts are kept intact during this incubation step.
    3. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 1 ml washing buffer, pH 8.0 and vortex for 2 sec. This step removes 45Ca or 54Mn bound at the surface of the membrane, because the washing buffer contains EGTA as chelating agent.
    4. Centrifuge at 16,000 x g (centrifuge 5418 R) for 1 min at 4 °C and remove supernatant. Resuspend pellet in 50 µl of 0.1% SDS and immediately transfer to 4 ml scintillation cocktail. Mix well and keep samples at room temperature until counting.

  7. Additional reaction for estimating uptake of 45Ca or 54Mn during 10 min incubation step on ice
    To start the import reaction, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of 45Ca working solution (step D2) or with 54Mn working solution (step D3), remove aluminum foil and immediately perform steps E2 to E4 and F2 to F4. The total incubation time is therefore 10 min on ice in the dark. Set up the reactions with three aliquots each corresponding to three replicates.

  8. Additional reaction for estimating background radiation
    To estimate background radiation, mix a 45 µl aliquot (= Intact chloroplasts, step C3) with 5 µl of 45Ca working solution (step D2) or with 54Mn working solution (step D3), remove aluminum foil and immediately perform steps E3 to E4. Set up the reaction with three aliquots each corresponding to three replicates.

  9. Determination of incorporated radioactivity 
    Collect scintillation vials form steps E4, F4, G and H and determine counts per minutes (cpm) in a liquid scintillation counter Tri-Carb® 2910 TR. Determine 45Ca values using program #5 and determine 54Mn values using program #8 with 5 min count time per individual scintillation vial.

Data analysis

  1. Collect cpm values from step I and insert values into an Excel sheet.
  2. Subtract cpm values for mean background radiation (step H) from all other cpm values (steps E4, F4 and G). Although the value derived after 10 min ice-incubation (step G) is not used for subsequent calculations, it should be clearly lower than the value obtained after 30 min incubation. If this is not the case, results can not be interpreted as ‘light dependent uptake’.
  3. Convert cpm values into relative units, e.g., take the mean cpm value after 30 min incubation time from step E4 and set it as a relative unit to ‘one’. Experiments performed with independent grown plants and isotopes can thus easily be compared.
  4. The thylakoid to stroma distribution in chloroplasts can be estimated from the amount of radioactivity separately detected in thylakoid fractions (step F4) and in intact chloroplast fractions (step E4). Set the cpm values obtained from intact chloroplast fraction (step E4) to 100% and calculate the percentage of cpm values from thylakoid fractions (step F4).
  5. The whole procedure (steps A to I) is described for three technical replicates (see step F1). It should be performed two to three times with independently grown plants (= independent experiments).

Notes

Arabidopsis growth can be performed as described in Steinberger et al., 2015. This protocol is also suitable for two-week old pea plants. For isolation of intact chloroplasts, it is important to avoid any starch granules, which accumulate during the light period. Starch granules inside chloroplasts might prevent accumulation of intact chloroplasts at the interface of the two Percoll layers. Therefore, dark adapt plants for at least 16 h; during the night starch degradation takes place.

Recipes

  1. 45Ca working solution (use a fresh preparation)
    4 µl of 45Ca stock solution
    76 µl of CaCl2 (15 µM)
  2. 54Mn working solution (use a fresh preparation)
    4 µl of 54Mn stock solution
    76 µl of MnSO4 (1.5 µM)
  3. 5x resuspension buffer, pH 8.4 (can be stored at 4 °C for a few days)
    0.4 M sorbitol
    20 mM Tricine/NaOH, pH 8.4
    2.5 mM EDTA
    5 mM MgCl2
  4. Homogenization buffer, pH 8.4 (can be stored at 4 °C for a few days)
    0.4 M sorbitol
    20 mM Tricine/NaOH, pH 8.4
    10 mM EDTA
    5 mM NaHCO3
    0.1% (w/v) BSA
    1 mM MgCl2
    1 mM MnCl2
  5. Import buffer, pH 8.0 (can be stored at 4 °C for a few days)
    0.33 M sorbitol
    50 mM Tricine/NaOH, pH 8.0
    5 mM MgCl2
  6. Washing buffer, pH 8.0 (can be stored at 4 °C for a few days)
    0.33 M sorbitol
    50 mM Tricine/NaOH, pH 8.0
    50 mM EGTA
  7. Lysis buffer, pH 7.6 (can be stored at 4 °C for a few weeks)
    10 mM HEPES/NaOH, pH 7.6
    5 mM MgCl2

Acknowledgments

We thank Gabi Burkhard for excellent technical assistance. This work was carried out in the laboratory of Prof. Dario Leister (Biozentrum der LMU München, Department Biologie I, Munich, Germany) and supported by funds from the Deutsche Forschungsgesellschaft. This protocol is adapted from Schneider et al., 2016.

References

  1. Ettinger, W. F., Clear A. M., Fanning, K. J., and Peck, M. L. (1999). Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane. Plant Physiol 119: 1379-1386.
  2. Porra, R. J. (2002). The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73(1-3): 149-156.
  3. Schneider, A., Steinberger, I., Herdean, A., Gandini, C., Eisenhut, M., Kurz, S., Morper, A., Hoecker, N., Ruhle, T., Labs, M., Flugge, U. I., Geimer, S., Schmidt, S. B., Husted, S., Weber, A. P., Spetea, C. and Leister, D. (2016). The evolutionarily conserved protein photosynthesis affected mutant71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis. Plant Cell 28(4): 892-910.
  4. Steinberger, I., Egidi, F., and Schneider, A. (2015). Chlorophyll fluorescence measurements in Arabidopsis wild-type and photosystem II mutant leaves. Bio-Protocol 5: e1532.

简介

测定Ca 2 + 和Mn 2 + 的相对分布是分析在叶绿体包膜或类囊体中钙和/或锰转运蛋白水平发生变化的突变体的重要工具膜。该方案中描述的方法允许使用同位素[ 45 Ca]和[ 54 Mn]定量分析叶绿体基质和类囊体之间钙和锰离子的相对分布,作为放射性示踪剂。为了避免非叶绿体膜系统的污染,该方法被设计用于分离拟南芥的纯和完整的叶绿体。通过Percoll梯度离心分离完整的叶绿体。然后在轻孵育步骤期间使叶绿体吸收[ 45 Ca]或[ 54 Mn]。孵化后,叶绿体保持完整或渗透/机械处理以释放类囊体。通过液体闪烁计数可以确定掺入量[ 45 Ca]或[ 54 Mn]的量,并计算相对分布。

背景 钙和锰是光系统II的结构组分,形成无机Mn 4 O 5 O 5 O 3簇,其中水氧化发生电子,质子和分子氧的结果。通过叶绿体包膜和类囊体膜的Ca 2+ / +和/或超氧化物质是使得植物细胞满足PSII对这些阳离子的高需求的基本过程。在先前的研究中,使用来自豌豆植物的分离的类囊体膜(Ettinger等人)分析了Ca 2 + / H + 1999)。在模拟植物中,拟南芥几乎没有任何Ca 2+ / + / / sup> + / / sup>止血活性在分离的类囊体膜中是可检测的(Schneider等人,2016),因此提出了允许类囊体膜系统驻留在其自然生理环境即叶绿体中的方案。此外,该方案允许待确定的叶绿体中Ca 2 + 和Mn 2 + 的相对分布。这里给出的方案已经用拟南芥以及豌豆植物进行了测试。

关键字:拟南芥, 叶绿体, 离子传输, 类囊体膜, 叶绿体被膜, 光合作用

材料和试剂

  1. Corex ®离心管(30ml)(Corning,USA)
    注意:本产品已停产。可替换物品:来自Krackeler Scientific的30ml玻璃管,目录号:6-45500-30。
  2. Miracloth(孔径:22-25μm)(EMD Millipore,目录号:475855)
  3. 移液头(10μl,200μl,1 ml)和"切断"移液管吸头(1 ml),用剪刀切割顶部(〜2 mm)
  4. Falcon管(50ml)(Greiner Bio One,目录号:227261)
  5. 2 ml管
  6. 铝箔
  7. 闪烁瓶(SARSTEDT,目录号:58.536)和推盖(SARSTEDT,目录号:65.816)
  8. 大约4周龄的拟南芥植物(见注释)
  9. Percoll(GE Healthcare,目录号:17-0891-01)
  10. 2
  11. 80%(v/v)丙酮(Carl Roth,目录号:9372.2)
  12. Ca储备溶液(1mCi [37MBq],CaCl 2> 10Ci/g)(PerkinElmer,目录号NEZ013001MC)
  13. (0.5N HCl> 20Ci/g)中的Mn储备溶液(200μCi[7.4MBq],MnCl 2)(PerkinElmer,目录号:NEZ040200UC)
  14. 氯化钙(CaCl 2)(Carl Roth,目录号:A119.1)
  15. 锰(II) - 硫酸一水合物(MnSO 4·H 2 O)(Carl Roth,目录号:4487.1)
  16. 0.1%(w/v)SDS(Carl Roth,目录号:CN30.4)
  17. Rotiszint ® Eco Plus闪烁鸡尾酒(Carl Roth,目录号:0016.3)
  18. 山梨醇(Carl Roth,目录号:6213.2)
  19. Tricine(Carl Roth,目录号:6977.5)
  20. 硝酸钠(NaOH)(Carl Roth,目录号:P031.2)
  21. EDTA(Carl Roth,目录号:8040.2)
  22. 氯化镁(MgCl 2)(Carl Roth,目录号:KK36.1)
  23. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  24. BSA(Carl Roth,目录号:8076.3)
  25. 氯化锰(II)脱水(MnCl 2)(EMD Millipore,目录号:105934)
  26. EGTA(Carl Roth,目录号:3054.2)
  27. HEPES
  28. 45 Ca工作溶液(见配方)
  29. 54 锰工作溶液(见配方)
  30. 5倍重悬浮缓冲液,pH 8.4(参见食谱)
  31. 均质缓冲液,pH 8.4(参见食谱)
  32. 导入缓冲液,pH 8.0(参见食谱)
  33. 洗涤缓冲液,pH 8.0(参见食谱)
  34. 裂解缓冲液,pH 7.6(参见食谱)

设备

  1. 冷室配有绿灯(500-530nm)
  2. 自动移液器控制器(BrandTech Scientific,型号:Accu-Jet ®)/10毫升玻璃移液器
  3. Waring搅拌机(Waring Laboratory Science,型号:7011S),带钢容器和四个剃刀刀片
  4. 具有转子JA-14和JS-13.1(Beckman Coulter,型号:AvantiTM J-25)的Beckman-Avanti TM J-25离心机
    注意:本产品已停产。可更换物品:Beckman Avanti J-E系列离心机(Beckman Coulter,型号:Avanti J-E)。
  5. 离心机类型4K15C用于Falcon管(Sigma Laborzentrifugen,型号:4K15C)或任何其他适用于Falcon管的离心机
  6. 用石英比色皿的UV/Vis分光光度计(Biochrom,型号:Ultrospec TM 2100)
  7. 光源Faser-Illuminator(Heinz Walz,型号:FL-460)设置为90μmol光子m -2 sec -1
  8. 涡旋振荡器(科学绝缘体,型号:Genie2)
  9. 离心机5418 R,转子FA 45-18-11用于2 ml管(Eppendorf,型号:5418 R)
  10. 液体闪烁计数器(PerkinElmer,型号:Tri-Carb< 2910TR)
  11. 同位素实验室,允许 54 锰处理,铅皮和铅屏蔽(约5厘米)

程序

  1. 制备Percoll梯度
    1. 将所有材料,试剂和缓冲液放在冷藏室中,以在4°C温度平衡。
    2. 通过混合4毫升Percoll,2毫升5x重悬浮缓冲液,pH8.4和4毫升H 2 O 2上清液来制备10ml Percoll溶液(40%)。
    3. 通过混合8ml Percoll和2ml 5x重悬浮缓冲液(pH 8.4)来制备10ml Percoll溶液(80%)。
    4. 填充到Corex管中,加入9ml 40%Percoll溶液,并使用10ml玻璃移液管和自动移液管控制器小心地将9ml 80%Percoll溶液底层保存在冰上直至进一步使用(图1)。 >

      图1. Percoll梯度的制备和完整叶绿体的分离(方法A和B)

  2. 完整叶绿体的分离
    1. 收获12克新鲜的叶子材料过夜暗适应拟南芥植物(见注释)。在配备绿灯(500-530 nm)的寒冷室内执行所有步骤。
    2. 向叶片材料中加入180ml均质缓冲液(pH8.4),并在Warring混合机中以18,000rpm(对应于按钮'Hi')均质化4×3秒。
    3. 将匀浆通过两层Miracloth过滤,并在4℃下在JA-14转子中以1,500×g离心5分钟。
    4. 删除上清液,并将小丸重新悬浮在1ml 1x重悬浮缓冲液pH 8.4中,仔细倒置管
    5. 通过在Percoll梯度顶部仔细地使用切断移液管尖端转移重悬的颗粒(=叶绿体悬浮液)(图1)。
    6. 密度梯度离心在JS-13.1转子中在4℃和7,000×g/g下进行15分钟,使用缓慢的加速度并且减速。离心后,叶绿体位于顶部馏分中,完整的叶绿体位于两个Percoll层的界面(图1)。>
    7. >去除破碎的叶绿体和上层溶液,并使用切断的移液管小瓶将完整的叶绿体转移到Falcon管中。
    8. 用25ml 1x重悬浮缓冲液pH8.4洗涤完整的叶绿体。该步骤去除残留的Percoll。在4℃下以1,500×g离心5分钟。
    9. 在500μl进口缓冲液pH 8.0(Ettinger等人,1999)中重悬沉淀(=完整叶绿体),转移到2ml管中,包裹在铝箔中并保持在冰上。

  3. 叶绿素浓度的测定
    1. 用990μl80%丙酮稀释10μl完整的叶绿体溶液,并在冰上和黑暗中孵育10分钟。
    2. 离心机16,000 xg(离心机5418 R)在室温下离心2分钟,并立即将上清液转移到石英比色皿中,以在750 nm(用于基线校正)测量,在紫外/可见光下为663 nm和646 nm,可见分光光度计。使用80%丙酮作为空白样品。
    3. 用下列公式计算叶绿素含量(Porra,2002):
      663 = 12.25×A 663 - 2.55 x A 646
      l>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> (Chl a + Chl b )x 100 = xxxμgChl/ml
      用进口缓冲液pH 8.0将叶绿素浓度调整到0.3mg/ml,并将样品分成45μl等分试样(=完整的叶绿体),包裹在铝箔中并保持在冰上。整个过程每个同位素实验需要15等份。

  4. 制备 45 Ca或 54 Mn工作溶液
    1. 执行同位素实验室的所有步骤。对于处理 54 Mn,必须戴铅笔,并用铅墙(5厘米)屏蔽放射性样品。在接下来的几天分别进行 45 Ca测定和 54 锰测定,例如,使用新鲜制备的叶绿体。
    2. 取4μl 45 Ca储备溶液,并用76μlCaCl 2(15μM)稀释(1:20)。混合均匀,该混合物相当于 45 Ca工作溶液,适用于15(+1)反应。
    3. 取4μl 54 Mn储备溶液,并用76μlMnSO 4(1.5μM)稀释(1:20)。混合均匀,该混合物对应于 54 Mn工作溶液,适用于15(+1)反应。

  5. 45 Ca或54Mn摄入叶绿体(图2)


    图2.实验装置的模式将完整的叶绿体与 45 Ca(A和B)或 54 Mn(C和D) )在90μmol光子中进行20分钟,其中m sec -1 。光孵化后,将样品在山梨醇中孵育,不涡旋以保持叶绿体完整(A和C),或样品不用山梨醇孵育,但涡旋释放类囊体(B和D)。使用EGTA作为螯合剂来复合二价阳离子,附着在膜的外面
    1. 为了开始进口反应,将45μl等分试样(=完整的叶绿体,步骤C3)与5μl的< sup>> Ca工作溶液(步骤D2)或/溶液(步骤D3),去除铝箔,并立即将样品暴露20分钟至90μmol光子,其中m -2 -1
      用三个重复的三个等分试样设置反应。
    2. 向每个等分试样中加入1ml进口缓冲液,pH 8.0,并在黑暗中孵育10分钟(总孵育时间为30分钟)。进口缓冲液含有山梨醇,因此在该孵育步骤中叶绿体保持完整
    3. 在4℃下离心16,000 x g(离心机5418R)1分钟,除去上清液。将沉淀重悬于1ml洗涤缓冲液(pH8.0)中并涡旋2秒。由于洗涤缓冲液含有EGTA作为螯合剂,所以该步骤除去在膜表面结合的 45 Ca或 54
    4. 在4℃下离心16,000 x g(离心机5418R)1分钟,除去上清液。将沉淀重悬于50μl0.1%SDS中,并立即转移至4 ml闪烁液中。混合均匀并将样品保持在室温直到计数。

  6. 45 Ca或 54摄入到叶绿体中,随后释放类囊体(图2)
    1. 为了开始进口反应,将45μl等分试样(=完整的叶绿体,步骤C3)与5μl的< sup>> Ca工作溶液(步骤D2)或/溶液(步骤D3),去除铝箔,并立即将样品暴露20分钟至90μmol光子,其中m -2 -1
      用三个重复的三个等分试样设置反应。
    2. 向每个等分试样中加入1ml进口缓冲液,pH 8.0,并在黑暗中孵育10分钟(总孵育时间为30分钟)。进口缓冲液含有山梨醇,因此在该孵育步骤中叶绿体保持完整
    3. 在4℃下离心16,000 x g(离心机5418R)1分钟,除去上清液。将沉淀重悬于1ml洗涤缓冲液(pH8.0)中并涡旋2秒。由于洗涤缓冲液含有EGTA作为螯合剂,所以该步骤除去在膜表面结合的 45 Ca或 54
    4. 在4℃下离心16,000 x g(离心机5418R)1分钟,除去上清液。将沉淀重悬于50μl0.1%SDS中,并立即转移至4 ml闪烁液中。混合均匀并将样品保持在室温直到计数。

  7. 在冰上10分钟温育步骤中估计摄取超氧化物或半胱氨酸的附加反应
    为了开始进口反应,将45μl等分试样(=完整的叶绿体,步骤C3)与5μl的< sup>> Ca工作溶液(步骤D2)或/溶液(步骤D3),去除铝箔,立即进行步骤E2至E4和F2至F4。因此,总孵化时间为黑暗中冰上10分钟。设置三个等分试样的反应,每个等分试样对应三次重复
  8. 估计背景辐射的附加反应
    为了估计背景辐射,将45μl等分试样(=完整叶绿体,步骤C3)与5μl的< sup>> Ca工作溶液(步骤D2)或与 54 Mn工作溶液(步骤D3),移除铝箔,立即进行步骤E3〜E4。设置三个等分试样的反应,每个等分试样对应三次重复
  9. 确定放射性的结合 
    收集闪烁瓶形成步骤E4,F4,G和H,并确定液体闪烁计数器Tri-Carb 2910 TR中的每分钟计数(cpm)。使用程序#5确定 45 Ca值,并使用程序#8以每个闪烁瓶5分钟计数时间确定 54 Mn值。

数据分析

  1. 从步骤I收集cpm值,并将值插入到Excel工作表中。
  2. 从所有其他cpm值(步骤E4,F4和G)减去平均背景辐射(步骤H)的cpm值。虽然在10分钟冰温度(步骤G)后得到的值不用于后续计算,但应明显低于30分钟孵育后获得的值。如果不是这样,结果不能解释为"轻度依赖性摄取"。
  3. 将cpm值转换为相对单位,例如,从步骤E4获取30分钟孵育时间后的平均cpm值,并将其设置为"1"的相对单位。可以容易地比较用独立的植物和同位素进行的实验。
  4. 类囊体到叶绿体中的基质分布可以从在类囊体组分(步骤F4)和完整的叶绿体组分中单独检测到的放射活性量估计(步骤E4)。将从完整的叶绿体级分(步骤E4)获得的cpm值设置为100%,并计算类囊细胞级分的cpm值的百分比(步骤F4)。
  5. 描述了三个技术重复的整个过程(步骤A至I)(参见步骤F1)。应该用独立生长的植物进行两到三次(=独立实验)。

笔记

拟南芥生长可以如Steinberger等人,2015所述进行。该方案也适用于两周龄的豌豆植物。为了分离完整的叶绿体,重要的是避免在光照期间积聚的任何淀粉颗粒。叶绿体内的淀粉颗粒可能会阻止完整叶绿体在两个Percoll层的界面积聚。因此,黑暗适应植物至少16小时;夜间淀粉降解发生。

食谱

  1. 45 Ca工作溶液(使用新鲜制剂)
    4μl 45 Ca储液溶液
    76μlCaCl 2(15μM)
  2. 54 Mn工作溶液(使用新鲜制剂)
    4μl 54 锰储备溶液
    76微升MnSO 4(1.5微米)
  3. 5倍重悬浮缓冲液,pH 8.4(可在4℃下储存几天)
    0.4 M山梨醇
    20mM Tricine/NaOH,pH8.4
    2.5 mM EDTA
    5mM MgCl 2
  4. 均质缓冲液,pH 8.4(可在4℃下储存几天)
    0.4 M山梨醇
    20mM Tricine/NaOH,pH8.4
    10 mM EDTA
    5mM NaHCO 3
    0.1%(w/v)BSA
    1mM MgCl 2
    1mM MnCl 2
  5. 进口缓冲液,pH 8.0(可在4℃下储存几天)
    0.33 M山梨醇
    50 mM Tricine/NaOH,pH 8.0
    5mM MgCl 2
  6. 洗涤缓冲液,pH 8.0(可在4℃下储存几天)
    0.33 M山梨醇
    50 mM Tricine/NaOH,pH 8.0
    50 mM EGTA
  7. 裂解缓冲液,pH 7.6(可在4℃下保存数周)
    10mM HEPES/NaOH,pH7.6。
    5mM MgCl 2

致谢

我们非常感谢Gabi Burkhard提供卓越的技术援助。这项工作是在德里·莱斯特(Prof.Dario Leister)的实验室(德国慕尼黑的生物制品公司生物技术研究所)进行的,并得到了德国富士施隆公司的资助。该协议由施耐德等人于2016年改编。

参考文献

  1. Ettinger,WF,Clear AM,Fanning,KJ和Peck,ML(1999)。  鉴定植物叶绿体类囊体膜中的Ca 2 + /H + antiport 植物生理 119:1379-1386。
  2. Porra,RJ(2002)。  开发和使用联立方程,以准确测定叶绿素a和/b> 和 b 。 149-156。
  3. Schneider,A.,Steinberger,I.,Herdean,A.,Gandini,C.,Eisenhut,M.,Kurz,S.,Morper,A.,Hoecker,N.,Ruhle,T.,Labs, Flugge,UI,Geimer,S.,Schmidt,SB,Husted,S.,Weber,AP,Spetea,C.和Leister,D。(2016)。进化上保守的蛋白质光合作用影响突变体71是拟南芥中类囊体膜上有效锰摄取所必需的。 植物细胞 28(4):892-910。
  4. Steinberger,I.,Egidi,F.和Schneider,A.(2015)。
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引用:Harms, A., Steinberger, I. and Schneider, A. (2017). Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts. Bio-protocol 7(1): e2094. DOI: 10.21769/BioProtoc.2094.
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