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
发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2094 浏览次数: 9212
评审: Dennis NürnbergRumen IvanovYuko Kurita
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
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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
- 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 . - Miracloth (pore size: 22-25 µm) (EMD Millipore, catalog number: 475855 )
- Pipette tips (10 µl, 200 µl, 1 ml) and ‘cut-off’ pipette tips (1 ml) with the top (~2 mm) cut with a scissors
- Falcon tubes (50 ml) (Greiner Bio One, catalog number: 227261 )
- 2 ml tubes
- Aluminum foil
- Scintillation vials (SARSTEDT, catalog number: 58.536 ) and push caps (SARSTEDT, catalog number: 65.816 )
- Arabidopsis plants, approximately 4 weeks old (see Notes)
- Percoll (GE Healthcare, catalog number: 17-0891-01 )
- H2Obidest
- 80% (v/v) acetone (Carl Roth, catalog number: 9372.2 )
- 45Ca stock solution (1 mCi [37 MBq], CaCl2 > 10 Ci/g) (PerkinElmer, catalog number NEZ013001MC )
- 54Mn stock solution (200 µCi [7.4 MBq], MnCl2 in 0.5 N HCl > 20 Ci/g) (PerkinElmer, catalog number: NEZ040200UC )
- Calciumchlorid (CaCl2) (Carl Roth, catalog number: A119.1 )
- Mangan(II)-sulfat monohydrate (MnSO4·H2O) (Carl Roth, catalog number: 4487.1 )
- 0.1% (w/v) SDS (Carl Roth, catalog number: CN30.4 )
- Rotiszint® Eco Plus scintillation cocktail (Carl Roth, catalog number: 0016.3 )
- Sorbitol (Carl Roth, catalog number: 6213.2 )
- Tricine (Carl Roth, catalog number: 6977.5 )
- Natriumhydroxid (NaOH) (Carl Roth, catalog number: P031.2 )
- EDTA (Carl Roth, catalog number: 8040.2 )
- Magnesiumchlorid (MgCl2) (Carl Roth, catalog number: KK36.1 )
- Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
- BSA (Carl Roth, catalog number: 8076.3 )
- Manganese(II) chloride dehydrate (MnCl2) (EMD Millipore, catalog number: 105934 )
- EGTA (Carl Roth, catalog number: 3054.2 )
- HEPES
- 45Ca working solution (see Recipes)
- 54Mn working solution (see Recipes)
- 5x resuspension buffer, pH 8.4 (see Recipes)
- Homogenization buffer, pH 8.4 (see Recipes)
- Import buffer, pH 8.0 (see Recipes)
- Washing buffer, pH 8.0 (see Recipes)
- Lysis buffer, pH 7.6 (see Recipes)
Equipment
- Cold room equipped with green light (500-530 nm)
- Automatic pipette controller (BrandTech Scientific, model: Accu-Jet® Pro ) and a 10 ml glass pipette
- Waring blender (Waring Laboratory Science, model: 7011S) with a steel container and four razor blades
- 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). - Centrifuge Type 4K15C for Falcon tubes (Sigma Laborzentrifugen, model: 4K15C) or any other centrifuge, which is adequate for Falcon tubes
- UV/Vis spectrophotometer (Biochrom, model: UltrospecTM 2100 ) with quartz cuvettes
- Light source Faser-Illuminator (Heinz Walz, model: FL-460 ) set at 90 µmol photons m-2 sec-1
- Vortex shaker (Scientific Industries, model: Genie2 )
- Centrifuge 5418 R with rotor FA 45-18-11 for 2 ml tubes (Eppendorf, model: 5418 R)
- Liquid scintillation counter (PerkinElmer, model: Tri-Carb® 2910TR )
- Isotope lab with permission for 54Mn handling, lead coat and lead shields (approximately 5 cm)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物细胞生物学 > 细胞器分离
植物科学 > 植物生物化学 > 其它化合物
生物化学 > 其它化合物 > 离子 > 钙
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