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Isolation of Chloroplast Inner and Outer Envelope Membranes
叶绿体内膜和外膜的分离   

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Abstract

The chloroplast is an important organelle found in plant cells that conduct photosynthesis. It is enclosed by a pair of closely spaced membranes, the double-membrane envelope, consisting of the inner membrane bounding the matrix or stroma and the outer membrane in contact with the cytoplasm. Like many bio-membranes, the chloroplast envelope plays an important role in mediating the complex interactions between the chloroplast and the cytoplasm. The envelope is also the site of various biosynthetic reactions, including the formation of the galactolipids, which are the major components of both envelope and the thylakoid membranes. The inner and outer envelope membranes have differences in both structure and function. For example, the outer membrane exhibits lower density of intramembranous particles than the inner membrane dose, suggesting that the protein content of the outer membrane is low. Also, the outer membrane is nonspecifically permeable to low molecular weight compounds, whereas the inner is impermeable to such compounds and contains several translocator systems for the transport of metabolites.
To prepare the envelope membranes, it is necessary to isolate intact chloroplasts first. Then the inner and outer envelope membranes are separated by: 1) the protease-treatment method and 2) the centrifuge method which based on the fact that the outer envelope is lighter and the inner membrane heavier. Both methods need to isolate the intact chloroplasts firstly. However, the centrifugal separation can get the pure inner and outer envelope preparations, which therefore are suitable to the subsequent analyses. Also, the centrifuge method can avoid the destruction of inner envelope polypeptides during the protease treatment, because some of the protease may gain access to the inner membrane. Moreover, the centrifuge method is easy to operate and to get the complete enveloped that contain less of the adhesion regions of the outer and inner envelope membranes. Here we describe a reliable method for isolation of the inner and outer envelope membranes of the chloroplasts from tobacco, which is the plant that relatively not easy to use for envelope isolation.

Keywords: Chloroplast envelope(叶绿体被膜), Chloroplast membrane(叶绿体膜), Tobacco(烟草)

Materials and Reagents

  1. 6-week old tobacco (Nicotiana tabacum) plants (3-week after sowing, the seedlings were transplanted and grew for another 3 weeks)
  2. 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) (Dojindo Molecular Technologies, catalog number: 345-01625 )
  3. 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (Dojindo Molecular Technologies, catalog number: 342-01375 )
  4. Sobitol (Wako Pure Chemical Industries, catalog number: 191-14735 )
  5. di-sodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA-2Na) (Nacalai tesque, catalog number: 15111-45 )
  6. Manganese chloride tetrahydrate (MnCl2.4H2O) (Nacalai tesque, catalog number: 13446-34-9 )
  7. Magnesium chloride hexahydrate (MgCl2.6H2O) (Nacalai tesque, catalog number: 7791-18-6 )
  8. Sodium hydroxide (Nacalai tesque, catalog number: 1310-73-2 )
  9. Sodium chloride (Nacalai tesque, catalog number: 7647-14-5 )
  10. Isoascorbic acid (Nacalai tesque, catalog number: 89-65-6 )
  11. Polyvinylpyrrolidone (Sigma-Aldrich, catalog number: PVP40 )
  12. Percoll (GE Healthecare, catalog number: 17-0891-01 )
  13. Pyrophosphate aci (Wako Pure Chemical Industries, catalog number: 163-05485 )
  14. Glutathione (Wako Pure Chemical Industries, catalog number: 071-02014 )
  15. Tricine (Nacalai Tesque, catalog number: 5704-04-1 )
  16. Sucrose (Nacalai tesque, catalog number: 57-50-1 )
  17. Acetone (Wako Pure Chemical Industries, catalog number: 016-00346 )
  18. Extraction buffer for homogenization (see Recipes)
  19. Gradient buffer (see Recipes)
  20. TE buffer (see Recipes)
  21. 40% (v/v) Percoll in gradient buffer (see Recipes)
  22. 90% (v/v) Percoll in gradient buffer (see Recipes)
  23. 0.2 M, 0.6 M and 1.2 M sucrose in TE buffer (see Recipes)
  24. Linear gradient of 0.6~1.2 M sucrose-TE buffer (see Recipes)

Equipment

  1. Juicer mixer (TESCOM, model: TM837 )
  2. Scissors
  3. pH meter (Horiba, model: F-71S )
  4. Refrigerator 4 °C and -20 °C
  5. Funnel
  6. Centrifuge tube (250 ml, 50 ml, and 10 ml for ultracentrifuge)
  7. Miracloth (Calbiochem, catalog number: 475855 ) and/or gauze
  8. Paint brush with soft hair
  9. Pasteur pipet
  10. Spectrophotometer (Shimadzu, model: MPS2000 )
  11. Density gradient device (round type, inner diameter: 10 mm, total volume: 10~15 ml) (Sanplateccorp)
  12. Magnetic stirrer (Pasolina, model: TR-300 )
  13. Super centrifuge (Hitachi, model: CR20F )
  14. Ultracentrifuge (Hitachi, model: CP 70MX )
  15. Syringe (1 ml volumn)

Procedure

  1. Before the experiment, the tobacco leaf should be shaded for 2~3 days before extraction to reduce the starch content.
  2. Collect ~100 g tobacco young leaves from 6-week old tobacco seedlings with scissors, and cut leaves into 3~4 cm pieces on ice.
  3. Homogenize leaves with extraction buffer by Mixer for several times (see Note 1).
  4. Filter the leaf homogenates by 8 layers gauze or by 2 layers gauze with 1 layer Miracloth, and gather the filtrate into two 250 ml centrifuge tube.
  5. Centrifuge at 2, 500 x g for 70 sec at 4 °C.
  6. Discard the supernatant and suspend the crude chloroplasts in 5 ml extraction buffer gently by paint brush with soft hair. Using paint brush is suggested because the crude chloroplasts are easily broken.
  7. Add the crude chloroplasts suspension on a 40% and 90% Percoll gradient, and centrifuge at 2, 500 x g for 20 min at 4 °C by a swing rotor (see Figure 1).


    Figure 1. Crude chloroplast after centrifuged by 40% and 90% Percoll. The upper layer is the broken chloroplasts, and the lower green layer is the intact chloroplasts, the small white-green in the bottom of the tube is the starch.

  8. The intact chloroplasts are recovered in the lower green layer, and transfer the intact chloroplast into a 50 ml centrifuge tube by Pasteur pipet.
  9. Add 3-volume gradient buffer and centrifuge at 2,500 x g for 70 sec at 4 °C for washing.
  10. Suspend the intact chloroplasts in 0.6 M sucrose in TE buffer, and the chlorophyll concentration is adjusted to 2 mg/ml (see Note 7).
  11. After keeping on ice for 10 min, the chloroplasts are ruptured by three freeze-thaw cycles in which the suspension is placed in a -20 °C freezer for 1.5 h and then at room temperature until thawed, and this freeze-thaw procedure is repeated for 3 times.
  12. The suspension of broken chloroplasts are adjusted to 0.2 M Sucrose by adding 2-volume TE buffer, and centrifuge at 4, 500 x g for 15 min at 4 °C to remove the most of the thylakoids and residual intact chloroplasts, whereas the envelope membranes remained in the supernatant fraction.
  13. Centrifuge the supernatant at 40,000 x g for 30 min at 4 °C to gather the envelope membranes.
  14. Suspend the envelope membranes by 0.2 M sucrose in TE buffer.
  15. Add the envelope suspension on a linear gradient of 0.6~1.2 sucrose solution and centrifuge at 113,000 x g for 14 h at 4 °C in a swing rotor.
  16. The fraction of inner envelope membranes is recovered from the lower, and the fraction of outer envelope membranes from the upper sucrose/sucrose interface of the gradients [photograph can be seen in supplemental Figure S1 of the Wang et al. (2014)]. Gather the inner and outer envelope membranes by 1 ml syringe gently.
  17. Store the envelope membranes at -20 °C until use (within one month is better).

Notes

  1. Cut the leaves into 3~4 cm pieces and divide all leaves into 3~4 parts. Put one part of leaves and extraction buffer (200~300 ml) into the Mixer, and use flash function of Mixer (3~5 sec) for 3~4 times. Do flash each time after adding the leaves until all leaves are added. Do not homogenize too much. After homogenization, some small pieces of leaves still can be seen.
  2. In our procedure, the extractions tube should always be kept on ice during steps 5-13 except specially noted.
  3. The jug of the Mixer should be put into -20 °C to make it cool before using.
  4. All the buffers should be stored at 4 °C.
  5. The isoascorbic acid and glutathione should be added just before using.
  6. For measuring the chlorophyll concentration, dilute the suspended chloroplast 100-fold by 80% acetone, mix well and centrifuge at 10,000 x g for 5 min, then the supernatant was measured by a spectrophotometer at 645 nm, 663 nm, the spectrophotometer was adjust to zero using 80% acetone at 720 nm. The total chlorophyll concentration (mg/ml) = 100 x (0.0202 x A645 + 0.00802 x A663).
  7. In addition to tobacco leaf, other plants like spinach, etc., can be used for extraction, and sufficient leaf should be collected until ~100 g of fresh weight.

Recipes

  1. Extraction buffer for homogenization
    0.33 M sorbitol
    50 mM MES-NaOH (pH 6.1)
    2 mM EDTA
    1 mM MnCl2
    1 mM MgCl2
    20 mM NaCl
    2 mM isoascorbic acid
    1% (w/v) polyvinypyrrolidone-40
  2. Gradient buffer
    0.33 M sorbitol
    50 mM HEPES-NaOH (pH 6.8)
    2 mM EDTA
    1 mM MnCl2
    1 mM MgCl2
    1 mM sodium pyrophosphate
    5 mM isoascorbic acid
    5 mM glutathione
  3. TE buffer (pH 7.5)
    10 mM tricine
    2 mM EDTA-2Na
  4. 40% (v/v) Percoll in gradient buffer
  5. 90% (v/v) Percoll in gradient buffer
  6. 0.2 M, 0.6 M and 1.2 M sucrose in TE buffer
  7. Linear gradient of 0.6~1.2 M sucrose-TE buffer
    This buffer is made by density gradient device (Equipment 11), by gradient mix the 0.6 M sucrose and 1.2 M sucrose in TE buffer solution.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 31200206), the West Light Foundation of the Chinese Academy of Sciences, and the Chinese Universities Scientific Fund (grant no. ZD2012023).

References

  1. Wang, S., Uddin, M. I., Tanaka, K., Yin, L., Shi, Z., Qi, Y., Mano, J., Matsui, K., Shimomura, N., Sakaki, T., Deng, X. and Zhang, S. (2014). Maintenance of chloroplast structure and function by overexpression of the rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE gene leads to enhanced salt tolerance in tobacco. Plant Physiol 165(3): 1144-1155.

简介

叶绿体是在进行光合作用的植物细胞中发现的重要细胞器。它被一对紧密间隔的膜包围,双膜包膜由包围基质或基质的内膜和与细胞质接触的外膜组成。像许多生物膜一样,叶绿体包膜在介导叶绿体和细胞质之间的复杂相互作用中起重要作用。包膜也是各种生物合成反应的位点,包括半乳糖脂的形成,半乳糖脂是包膜和类囊体膜的主要成分。内封套膜和外封套膜在结构和功能上都有差异。例如,外膜表现出比内膜剂量更低的膜内颗粒密度,表明外膜的蛋白质含量低。此外,外膜对于低分子量化合物是非特异性可渗透的,而内部膜对于这些化合物是不可渗透的,并且包含用于运输代谢物的几种易位体系统。
为了制备包膜,必须首先分离完整的叶绿体。然后通过以下方式分离内外膜:1)蛋白酶处理方法和2)基于外壳更轻且内膜更重的事实的离心方法。两种方法都需要首先分离完整的叶绿体。然而,离心分离可以得到纯的内外封套制剂,因此适合于随后的分析。此外,离心法可以避免在蛋白酶处理期间内皮多肽的破坏,因为一些蛋白酶可以进入内膜。此外,离心方法容易操作并且获得包含较少外部和内部信封膜的粘合区域的完全包封。在这里,我们描述了一种可靠的方法,用于从烟草中分离叶绿体的内和外包膜,这是相对不容易用于包膜分离的植物。

关键字:叶绿体被膜, 叶绿体膜, 烟草

材料和试剂

  1. 6周龄烟草(烟草)植物(播种后3周,移植幼苗并再生长3周)
  2. 2-(N-吗啉代)乙磺酸一水合物(MES)(Dojindo Molecular Technologies,目录号:345-01625)
  3. 2- [4-(2-羟乙基)-1-哌嗪基]乙磺酸(HEPES)(Dojindo Molecular Technologies,目录号:342-01375)
  4. Sobitol(Wako Pure Chemical Industries,目录号:191-14735)
  5. 二钠乙二胺四乙酸二钠二水合物(EDTA-2Na)(Nacalai tesque,目录号:15111-45)
  6. 氯化锰四水合物(MnCl 2·6H 2 O·4H 2 O)(Nacalai tesque,目录号:13446-34-9)
  7. 氯化镁六水合物(MgCl 2·6H 2 O·6H 2 O)(Nacalai tesque,目录号:7791-18-6)
  8. 氢氧化钠(Nacalai tesque,目录号:1310-73-2)
  9. 氯化钠(Nacalai tesque,目录号:7647-14-5)
  10. 异抗坏血酸(Nacalai tesque,目录号:89-65-6)
  11. 聚乙烯吡咯烷酮(Sigma-Aldrich,目录号:PVP40)
  12. Percoll(GE Healthecare,目录号:17-0891-01)
  13. 焦磷酸盐(Wako Pure Chemical Industries,目录号:163-05485)
  14. 谷胱甘肽(Wako Pure Chemical Industries,目录号:071-02014)
  15. Tricine(Nacalai Tesque,目录号:5704-04-1)
  16. 蔗糖(Nacalai tesque,目录号:57-50-1)
  17. 丙酮(Wako Pure Chemical Industries,目录号:016-00346)
  18. 用于均化的提取缓冲液(参见配方)
  19. 渐变缓冲区(参见配方)
  20. TE缓冲区(参见配方)
  21. 40%(v/v)Percoll的梯度缓冲液(见配方)
  22. 90%(v/v)Percoll的梯度缓冲液(见配方)
  23. 0.2 M,0.6 M和1.2 M蔗糖的TE缓冲液(见配方)
  24. 0.6〜1.2M蔗糖-TE缓冲液的线性梯度(参见配方)

设备

  1. 榨汁混合器(TESCOM,型号:TM837)
  2. 剪刀
  3. pH计(Horiba,型号:F-71S)
  4. 冰箱4°C和-20°C
  5. 漏斗
  6. 离心管(250 ml,50 ml和10 ml超速离心机)
  7. Miracloth(Calbiochem,目录号:475855)和/或纱布
  8. 油漆刷与软毛
  9. 巴斯德移液器
  10. 分光光度计(Shimadzu,型号:MPS2000)
  11. 密度梯度装置(圆形,内径:10mm,总体积:10〜15ml)(Sanplateccorp)
  12. 磁力搅拌器(Pasolina,型号:TR-300)
  13. 超离心机(日立,型号:CR20F)
  14. 超速离心机(日立,型号:CP 70MX)
  15. 注射器(1ml体积)

程序

  1. 实验前,应在提取前将烟叶遮蔽2〜3天,以减少淀粉含量
  2. 用剪刀从6周龄烟草幼苗中收集〜100g烟草幼叶,并在冰上将叶切成3〜4cm的块。
  3. 用混合器将叶子用提取缓冲液均质化几次(见注1)
  4. 通过8层纱布或2层纱布和1层Miracloth过滤叶匀浆,并将滤液收集到两个250ml离心管中。
  5. 在4℃下以2,500×g离心70秒钟
  6. 弃去上清液,并悬浮在5ml提取缓冲液中轻轻地用油漆刷与柔软的头发的粗叶绿体。 建议使用油漆刷,因为粗制叶绿体容易破碎。
  7. 将粗制叶绿体悬浮液添加到40%和90%Percoll梯度上,并且通过摆动转子在4℃下在2,500×g离心20分钟(参见图1)。


    图1.通过40%和90%Percoll离心后的粗制叶绿体。上层是破碎的叶绿体,下面的绿色层是完整的叶绿体,在底部的小白绿色管是淀粉。

  8. 完整的叶绿体在较低的绿色层中回收,并通过巴斯德吸管将完整的叶绿体转移到50ml离心管中。
  9. 加入3体积梯度缓冲液,在4℃下以2,500×g离心70秒钟进行洗涤。
  10. 将完整叶绿体悬浮在TE缓冲液中的0.6M蔗糖中,并将叶绿素浓度调节至2mg/ml(见注7)。
  11. 在冰上保持10分钟后,通过三个冻融循环破坏叶绿体,其中将悬浮液置于-20℃的冷冻器中1.5小时,然后在室温下直至解冻,并重复该冻融程序3次。
  12. 通过加入2体积TE缓冲液将破碎的叶绿体的悬浮液调节至0.2M蔗糖,并在4,500℃下在4,500×g离心15分钟以除去大部分类囊体和残留的完整叶绿体,而包膜保留在上清液部分中
  13. 在4℃下以40,000×g离心上清液30分钟以收集包膜。
  14. 用0.2M蔗糖在TE缓冲液中悬浮包膜
  15. 将包封悬浮液在0.6〜1.2蔗糖溶液的线性梯度上添加,并在摇摆转子中在4℃下在113,000×g离心14小时。
  16. 内包膜的部分从下层回收,并且来自梯度的上部蔗糖/蔗糖界面的外包膜的部分[照片可见于Wang等人的补充图S1中。 >(2014)]。用1ml注射器轻轻地收集内外膜
  17. 将信封膜存放于-20°C直至使用(一个月内更好)。

笔记

  1. 将叶片切成3〜4厘米,将所有叶片分成3〜4份。 将一部分叶子和提取缓冲液(200〜300ml)放入混合器中,使用混合器的闪光功能(3〜5秒)3〜4次。 在添加叶子后,每次都闪烁,直到添加所有叶子。 不要均匀化太多。 在均化后,仍然可以看到一些小片叶。
  2. 在我们的程序中,除了特别注明,提取管应始终保持在冰上的步骤5-13。
  3. 搅拌机的水壶应放在-20°C以使其冷却后使用。
  4. 所有缓冲液应储存在4°C
  5. 在使用前应加入异抗坏血酸和谷胱甘肽。
  6. 为了测量叶绿素浓度,用80%丙酮稀释悬浮的叶绿体100倍,充分混合并在10,000×g离心5分钟,然后通过分光光度计在645nm,663nm处测量上清液 ,使用720%的80%丙酮将分光光度计调节至零。 总叶绿素浓度(mg/ml)= 100×(0.0202×A 645+ 0.00802×A 663)。
  7. 除了烟叶,其他植物如菠菜等可用于提取,并且应当收集足够的叶子,直到约100g鲜重。

食谱

  1. 用于匀浆的提取缓冲液
    0.33M山梨醇 50mM MES-NaOH(pH6.1) 2mM EDTA 1mM MnCl 2
    1mM MgCl 2
    20mM NaCl 2mM异抗坏血酸 1%(w/v)聚乙烯吡咯烷酮-40
  2. 渐变缓冲区
    0.33M山梨醇 50mM HEPES-NaOH(pH 6.8)
    2mM EDTA 1mM MnCl 2
    1mM MgCl 2
    1mM焦磷酸钠
    5mM异抗坏血酸 5mM谷胱甘肽
  3. TE缓冲液(pH7.5)
    10 mM tricine
    2mM EDTA-2Na
  4. 40%(v/v)Percoll的梯度缓冲液中
  5. 90%(v/v)Percoll的梯度缓冲液中
  6. 0.2 M,0.6 M和1.2 M蔗糖的TE缓冲液中
  7. 0.6〜1.2M蔗糖-TE缓冲液的线性梯度 该缓冲液通过密度梯度装置(设备11),通过在TE缓冲溶液中梯度混合0.6M蔗糖和1.2M蔗糖制备。

致谢

这项工作得到了中国国家自然科学基金(拨款号31200206),中国科学院西光基金会和中国高校科学基金(授予号ZD2012023)的支持。

参考文献

  1. Wang,S.,Uddin,MI,Tanaka,K.,Yin,L.,Shi,Z.,Qi,Y.,Mano,J.,Matsui,K.,Shimomura,N.,Sakaki, ,X。和Zhang,S。(2014)。 通过过表达水稻MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE基因来维持叶绿体结构和功能,从而提高烟草的耐盐性 。 Plant Physiol 165(3):1144-1155。
  • English
  • 中文翻译
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: © 2015 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. Wang, S., Yin, L., Mano, J. and Tanaka, K. (2015). Isolation of Chloroplast Inner and Outer Envelope Membranes. Bio-protocol 5(4): e1405. DOI: 10.21769/BioProtoc.1405.
  2. Wang, S., Uddin, M. I., Tanaka, K., Yin, L., Shi, Z., Qi, Y., Mano, J., Matsui, K., Shimomura, N., Sakaki, T., Deng, X. and Zhang, S. (2014). Maintenance of chloroplast structure and function by overexpression of the rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE gene leads to enhanced salt tolerance in tobacco. Plant Physiol 165(3): 1144-1155.
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