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Transmission Electron Microscopy for Tobacco Chloroplast Ultrastructure
透射电镜观察烟草叶绿体的超微结构   

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Abstract

The chloroplast is the site of photosynthesis that enabled and sustains aerobic life on Earth. Chloroplasts are relatively large organelles with a diameter of ~5 μm and width of ~2.5 μm, and so can be readily analysed by electron microscopy. Each chloroplast is enclosed by two envelope membranes, which encompass an aqueous matrix, the stroma and the thylakoids. Components of stroma include starch granules and plastoglobuli, which can be observed by electron microscopy. And the thylakoids consist of stromal thylakoid, granal thylakoid and as well as granum (a stack of thylakoids). These structure components are quite sensitive to developmental changes and environmental variations, such as drought, salinity, cold, high temperature and others. Transmission electron microscopy (TEM) is a powerful technique for monitoring the effects of various changing parameters or treatments on the development and differentiation of these important organelles. Here we describe a reliable method for the analysis of plastid ultrastructure in tobacco plant by TEM.

Keywords: Chloroplast(叶绿体), Ultrastructure(超微结构), Tobacco(烟草)

Materials and Reagents

  1. Tobacco (Nicotiana tabacum) plants (about 6-week grew on MS agar plates, 3-week grew in 1/4 Hoagland solution, and 2~3 weeks grew on soil)
  2. Glutaraldehyde (Wako Pure Chemical Industries, catalog number: 071-01931 )
  3. Osmium tetroxide (Nisshin EM Corporation, catalog number: 300 )
  4. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
  5. Propylene oxide (Wako Pure Chemical Industries, catalog number: 165-05026 )
  6. Quetol 812 set (Nisshin EM Corporation, catalog number: 340 )
  7. Uranyl acetate (Wako Pure Chemical Industries, catalog number: 6159-44-0 )
  8. 3% (w/v) lead citrate (Wako Pure Chemical Industries, catalog number: 121-01722 )
  9. Dodecenyl succinic anhydride (DDSA)
  10. Methyl nadic anhydride (MNA)
  11. DMP
  12. 0.1 M phosphate buffered saline (see Recipes)
  13. 1% osmium tetroxide (see Recipes)
  14. Quetol-821 resin (see Recipes)
  15. Hoagland solution (see Recipes)

Equipment

  1. Razor blade
  2. Lens tissue
  3. Scissors
  4. Tweezers
  5. Needle and thread
  6. Glass tube
  7. Vacuum equipment
  8. Balance
  9. Petri dish
  10. Oven at 60 °C
  11. Plastic flat embedding mold (catalog number: 70900 )
  12. Beaker
  13. Ultramicrotome (RMC, model: MT-7000 )
  14. Copper grid
  15. Transmission electron microscope (JEOL, model: JEM-100CX II )

Procedure

  1. Cut and trim tobacco leaf with a razor blade to 2 mm x 4 mm size, cover the leaf with lens tissue and tie it with needle and spread which can protect the sample and facilitate the fixation and the following steps (because the sample is easily floating and covered with lens tissue will facilitate sinking during fixation), then put it into 2% glutaraldehyde in a glass tube.
  2. Vacuum the glutaraldehyde tube by vacuum equipment until no air bubble is coming out, and exhaust slowly to let the fix solution go into leaf tissue.
  3. Fix in 2% glutaraldehyde for 2 h at room temperature.
  4. Wash with 0.1 M PBS for 4~5 times and each time for 20~30 min.
  5. Fix in 1% osmium tetroxide for 2 h.
  6. Wash with distilled water for 30 min during which change the distilled water 5~8 times.
  7. Add 50% ethanol and incubate for 10 min.
  8. Discard 50% ethanol, add 70% ethanol and seal with Parafilm, keep overnight.
  9. Discard 70% ethanol, add 90% ethanol for 10 min.
  10. Discard 90% ethanol, wash with 100% ethanol for 3 times and each time for 10 min.
  11. Add propylene oxide for 2 times and each time for 5 min.
  12. Put the leaf sample into propylene oxide: Quetol 812 resin = 1:1 (v/v) and covered with aluminum foil overnight.
  13. Change to Quetol 812 resin and keep for 24 h.
  14. Embed the leaf sample horizontally with Quetol 812 resin on the Plastic flat embedding mold, and put into 60 °C for 48 h.
  15. Cut to thin sections (70~100 nm) with a diamond knife on an ultramicrotome, and the thin sections are gathered on a copper grid which is specially used for transmission electron microscope observation (Figure 1).


    Figure 1. A picture of copper grid with sample sections. The red circle indicated the sample sections.

  16. Stain with 2% (w/v) uranyl acetate for 40 min by putting copper grid with the sample (the sample side is downward) in staining solution and keep for 40 min, the following steps 16-18 are same.
  17. Wash with distilled water in a beaker for 3 times and each time for 10 min gently.
  18. Stain with 3% (w/v) lead citrate for 2~3 min.
  19. Wash with distilled water in a beaker for 3 times and each time for 10 min gently.
  20. Put on filter paper to make it dry and keep into the case.
  21. Observe the samples on a transmission electron microscope at 80 kv, and take photos. The sample image of chloroplast can be found in Figure 6 in the Wang et al. (2014) .

Notes

  1. In step 2, be sure to exhaust slowly to let the fix solution go into the sample tissue.
  2. During ethanol stepcoppers, keep a small amount of former solution before add new solution, this can avoid the dry of tissue edge.
  3. When prepare the resin, be sure to mix well before adding DMP-30 accelerator. And the ratio of each reagent is important, but the total amount can be adjusted.
  4. In steps 15 and 17, stain with uranyl acetate, drop a few drops on a Parafilm in a petridish, and put the copper disc on the drop.

Recipes

  1. 0.2 M phosphate buffered saline (pH 7.0)
    Solution A: 0.2 M Na2HPO4, Na2HPO4.12H2O 7.164 g in 100 ml distilled water
    Solution B: 0.2 M NaH2PO4, NaH2PO4.2H2O 3.121 g in 100 ml distilled water
    Put 61 ml solution A and 39 ml solution B together and mix well to make 0.2 M phosphate buffered saline at pH 7.0
  2. 1% osmium tetroxide
    4% osmium tetroxide: Distilled water: 0.2 M PBS= 1:1:2
  3. Quetol-821 resin (in the case of prepare 102 g)
    Quetol-812
    48 g
    DDSA
    19 g
    MNA
    33 g
    DMP
    302 g
  4. Hoagland solution
    Note: Recipe can be found in Epstein (1972).

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. Epstein, E. (1972). Mineral nutrition of plants: principles and perspectives.
  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.

简介

叶绿体是光合作用的位点,其使得和维持地球上的有氧生命。叶绿体是相对大的细胞器,直径为〜5μm,宽度为〜2.5μm,因此可以容易地通过电子显微镜分析。每个叶绿体被两个包膜包封,其包含水性基质,基质和类囊体。基质的组分包括淀粉颗粒和塑性球体,其可以通过电子显微镜观察。类囊体由基质类囊体,颗粒类囊体以及颗粒(一叠类囊体)组成。这些结构组分对发展变化和环境变化(如干旱,盐度,寒冷,高温等)相当敏感。透射电子显微镜(TEM)是一种强大的技术,用于监测各种变化的参数或治疗对这些重要细胞器的发育和分化的影响。在这里我们描述一种可靠的方法通过TEM分析烟草植物的质体超微结构。

关键字:叶绿体, 超微结构, 烟草

材料和试剂

  1. 烟草(Nicotiana tabacum)植物(在MS琼脂平板上生长约6周,在1/4 Hoagland溶液中生长3周,在土壤上生长2〜3周)
  2. 戊二醛(Wako Pure Chemical Industries,目录号:071-01931)
  3. 四氧化锇(Nisshin EM Corporation,目录号:300)
  4. 乙醇(Wako Pure Chemical Industries,目录号:057-00456)
  5. 环氧丙烷(Wako Pure Chemical Industries,目录号:165-05026)
  6. Quetol 812组(Nisshin EM Corporation,目录号:340)
  7. 铀乙酸酯(Wako Pure Chemical Industries,目录号:6159-44-0)
  8. 3%(w/v)柠檬酸铅(Wako Pure Chemical Industries,目录号:121-01722)
  9. 十二碳烯基琥珀酸酐(DDSA)
  10. 甲基纳迪克酸酐(MNA)
  11. DMP
  12. 0.1 M磷酸盐缓冲盐水(见配方)
  13. 1%四氧化锇(见配方)
  14. Quetol-821树脂(参见配方)
  15. Hoagland解决方案(参见配方)

设备

  1. 剃刀刀片
  2. 镜片组件
  3. 剪刀
  4. 镊子
  5. 针和线
  6. 玻璃管
  7. 真空设备
  8. 余额
  9. 培养皿
  10. 烤箱在60°C时
  11. 塑料平型嵌入模具(目录号:70900)
  12. 烧杯
  13. 超微切片机(RMC,型号:MT-7000)
  14. 铜网格
  15. 透射电子显微镜(JEOL,型号:JEM-100CX II)

程序

  1. 用刀片切割和修剪烟叶,尺寸为2mm×4mm,用透镜薄纸覆盖叶片,并用针头和展开结扎,可以保护样品,便于固定和以下步骤(因为样品容易漂浮 并用镜片组织覆盖将有助于在固定期间下沉),然后将其放入玻璃管中的2%戊二醛中。
  2. 通过真空设备真空戊二醛管,直到没有气泡出来,缓慢排气,让固定溶液进入叶组织。
  3. 在室温下在2%戊二醛中固定2小时
  4. 用0.1M PBS洗涤4〜5次,每次20〜30分钟
  5. 在1%四氧化锇中固定2小时
  6. 用蒸馏水清洗30分钟,在此期间更换蒸馏水5〜8次
  7. 加入50%乙醇,孵育10分钟
  8. 弃去50%乙醇,加入70%乙醇并用Parafilm密封,保持过夜
  9. 弃去70%乙醇,加入90%乙醇10分钟
  10. 弃去90%乙醇,用100%乙醇洗涤3次,每次10分钟
  11. 加入环氧丙烷2次,每次5分钟
  12. 将叶样品放入环氧丙烷:Quetol 812树脂= 1:1(v/v)中并用铝箔覆盖过夜。
  13. 更换为Quetol 812树脂并保持24小时
  14. 将树叶样品用Quetol 812树脂水平嵌入塑料平面嵌入模具中,并在60℃下放置48小时。
  15. 用超薄切片机上的金刚石刀切成薄的部分(70〜100nm),将薄切片聚集在专门用于透射电子显微镜观察的铜网上(图1)。


    图1.带有样品部分的铜网格图片。红色圆圈表示样品部分。

  16. 用2%(w/v)乙酸双氧铀染色40分钟,方法是在样品中放置铜网(样品面朝下),并保持40分钟,以下步骤16-18相同。
  17. 用蒸馏水在烧杯中洗涤3次,每次10分钟
  18. 用3%(w/v)柠檬酸铅染色2〜3分钟
  19. 用蒸馏水在烧杯中洗涤3次,每次10分钟
  20. 放在滤纸上,使其干燥并保持在盒子中。
  21. 在80kv的透射电子显微镜下观察样品,并拍照。 叶绿体的样品图像可以在Wang等人(2014)的图6中找到。

笔记

  1. 在步骤2中,确保缓慢排气,以使固定溶液进入样品组织。
  2. 在乙醇步骤中,在添加新溶液之前保留少量的前溶液,这可以避免组织边缘的干燥。
  3. 当制备树脂时,在添加DMP-30促进剂之前一定要混合均匀。 每种试剂的比例很重要,但总量可以调整。
  4. 在步骤15和17中,用乙酸双氧铀染色,在培养皿中的石蜡膜上滴几滴,并将铜盘放在液滴上。

食谱

  1. 0.2M磷酸盐缓冲盐水(pH7.0) 溶液A:0.2M Na 2 HPO 4水溶液,Na 2 HPO 4水溶液。 12H 2 O 7.164g在100ml蒸馏水中的溶液 溶液B:0.2M NaH 2 PO 4,NaH 2 PO 4,pH 4。 2H 2 O 3.121g在100ml蒸馏水中的溶液 将61ml的溶液A和39ml的溶液B放在一起,充分混合以制备pH7.0的0.2M磷酸盐缓冲盐水
  2. 1%四氧化锇
    4%四氧化锇:蒸馏水:0.2M PBS = 1:1:2
  3. Quetol-821树脂(在制备102g的情况下)
    Quetol-812
    48克
    DDSA
    19克
    MNA
    33克
    DMP
    302克
  4. Hoagland解决方案
    注意:食谱可以在Epstein(1972)找到。

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. Epstein,E。(1972)。 植物的矿物营养:原则和观点。
  2. 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。
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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。
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. Yin, L., Wang, S., Shimomura, N. and Tanaka, K. (2015). Transmission Electron Microscopy for Tobacco Chloroplast Ultrastructure. Bio-protocol 5(4): e1404. DOI: 10.21769/BioProtoc.1404.
  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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