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Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain
染色法检测拟南芥胚囊中线粒体膜电位   

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Abstract

The aim of this experiment is to study mitochondrial functional status in Arabidopsis embryo sacs using the membrane potential indicator JC-1. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibrium of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol, a distinctive feature of the early stages of programmed cell death.
JC-1 is a lipophilic dye that can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. In healthy cells with high mitochondrial potential, JC-1 spontaneously forms complexes with intense red fluorescence. On the other hand, in mitochondria with low mitochondrial potential, JC-1 remains in the monomeric form, which exhibits only green fluorescence (Martin et al., 2013; Hauser et al., 2006).
This protocol could be used in isolated mitochondria, and in a variety of cell types and different tissues of plants and other organism.

Keywords: Mitochondrial membrane potential(线粒体膜电位), Female gametophyte(雌配子体), Arabidopsis thaliana(拟南芥), Plant embryo sac(植物胚囊), JC-1(JC-1)

Materials and Reagents

  1. Flowers at different developmental stages from an Arabidopsis inflorescence
  2. JC-1 dye (Life Technologies, Molecular Probes®, catalog number: T3168 )
  3. DMSO 99.9% (Sigma-Aldrich, catalog number: D8418 )
  4. Stock solution (10 mg/ml of JC-1 in DMSO)
  5. Working solution (10 µg/ml of JC-1 in buffer A)
  6. Buffer A (20 mM HEPES buffer, pH 7.2) (Sigma-Aldrich, catalog number: H3375 ) (see Recipes)

Equipment

  1. Confocal microscope (Nikon Eclipse C1 Plus Confocal microscope, using EZ-C1 3.80 imaging software and Ti-Control)
  2. Dissecting microscope (Nikon Corporation, model: SMZ800 )
  3. Coverslip (18 x 18 mm)
  4. Microscopic slide (26 x 76 mm)
  5. 1 ml insulin syringe with the 0.3 x 13 mm needle (BD)
  6. Needle point tweezers

Software

  1. NIH ImageJ software 1.47 for Windows (http://rsb.info.nih.gov/ij/)

Procedure

  1. Pistils isolation (Figure 1, A-E).
    Using a pair of tweezers, take flowers, at different developmental stages from an Arabidopsis inflorescence (Alvarez-Buylla et al., 2010). On a microscopic slide and under a dissecting microscope, remove sepals, petals, anthers and stigma using the needles of two 1 ml insulin syringes (0.3 x 13 mm) and make longitudinal superficial cuts on pistils at each side of the septum (Figure 1).
  2. Pistils stain (Figure 1, F-G).
    1. Submerge the pistils in 100 µl of a solution containing 10 µg/ml of JC-1 in buffer A.
    2. Incubate for 30 min at room temperature without shaking. Protect from light, as the dye is photosensitive.
    3. Gently wash the pistils three times with buffer A.
  3. Sample preparation for microscopy (Figure 1, H-L).
    1. On a microscopic slide under a dissecting microscope, use the needles of two 1 ml insulin syringes (0.3 x 13 mm) to dissect the pistils exposing the ovules
    2. Add a drop of buffer A and cover the sample with a coverslip.
    3. Immediately observe under a confocal microscope. The intensities of green (excitation/emission wavelength = 485/538 nm) and red (excitation/emission wavelength = 485/590 nm) are analyzed.


      Figure 1. Schematic illustration showing the steps required for embryo sac mitochondrial staining

  4. Image analysis (Power Point 1). The ratio of red to green fluorescence of JC-1 images is calculated using NIH ImageJ software.
    1. The outline of each embryo sac is delimited using the freehand tool to create a region of interest (ROI) and saved using the ROI manager tool (Analyze>tools>ROI manager>Add).
    2. In the “Analyze” menu, select “set measurements” and click on area and “mean gray value”.
    3. Using the image Menu, select “color” and then “split channels”.
    4. Close the image in the blue channel. On the green channel, go to the analyze>tools>ROI manager and select the ROI added before. The ROI will appear on the image. Still in the ROI manager go to more>multi Measure. The result will appear in a new window.
    5. Repeat this step with the image in the red channel.
    6. Copy the results and paste them in an excel worksheet.
    7. Calculate the red to green fluorescence ratio for each ROI.

Recipes

  1. Buffer A
    20 mM HEPES buffer (pH 7.2)
    For 1 L of 1 M HEPES buffer:
    Dissolve 238.3 g HEPES (free acid) in 500 ml of ddH2O
    Stir while adjusting the pH 7.2 with 0.5 N NaOH
    Bring up the volume to 1 L with ddH2O to prepare 1 L of 20 mM HEPES buffer (Buffer A)
    Add 20 ml of 1 M HEPES buffer in 980 ml of ddH2O

Acknowledgments

This protocol was adapted from Hauser et al. (2006). This work was supported by The Howard Hughes Medical Institute (HHMI), National Scientific and Technical Research Council (CONICET), National Agency for Promotion of Science and Technology (AGENCIA) and National University of Mar del Plata (UNMdP). We are grateful to the Editorial Committee of Bio-protocol for kindly inviting us to write this protocol.

References

  1. Alvarez-Buylla, E. R., Benitez, M., Corvera-Poire, A., Chaos Cador, A., de Folter, S., Gamboa de Buen, A., Garay-Arroyo, A., Garcia-Ponce, B., Jaimes-Miranda, F., Perez-Ruiz, R. V., Pineyro-Nelson, A. and Sanchez-Corrales, Y. E. (2010). Flower development. Arabidopsis Book 8: e0127.
  2. Hauser, B. A., Sun, K., Oppenheimer, D. G. and Sage, T. L. (2006). Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion. Planta 223(3): 492-499.
  3. Martin, M. V., Fiol, D. F., Sundaresan, V., Zabaleta, E. J. and Pagnussat, G. C. (2013). oiwa, a female gametophytic mutant impaired in a mitochondrial manganese-superoxide dismutase, reveals crucial roles for reactive oxygen species during embryo sac development and fertilization in Arabidopsis. Plant Cell 25(5): 1573-1591.

简介

该实验的目的是使用膜电位指示剂JC-1研究拟南芥胚胎囊中的线粒体功能状态。假定膜电位的变化是由于线粒体通透性转换孔(MPTP)的打开,允许离子和小分子通过。所产生的离子平衡又导致呼吸链的解耦和细胞色素c释放到细胞溶质中,这是程序性细胞死亡的早期阶段的显着特征.JC-1是一种亲脂性染料,其可以选择性地进入线粒体并随着膜电位增加可逆地将颜色从绿色改变为红色。在具有高线粒体电位的健康细胞中,JC-1自发形成具有强烈红色荧光的复合物。另一方面,在具有低线粒体电位的线粒体中,JC-1保持单体形式,其仅显示绿色荧光(Martin等人,2013; Hauser等人, em,2006)。
此协议可用于孤立的线粒体,以及各种细胞类型和植物和其他生物体的不同组织。

关键字:线粒体膜电位, 雌配子体, 拟南芥, 植物胚囊, JC-1

材料和试剂

  1. 花在不同发育阶段的拟南芥花序
  2. JC-1染料(Life Technologies,Molecular Probes ,目录号:T3168)
  3. DMSO 99.9%(Sigma-Aldrich,目录号:D8418)
  4. 储备溶液(10mg/ml的JC-1的DMSO溶液)
  5. 工作溶液(10μg/ml的JC-1在缓冲液A中)
  6. 缓冲液A(20mM HEPES缓冲液,pH7.2)(Sigma-Aldrich,目录号:H3375)(参见配方)

设备

  1. 共聚焦显微镜(Nikon Eclipse C1 Plus共焦显微镜,使用EZ-C1 3.80成像软件和Ti-Control)
  2. 解剖显微镜(尼康公司,型号:SMZ800)
  3. 盖片(18 x 18毫米)
  4. 显微镜载玻片(26×76mm)
  5. 1ml胰岛素注射器和0.3×13mm针头(BD)
  6. 针尖镊子

软件

  1. 用于Windows的NIH ImageJ软件1.47( http://rsb.info.nih.gov/ij/

程序

  1. 雌蕊隔离(图1,A-E) 使用一对镊子,在不同发育阶段从拟南芥花序中获取花(Alvarez-Buylla等人,2010)。 在显微镜载玻片上和在解剖显微镜下,使用两个1ml胰岛素注射器(0.3×13mm)的针去除萼片,花瓣,花药和柱头,并在隔膜的每一侧的雌蕊上进行纵向浅表切口(图1) 。
  2. 雌蕊染色(图1,F-G)。
    1. 将雌蕊浸在100μl含有10μg/ml JC-1的缓冲液A溶液中。
    2. 在室温下孵育30分钟,无振荡。 避光,因为染料是光敏的。
    3. 用缓冲液A轻轻洗涤雌蕊三次。
  3. 显微镜的样品制备(图1,H-L)。
    1. 在解剖显微镜下的显微镜下,使用的针   两个1ml胰岛素注射器(0.3×13mm)以解剖露出的雌蕊   胚珠
    2. 添加一滴缓冲液A,并用盖玻片覆盖样品
    3. 立即在共聚焦显微镜下观察。 强度 绿色(激发/发射波长= 485/538nm)和红色 (激发/发射波长= 485/590nm)

      图1.示意图显示胚囊线粒体染色所需的步骤

  4. 图片分析( Power Point 1 )。 使用NIH ImageJ软件计算JC-1图像的红色与绿色荧光的比率。
    1. 每个胚囊的轮廓使用徒手工具分隔 创建感兴趣区域(ROI)并使用ROI管理器工具保存 (分析>工具> ROI经理>添加)。
    2. 在"分析"菜单中,选择"设置测量",然后单击区域和"平均灰度值"
    3. 使用图像菜单,选择"颜色",然后选择"拆分频道"
    4. 关闭蓝色通道中的图像。 在绿色通道上,转到 分析>工具> ROI经理并选择之前添加的ROI。 投资回报率   将出现在图像上。 仍然在ROI经理去更多>多 测量。 结果将显示在新窗口中。
    5. 对红色通道中的图像重复此步骤。
    6. 复制结果并将其粘贴到excel工作表中。
    7. 计算每个ROI的红色到绿色荧光比率。

食谱

  1. 缓冲区A
    20mM HEPES缓冲液(pH7.2) 对于1升1 M HEPES缓冲液:
    将238.3g HEPES(游离酸)溶解在500ml ddH 2 O中。
    搅拌,同时用0.5N NaOH调节pH7.2 用ddH 2 O将体积调至1L,以制备1L的20mM HEPES缓冲液(缓冲液A)
    在980ml ddH 2 O中加入20ml 1M HEPES缓冲液

致谢

该方案改编自Hauser等人(2006)。 这项工作得到霍华德休斯医学院(HHMI),国家科学技术研究委员会(CONICET),国家科学技术促进局(AGENCIA)和马德普拉塔国立大学(UNMdP)的支持。 我们感谢生物协议编辑委员会友好地邀请我们编写这个协议。

参考文献

  1. Alvarez-Buylla,ER,Benitez,M.,Corvera-Poire,A.,Chaos Cador,A.,de Folter,S.,Gamboa de Buen,A.,Garay-Arroyo,A.,Garcia-Ponce, ,Jaimes-Miranda,F.,Perez-Ruiz,RV,Pineyro-Nelson,A.and Sanchez-Corrales,YE(2010)。 花卉开发。拟南芥书 8:e0127。
  2. Hauser,B.A.,Sun,K.,Oppenheimer,D.G。和Sage,T.L。(2006)。 线粒体膜电位的变化和活性氧簇的积累先于胚珠流产期间的超微结构变化。 223(3):492-499
  3. Martin,M.V.,Fiol,D.F.,Sundaresan,V.,Zabaleta,E.J.and Pagnussat,G.C。(2013)。 oiwa,一种线粒体锰超氧化物歧化酶中受损的女性配子体突变,揭示了活性氧的关键作用 植物细胞 25(5):1573-1591。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martin, M. V., Fiol, D. F., Zabaleta, E. J. and Pagnussat, G. C. (2014). Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain. Bio-protocol 4(10): e1128. DOI: 10.21769/BioProtoc.1128.
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