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Measurement of Mitochondrial Respiration Rate in Maize (Zea mays) Leaves
玉米叶线粒体呼吸率的测定   

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Abstract

Mitochondria play essential roles in plant growth and development as they host the oxidative phosphorylation pathways, tricarboxylic acid cycle and other important metabolisms. Disruption of mitochondrial functions frequently leads to embryo lethality. Moreover, mitochondria play roles in programmed cell death, pathogen and stress responses in plants. In contrast to animal mitochondria, plant mitochondria possess an additional electron transport pathway, the cyanide-resistant alternative pathway catalyzed by a single alternative oxidase (AOX). Unlike cytochrome pathway that is coupled to oxidative phosphorylation via proton translocation, electron transport from ubiquinol to AOX is non-phosphorylating. It releases the energy as heat. Chlorolab II liquid-phase oxygen electrode (Hansatech) is a high-precise Clark type oxygen electrode, which is equipped with the powerful WINDOWS software and could record the oxygen changes in real time. Its electrode disc comprises a central platinum cathode and a concentric silver anode. The electrode disc is connected to an electrode control unit which applies a small polarising voltage between the platinum and silver electrodes. In the presence of oxygen, a small current is generated proportional to oxygen content in the sample. It could respond sensitively and rapidly to small changes of oxygen content in the sample. This protocol describes how to measure the mitochondrial total respiration rate, cytochrome pathway capacity as well as alternative pathway capacity in maize leaves with chlorolab II oxygen electrode.

Keywords: Mitochondrial total respiration rate(线粒体总呼吸速率), Cytochrome pathway capacity(细胞色素途径容量), Alternative pathway capacity(替代途径的能力), Chlorolab II liquid-phase oxygen electrode(chlorolab II液相氧电极)

Materials and Reagents

  1. Leaves of maize seedlings
  2. Potassium cyanide (KCN) (Sigma-Aldrich, catalog number: 207810 )
  3. Salicylhydroxamic acid (SHAM) (Sigma-Aldrich, catalog number: S607 )
  4. Sodium dithionite (Sigma-Aldrich, catalog number: 71699 )
  5. Reaction medium buffer (see Recipes)
  6. 0.2 M KCN (see Recipes)
  7. 0.2 M SHAM (see Recipes)

Equipment

  1. Chlorolab II liquid-phase oxygen electrode (Hansatech)
  2. Thermostatic water bath
  3. Circulating water pump
  4. Microsyringe (50 μl)

Procedure

  1. The maize seedlings were cultured in potting soil under the growth conditions (at 22 ± 2 °C with a 16-h light / 8-h dark photoperiod and light density of 150 µmol/m2/s) for 2 weeks.
  2. Before measurement, the maize seedlings were kept in the dark for about 2 h in order to reduce the influence of photosynthesis.
  3. Open the thermostatic water bath and set its temperature to 25 °C; meanwhile, put the reaction medium buffer (about 200 ml) and circulating water pump into it.
  4. Clean the oxygen electrode: Dip a swab into the rapid electrode disc polish (provided with the equipment), and clean the anode and cathode of the electrode with it (Figure 1A); then wash the electrode with distilled water, and remove the surface water with an absorbent paper.


    Figure 1. Preparation of electrode disc. A. Clean the electrode disc. B. Install the electrode membrane. C. Install the electrode disc.

  5. Install the electrode membrane on the basis of instructions (Figure 1B):
    1. Place a small drop of electrolyte (the half saturation KCl) on top of the dome of the electrode disc (cathode);
    2. Place a piece of 1.5 cm wide and 3.5 cm long salt-bridge paper over the electrolyte, and ensure that at least one corner is in the electrode well to act as a wick;
    3. Cover this with a piece of electrode membrane (P. T. F. E membrane), which is selectively permeable to oxygen molecules;
    4. Place the small O-ring over the dome of the electrode disc with applicator tool;
    5. Add several drops of electrolyte into the electrode groove (anode).
      Notes: The electrode membrane surface should be smooth and free of air bubbles.
  6. Install the electrode disc (Figure 1C): Holding the electrode chamber vertically, place the electrode into it so that the membrane-covered dome forms the floor of the electrode chamber.
  7. The balance of oxygen electrode: Add 50 ml of distilled water into the beaker, and fully stir it for a few minutes in order to sufficiently dissolve O2; put 2 ml of the air-saturated distilled water and the magnetic follower in the reaction chamber and start the oxygen electrode; connect circulating water pump with it to keep the temperature constant in the reaction chamber. The oxygen electrode should be balanced for about 30 min before calibration (Figure 2A).
  8. The calibration of the oxygen electrode:
    1. Establish “air line” in the reaction chamber.
      Press successively “Calibrate” - “Oxygen” - “New” on the toolbar; then input the actual temperature and the atmospheric pressure in the calibration window and press “OK”; the oxygen concentration of distilled water in the chamber will be recorded in real time; when trace is stable, press “stop”.
    2. Establish “zero oxygen line” in the reaction chamber.
      When the screen displays “Establish zero oxygen in the chamber”, add the high concentration of sodium dithionite solution (prepared when using) into the reaction chamber with microsyringe, then press “OK”; at this time, the oxygen in distilled water will be consumed by sodium dithionite; When the oxygen concentration decreases nearly to zero and the trace is stable, the screen will display again “Establish air line in the chamber”; discard the solution in the reaction chamber and wash the reaction chamber, the cover and the magnetic follower with distilled water repeatedly; then add 2 ml of the air-saturated distilled water into the chamber, and press “OK”; when trace is stable, press “stop”. The calibration is completed.


      Figure 2. Respiration rate determination. A. Balance of oxygen electrode. B. Place the leaves into the reactive chamber and balance for 5 min. C. Put the electrode chamber cover and start recording respiration rate. D. Add the inhibitor with microsyringe in the process of determination.

  9. During measurement, about 0.15 g of maize seedling leaves were cut into the same size fragments (about 2 mm2), and O2 consumption rate was measured in the reaction medium buffer at 25 °C in the dark (Figure 2B).
  10. Measurement procedure: add 2 ml of the reaction medium buffer and leaves into the reaction chamber; balance for 5 min and then start recording O2 consumption for 2-3 min; stop recording and add 20 µl 0.2 M KCN with microsyringe into the reaction chamber, 1 min later, restart recording for 2-3 min; stop recording and add 20 µl 0.2 M SHAM, 2 min later, restart recording for 2-3 min. To avoid oxygen-limiting conditions inside the reaction chamber, all measurements were terminated before O2 reached about 50 to 60% of air saturation levels (Figure 2C and D).
  11. Respiration rate calculation: In the experiment, KCN and SHAM were used to inhibit the activity of cytochrome c oxidase and alternative oxidase, respectively. Mitochondrial total respiration (TP) was defined as O2 consumption rate in the absence of any additions. Residual respiration (RP) was defined as O2 consumption rate in the presence of 2 mM KCN and 2 mM SHAM. Cytochrome pathway capacity (CP) was defined as TP minus the values of O2 consumption rate in the presence of 2 mM KCN. Alternative pathway capacity was defined as TP minus both CP and RP (Figure 3).


    Figure 3. Calculation of the respiration rate. After the oxygen electrode was calibrated, the computer could display the actual O2 concentration in the reaction chamber in real time (Y-axis). Thus, the respiration rate could be calculated on the basis of the volume of reaction buffer in the reaction chamber, the changes of oxygen concentration, the reaction time and the fresh weight (FW) of leaves. The respiration rate can be expressed as µmol O2/min/g FW.

Recipes

  1. Reaction medium buffer
    10 mM HEPES
    10 mM MES
    2 mM CaCl2
    Adjust pH to 6.8 with KOH
  2. 0.2 M KCN
    Dissolved in distilled water
  3. 0.2 M SHAM
    Dissolved in dimethyl sulfoxide (DMSO)

Acknowledgments

This work was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region to B.-C.T. (Project no. 473512 and 473611), and a grant from the National Natural Science Foundation of China to B.-C.T. (Project No. 31170298).

References

  1. Li, X. J., Zhang, Y. F., Hou, M., Sun, F., Shen, Y., Xiu, Z. H., Wang, X., Chen, Z. L., Sun, S. S., Small, I. and Tan, B. C. (2014). Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa). Plant J 79(5): 797-809.

简介

线粒体在植物生长和发育中起重要作用,因为它们宿主氧化磷酸化途径,三羧酸循环和其他重要代谢。线粒体功能的破坏经常导致胚胎致死。此外,线粒体在程序性细胞死亡,病原体和植物应激反应中发挥作用。与动物线粒体相反,植物线粒体具有额外的电子传递途径,由单一备选氧化酶(AOX)催化的氰化物抗性替代途径。与通过质子易位与氧化磷酸化偶联的细胞色素途径不同,从泛素到AOX的电子转运是非磷酸化的。它释放能量作为热。 Chlorolab II液相氧电极(Hansatech)是一种高精度的Clark型氧电极,配有强大的WINDOWS软件,可以实时记录氧变化。其电极盘包括中心铂阴极和同心银阳极。电极盘连接到在铂和银电极之间施加小的极化电压的电极控制单元。在氧的存在下,产生与样品中的氧含量成比例的小电流。它可以敏感地和快速地对样品中氧含量的小变化作出响应。该协议描述如何测量线粒体总呼吸率,细胞色素通路能力以及具有chlorolab II氧电极的玉米叶中的替代途径能力。

关键字:线粒体总呼吸速率, 细胞色素途径容量, 替代途径的能力, chlorolab II液相氧电极

材料和试剂

  1. 玉米幼苗叶片
  2. 氰化钾(KCN)(Sigma-Aldrich,目录号:207810)
  3. 水杨酰羟肟酸(SHAM)(Sigma-Aldrich,目录号:S607)
  4. 连二亚硫酸钠(Sigma-Aldrich,目录号:71699)
  5. 反应介质缓冲液(参见配方)
  6. 0.2 M KCN(参见配方)
  7. 0.2 M SHAM(参见配方)

设备

  1. Chlorolab II液相氧电极(Hansatech)
  2. 恒温水浴
  3. 循环水泵
  4. 微量注射器(50μl)

程序

  1. 在生长条件下(在22±2℃,16小时光/8小时黑暗光周期和光密度150μmol/m 2/s)在盆栽土壤中培养玉米幼苗, 为期2周。
  2. 在测量之前,将玉米幼苗在黑暗中保持约2小时,以减少光合作用的影响。
  3. 打开恒温水浴,将其温度设置为25°C; 同时,将反应介质缓冲液(约200ml)和循环水泵放入其中
  4. 清洁氧电极:将棉签浸入快速电极盘抛光(随设备提供),并用其清洁电极的阳极和阴极(图1A);然后用蒸馏水清洗电极,并用吸水纸除去表面水

    图1.准备电极盘。 A.清洁电极盘。 B.安装电极膜。 C.安装电极盘。

  5. 根据说明安装电极膜(图1B):
    1. 将一小滴电解质(半饱和KCl)放在电极盘(阴极)的圆顶上;
    2. 放一块1.5厘米宽和3.5厘米长的盐桥纸 电解质,并确保至少一个角落在电极中  很好地作为灯芯;
    3. 用一片电极膜(P.T.F.E膜)覆盖,其对氧分子是选择性可渗透的;
    4. 将小O形环放置在电极盘的圆顶上,用施加器工具;
    5. 将几滴电解液加入电极槽(阳极)。
      注意:电极膜表面应平滑,无气泡。
  6. 安装电极盘(图1C):垂直握住电极室,将电极放入其中,使膜覆盖的圆顶形成电极室的底板。
  7. 氧电极的平衡:将50ml蒸馏水加入烧杯中,并充分搅拌几分钟以充分溶解O 2;将2ml空气饱和的蒸馏水和磁性随动物放入反应室中并启动氧电极;连接循环水泵,使反应室内的温度保持恒定。氧气电极应在校准前平衡约30分钟(图2A)
  8. 氧电极的校准:
    1. 在反应室中建立"空气管路"。
      连续按 "Calibrate" - "Oxygen" - "New"; 然后输入实际 温度和校准窗口中的大气压力 按"确定"; 腔室中蒸馏水的氧浓度 将实时记录; 当轨迹稳定时,按"停止"。
    2. 在反应室中建立"零氧气管线"。
      当屏幕显示"在腔室中建立零氧气"时,添加 高浓度的连二亚硫酸钠溶液(制备时 使用)用微量进样器注入反应室,然后按"确定"; 在 这一次,蒸馏水中的氧气将被钠消耗 连二亚硫酸盐 当氧浓度几乎降低到零时 轨迹稳定,屏幕会再次显示"建立空气管路 在房间";丢弃反应室中的溶液并洗涤 反应室,盖和磁性随动器蒸馏  水反复;然后加入2ml空气饱和的蒸馏水 进入腔室,然后按"确定";当轨迹稳定时,按"停止"。 校准完成。


      图2.呼吸频率 测定。 A.氧电极的平衡。 B.把叶子放进去 反应室和平衡5分钟。 C.放电极室  覆盖并开始记录呼吸率。 D.加入抑制剂 微量注射器在测定过程中

  9. 在测量期间,将约0.15g玉米幼苗叶切成相同大小的片段(约2mm 2),并在反应介质缓冲液中测量O 2消耗速率在25℃在黑暗中(图2B)。
  10. 测量程序:加入2ml反应介质缓冲液并离开反应室;平衡5分钟,然后开始记录O 2消耗2-3分钟;停止记录,并加入20μl0.2 M KCN与微量进样器的反应室,1分钟后,重新开始记录2-3分钟;停止记录并加入20μl0.2M SHAM,2分钟后,重新开始记录2-3分钟。为了避免反应室内的氧限制条件,在O 2达到空气饱和水平的约50至60%(图2C和D)之前终止所有测量。
  11. 呼吸速率计算:在实验中,KCN和SHAM分别用于抑制细胞色素氧化酶和替代氧化酶的活性。线粒体总呼吸(TP)定义为在没有任何添加的情况下O 2消耗率。剩余呼吸(RP)定义为在2mM KCN和2mM SHAM存在下的O 2消耗速率。细胞色素途径能力(CP)定义为TP减去在2mM KCN存在下O 2消耗速率的值。替代途径能力定义为TP减去CP和RP(图3)。


    图3.呼吸速率的计算。在校准氧电极后,计算机可以实时显示反应室中的实际O 2浓度(Y轴)。因此,呼吸速率可以基于反应室中反应缓冲液的体积,氧浓度的变化,反应时间和叶的鲜重(FW)计算。呼吸速率可以表示为μmolO 2次/min/g FW

食谱

  1. 反应介质缓冲液
    10 mM HEPES
    10 mM MES
    2mM CaCl 2 2 / 用KOH调节pH至6.8,
  2. 0.2 M KCN
    溶于蒸馏水中
  3. 0.2 M SHAM
    溶于二甲基亚砜(DMSO)中

确认

这项工作得到香港特别行政区研究资助局给予B.C.T.的资助。 (项目编号473512和473611),以及中国国家自然科学基金会给B.-C.T.的资助。 (项目编号31170298)。

参考文献

  1. 中国农业大学学报(自然科学版) )。 小核1编码线粒体nad7转录本编辑和玉米种子发育所需的pentatricopeptide重复蛋白(< 79(5):797-809。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X., Chang, N., Bi, Y. and Tan, B. (2015). Measurement of Mitochondrial Respiration Rate in Maize (Zea mays) Leaves. Bio-protocol 5(10): e1483. DOI: 10.21769/BioProtoc.1483.
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