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Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces
化学发光检测植物叶片中的氧化迸发   

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参见作者原研究论文

本实验方案简略版
The Plant Cell
May 2014

Abstract

The production of 'reactive oxygen species' (ROS), also termed oxidative burst, is a typical cellular response of animals and plants to diverse biotic and abiotic stresses. Here, we outline the detection of the ROS-burst in plant leaf pieces using a luminol-based bioassay which allows for the detection of chemiluminescence. The assay was originally described by Keppler et al. (1989) and subsequently adapted for other plant cells and tissues (Felix et al., 1999) and also used in recent publications (Albert et al., 2013; Albert et al., 2010; Butenko et al., 2014; Halter et al., 2014). In this protocol we outline a standardized version of this assay including remarks and recommendations for data evaluation and interpretation of results.

Materials and Reagents

  1. Plant leaf pieces (any plant of interest, e.g. Arabidopsis or N. benthamiana transiently expressing a construct of interest)
  2. Water
  3. 200 µM luminol L-012 (Wako Chemicals USA, catalog number: 075-05111 ; http://www.wako-chem.co.jp/english/labchem/journals/wpu_bio1/10.htm)
  4. 10 µg/ml peroxidase, horseradish peroxidase (AppliChem, catalog number: A3791 )
  5. MAMPs or any elicitors of interest
  6. Known MAMP as positive control (e.g. flg22, chitin-oligomers, etc.)
  7. 10x luminol master mix (see Recipes)

Equipment

  1. Scissors
  2. Petridishes
  3. Small spatula
  4. 96 well plates, white, flat-bottomed (e.g., LIA-plate, whit, 96-well, flat-bottom, Greiner Bio-One GmbH) or suitable cuvettes for single cell instruments
  5. Luminometer, either a 96 well plate reader (e.g., Centro LB 960, Microplate Luminometer, BERTHOLD TECHNOLOGIES) or a single cell instrument

Procedure

  1. Preparation of plant samples, one day before oxidative burst measurements
    1. Label petridishes appropriately and fill half with deionized water.
    2. Cut leaves (younger or highly differentiated) of plants [important: plants should not flower, and should not be too old (about 4-6 weeks)] with scissors and cut off the edge of the leaves with scissors.
    3. Cut the remaining part of the leaf in small, equally sized pieces (e.g. 3 x 3 mm, pieces should easily fit into the wells of a 96-well plate). It is recommended that you cut about 2x to 3x more leaf pieces than you need.
    4. Float leaf pieces on the water in the petridishes (orientation does not matter), avoid submersion!
    5. Cover the samples and incubate overnight (or > 8 h) at room temperature, avoid shaking.

  2. Preparation of samples prior to luminescence measurements, next day
    1. Depending on the luminometer (single cell or multi-well plate reader) 90 µl water + 10 µl luminol master mix (10x) are pipetted into each well of a new 96-well plate. For single cell luminometers this might be scaled up 2x to 3x, according to the size of the measurement cuvette.
    2. Carefully add one leaf piece per well, avoid mechanical damage and wounding! If the pieces are very small one could also use two per well. Use a small spatula, no forceps! Check if all leaf pieces are in the center of the well prior to starting the measurement.
    3. Per planned treatment prepare about 3-4 replicates (each in an extra well); do not forget negative (e.g. untreated) and positive controls; all leaf pieces should have the equal size!

  3. Luminometer measurements
    Luminometer settings (96-well reader): Measurement duration per well = 1 sec (could be shorter, depending on the limits of your machine); cycle time might be set according to the number of total samples or used wells and the duration of one measurement.
    Note: Some software/programs calculate the cycle time depending on the measurement duration and number of samples. Repetitions of measurement cycles/total time should be set to appropriately monitor signals for 20 min up to 60 min. It is recommended that in maximum only half of the plate is occupied with samples since the time difference between the measurement of the first and the last sample is too long. One round of measurement should be less than 60 sec. If you use a single cell luminometer you might just use the continuous mode or use a comparable program such as described above.
    1. Before the induction of a ROS-burst with MAMPs/elicitors, the background level has to be measured to ensure constant values of emitted light over time. Usually a measurement for about 5-10 min is sufficient to control for a stable non-oscillating baseline. If the baseline is not constant within this time the background measurement might be increased up to 60 min.
    2. Interrupt the background measurements and add your MAMP or any triggering substance (usually 1 µl) slightly shake plate horizontally on the table, and continue/re-start the measurement. The elicitors can be added by using an injector integrated in the machine, or by pipetting directly into the wells. Measurement cycles (30-60 sec, each) can be repeated to a total time of 20-60 min or longer if necessary.

Representative data

  1. Data handling and evaluation
    The monitored ROS-burst is measured as emitted light due to the oxidation of luminol and is given in RLU (relative light units). Since RLU is not a clearly defined unit it can vary from machine to machine. Thus it is difficult to compare the results obtained with different machines. The measurement of the oxidative burst over time results in a kinetics curve as it is shown in the exemplary graph of Figure 1. For experiments it is important to use negative and positive controls, and leaf pieces of different plants should have the same size! Replicates (n ≥ 3) with equally sized leaf pieces are always necessary to perform statistics.


    Figure 1. Schematic graph of a ROS-burst kinetics curve; ROS-burst was detected as emitted light in the luminol-based assay via a luminometer. Treatment of samples with a MAMP (e.g. flg22; 1 µl of a 10 µM stock solution; final concentration 100 nM) leads to a nice ROS-burst with a maximum peak at ~15 min. A lag-phase of 2-10 min is often observed. The negative control (e.g. water, BSA, etc.) does not cause a ROS-burst and the corresponding curve shows no peak.

    To get an idea about the sensitivity of a system for a certain MAMP-trigger it is indispensable to measure the oxidative burst in a MAMP-dose-dependent manner. An appropriate amount of samples is treated with different doses of a MAMP, and the ROS-burst is recorded as described above. The obtained kinetics curves (examples in Figure 2 left panel) can be processed to a classical dose-response curve as shown in Figure 2 (right panel). In this way the EC50 can be determined that directly indicates the sensitivity of a biological system for a treatment with any trigger.


    Figure 2. Dose-dependence of the ROS-burst response. Left panel: Samples were treated with different concentrations of a MAMP as indicated and ROS was recorded as emitted light over time. The highest concentrations (1,000 and 100 nM) give similar curves, indicating the system is saturated. The lower doses with concentrations of 10 and 1 nM result in curves with smaller amplitudes and a dose of 0.1 nM triggers no increase in ROS over the levels found in control treatment (water, 0 nM) Right panel: The maximum values from the left were plotted against the corresponding concentration and the graph shows a classical dose-response-curve. The EC50 of ~10 nM (half maximum effective concentration) was exemplary determined as indicated in the graph.

Recipes

  1. 10x luminol master mix
    200 µM luminol L-012
    10 µg/ml peroxidase, horseradish peroxidase

Acknowledgments

R.A.’s work was supported by Grant 348256/F20 from the Research Council of Norway; and Grant 216856 from the Research Council of Norway and the Deutscher Akademischer Austausch Dienst. M. A. was supported by the Deutsch Forschungsgemeinschaft (AL1426/1-1). The method was recently applied in Butenko et al. (2014).

References

  1. Albert, M., Jehle, A. K., Furst, U., Chinchilla, D., Boller, T. and Felix, G. (2013). A two-hybrid-receptor assay demonstrates heteromer formation as switch-on for plant immune receptors. Plant Physiol 163(4): 1504-1509.
  2. Albert, M., Jehle, A. K., Mueller, K., Eisele, C., Lipschis, M. and Felix, G. (2010). Arabidopsis thaliana pattern recognition receptors for bacterial elongation factor Tu and flagellin can be combined to form functional chimeric receptors. J Biol Chem 285(25): 19035-19042.
  3. Butenko, M. A., Wildhagen, M., Albert, M., Jehle, A., Kalbacher, H., Aalen, R. B. and Felix, G. (2014). Tools and strategies to match peptide-ligand receptor pairs. Plant Cell 26(5): 1838-1847.
  4. Felix, G., Duran, J. D., Volko, S. and Boller, T. (1999). Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3): 265-276.
  5. Halter, T., Imkampe, J., Mazzotta, S., Wierzba, M., Postel, S., Bücherl, C., Kiefer, C., Stahl, M., Chinchilla, D. and Wang, X. (2014). The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity. Curr Biol 24(2): 134-143.
  6. Keppler, L. D., Baker, C. J. and Atkinson, M. M. (1989). Active oxygen production during a bacteria-induced hypersensitive reaction in tobacco suspension cells. Phytopathology 79(9): 974-978.

简介

"活性氧"(ROS)的产生,也称为氧化爆发,是动物和植物对多种生物和非生物胁迫的典型细胞应答。 在这里,我们概述使用基于鲁米诺的生物测定法允许检测化学发光的植物叶片中的ROS突发的检测。 该测定最初由Keppler等人(1989)描述,随后适用于其他植物细胞和组织(Felix等人,1999),并且也用于最近的出版物 (Albert et al。,2013; Albert et al。,2010; Butenko et al。,2014; Halter et al。 ,2014)。 在本协议中,我们概述了该测定的标准化版本,包括对数据评估和结果解释的评论和建议。

材料和试剂

  1. 植物叶片(任何感兴趣的植物,例如瞬时表达感兴趣的构建体的拟南芥或本生烟草)

  2. 200μM鲁米诺L-012(Wako Chemicals USA,目录号:075-05111; http://www.wako-chem.co.jp/english/labchem/journals/wpu_bio1/10.htm
  3. 10μg/ml过氧化物酶,辣根过氧化物酶(AppliChem,目录号:A3791)
  4. MAMP或任何感兴趣的激励者
  5. 已知的MAMP作为阳性对照(例如 flg22,几丁质寡聚体,等)
  6. 10x鲁米诺主混合物(见配方)

设备

  1. 剪刀
  2. 培训
  3. 小铲
  4. 96孔板,白色平底(例如,LIA-板,惠特,96孔,平底,Greiner Bio-One GmbH)或适用于单细胞仪器的试管
  5. 发光计,96孔板读数器(例如Centro LB 960,微孔板发光计,BERTHOLD TECHNOLOGIES)或单细胞仪器

程序

  1. 准备植物样品,在氧化爆发测量前一天
    1. 标签适当地铺在一起,用去离子水填满一半
    2. 切叶(更年轻或高度分化)的植物[重要: 植物不应该花,不应太老(约4-6周)] 用剪刀和剪刀切掉叶子的边缘
    3. 将小叶的剩余部分切成小块,大小相同(例如 3   ×3mm,片应当容易地装配到96孔板的孔中)。 它   建议你剪切比你多大约2到3倍的叶片 需要。
    4. 浮石叶片在水中的培养皿(方向无关紧要),避免浸入!
    5. 覆盖样品并在室温下孵育过夜(或> 8小时),避免摇动。

  2. 在发光测量之前制备样品,第二天
    1. 根据光度计(单细胞或多孔板读数器)90   μl水+10μl鲁米诺主混合物(10x)移液到每个孔中 的新的96孔板。 对于单电池发光计,这可能是 根据测量比色杯的大小放大2倍至3倍。
    2. 小心地每孔添加一片叶片,避免机械损坏和 伤人! 如果碎片非常小,也可以每孔使用两个。 使用小铲,没有镊子! 检查是否所有叶片都在 中心,然后开始测量
    3. 每计划   处理准备约3-4次重复(每次在额外的孔中); 不要 忘记阴性(例如未治疗)和阳性对照;所有叶片 应该有相同的大小!

  3. 光度计测量
    光度计设置(96孔读数器):每孔测量持续时间= 1秒(可能更短,取决于您的机器的限制);循环时间可以根据总样品或使用的孔的数量以及一次测量的持续时间来设定。
    注意:一些软件/程序根据测量持续时间和样品数量计算周期时间。重复测量周期/总时间应设置为适当监测信号20分钟至60分钟。建议最多只有一半的板被样品占用,因为第一个样品和最后一个样品的测量之间的时间差太长。一轮测量应小于60秒。如果您使用单电池光度计,您可能只是使用连续模式或使用类似的程序,如上所述。
    1. 在用MAMP /诱导子诱导ROS突发之前,背景  必须测量电平以确保发射光的恒定值 随着时间的推移。通常测量约5-10分钟就足够了 控制稳定的非振荡基线。如果基线不是 常数在这个时间内背景测量可能会增加 最多60分钟。
    2. 中断背景测量并添加您的 MAMP或任何触发物质(通常1μl)轻微摇动平板 水平地放在工作台上,并继续/重新开始测量。 的 可以通过使用集成在机器中的注射器来添加引出器, 或通过直接移液到孔中。 测量周期(30-60秒, 每个)可以重复到总时间20-60分钟或更长时间 必要。

代表数据

  1. 数据处理和评估
    由于鲁米诺的氧化,监测的ROS突发被测量为发射光,并且以RLU(相对光单位)给出。由于RLU不是一个明确定义的单位,它可以随机器而变化。因此,难以比较用不同机器获得的结果。氧化爆发随时间的测量导致动力学曲线,如图1的示例性曲线图所示。对于实验,重要的是使用阴性和阳性对照,不同植物的叶片应具有相同的尺寸。重复(n≥3)具有相同大小的叶片总是需要执行统计

    图1. ROS突发动力学曲线的示意图;通过发光计在基于发光氨的测定中检测ROS-爆发作为发射光。用MAMP(例如flg22)处理样品;1μl10μM储备溶液;最终浓度100 nM)导致良好的ROS爆发,在〜15分钟具有最大峰。通常观察到2-10分钟的滞后期。阴性对照(例如水,BSA,等)不引起ROS突发,并且相应的曲线不显示峰。

    为了获得关于系统对于某一MAMP-触发的灵敏度的想法,以MAMP-剂量依赖性方式测量氧化爆发是不可缺少的。用不同剂量的MAMP处理适当量的样品,并如上所述记录ROS脉冲。获得的动力学曲线(图2左图中的实施例)可以加工成如图2所示的经典剂量 - 反应曲线(右图)。以这种方式,可以确定EC 50 ,其直接指示生物系统对于具有任何触发的治疗的灵敏度。


    图2.ROS-爆发响应的剂量依赖性。左图:用所示的不同浓度的MAMP处理样品,随着时间记录ROS作为发射光。最高浓度(1,000和100nM)给出类似的曲线,表明系统是饱和的。浓度为10和1nM的较低剂量产生具有较小幅度的曲线,并且0.1nM的剂量不会引起ROS增加超过对照处理中发现的水平(水,0nM)。右图:左边的最大值相对于相应的浓度绘图,并且该图显示了经典的剂量 - 反应曲线。 〜10nM(半数最大值)的EC 50 有效浓度)是如图所示的示例性测定

食谱

  1. 10x鲁米诺主混合物
    200μM鲁米诺L-012
    10μg/ml过氧化物酶,辣根过氧化物酶

致谢

挪威研究委员会的Grant 348256/F20支持R.A.的工作; 和来自挪威研究委员会和Deutscher Akademischer Austausch Dienst的Grant 216856。 M. A.由Deutsch Forschungsgemeinschaft(AL1426/1-1)支持。 该方法最近应用于Butenko等人(2014)。 有效浓度)是如图所示的示例性测定

食谱

  1. 10x鲁米诺主混合物
    200μM鲁米诺L-012
    10μg/ml过氧化物酶,辣根过氧化物酶

致谢

挪威研究委员会的Grant 348256/F20支持R.A.的工作; 和来自挪威研究委员会和Deutscher Akademischer Austausch Dienst的Grant 216856。 M. A.由Deutsch Forschungsgemeinschaft(AL1426/1-1)支持。 该方法最近应用于Butenko等人(2014)。... 有效浓度)是如图所示的示例性测定

食谱

  1. 10x鲁米诺主混合物
    200μM鲁米诺L-012
    10μg/ml过氧化物酶,辣根过氧化物酶

致谢

挪威研究委员会的Grant 348256/F20支持R.A.的工作; 和来自挪威研究委员会和Deutscher Akademischer Austausch Dienst的Grant 216856。 M. A.由Deutsch Forschungsgemeinschaft(AL1426/1-1)支持。 该方法最近应用于Butenko等人(2014)。......

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Albert, M., Butenko, M. A., Aalen, R. B., Felix, G. and Wildhagen, M. (2015). Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces. Bio-protocol 5(6): e1423. DOI: 10.21769/BioProtoc.1423.
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sridivya vungarala
centre for cellular and molecular biology
what is the reading of a well without any leaf disc in luminol solution.
5/12/2015 1:03:49 AM Reply
Markus Albert
Center for Plant Molecular Biology, University of Tübingen, Germany

This has to be compared individually!
RLU (relative light units) are not properly defined and vary depending on the individual machine. Thus, one have to set up a few wells in each individual experiment without leaf discs.

5/12/2015 1:11:53 AM


sridivya vungarala
centre for cellular and molecular biology

Wwhat is the reading(range in RLU) when u add just luminol (as a blank i.e, without any sample) in chemiluminescence assay.

6/11/2015 7:24:03 AM