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Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves
鲁米诺反应检测拟南芥叶片中免疫激发产生的过氧化氢   

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Abstract

In Arabidopsis thaliana, one of the very early immune-related responses induced after elicitor perception is the oxidative burst, i.e., reactive oxygen species (ROS) generation including superoxide anion and hydrogen peroxide (H2O2). ROS production plays different roles in a wide range of biotic and abiotic stress responses, including the closure of stomata and the regulation of cell expansion. In particular, elicitor-induced H2O2 is produced mainly by the membrane localized NAD(P)H oxidases RESPIRATORY BURST OXIDASE HOMOLOGUE D and F. In this protocol, we describe a simple and reproducible luminol/peroxidase-based assay to detect and evaluate immunity-related accumulation of H2O2 produced in Arabidopsis leaf discs treated with immunity elicitors, such as oligogalacturonides (OGs), flagellin (flg22) or the elongation factor-thermo-unstable (EF-Tu - elf18). This method is based on the detection of the luminescence released by excited-luminol molecules generated after the horseradish peroxidase (HRP)-catalyzed oxidation of luminol molecules in the presence of H2O2. Levels as well as duration of the luminescence are proportional to the amount of H2O2 produced by elicited leaf discs.

Materials and Reagents

  1. Petri plates (90 x 15 mm)
  2. 96-well luminometer plate (Thermo Fisher Scientific, catalog number: 136101 )
  3. Aluminum foil
  4. At least two Arabidopsis thaliana (e.g., ecotype Columbia-0) plants for each elicitor treatment
  5. Luminol (Sigma-Aldrich, catalog number: 123072 )
  6. Peroxidase from horseradish (Sigma-Aldrich, catalog number: P8125 )
  7. Sterile distilled water
  8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 41640 )
  9. Flg22 (QRLSTGSRINSAKDDAAGLQIA) and elf18 (ac-SKEKFERTKPHVNVGTIG)-EZBiolab (http://www.ezbiolab.com/)
  10. OGs with an average degree of polymerization of 10 to 16 prepared as previously described (Bellincampi et al., 2000)
  11. 500x luminol stock solution (LSS) (see Recipes)
  12. 500x horseradish peroxidase stock solution (HPSS) (see Recipes)
  13. 1 mM flg22 stock solution (see Recipes)
  14. 1 mM elf18 stock solution (see Recipes)

Equipment

  1. Arabidopsis growth chamber
  2. Surgical blade
  3. Cork borer set (0.125 cm2 area) (Sigma-Aldrich, catalog number: Z165220 )
  4. Plastic bur block
  5. Flat tweezer
  6. GloMax®-Multi+ Detection System with dual injectors (Promega Corporation)
  7. Microsart® e.jet laboratory vacuum pump (Sartorius AG, catalog number: 16612 )
  8. Plastic or glass desiccator

Procedure

  1. Preparation of plant material
    Note: At least 12 leaf discs are used for each elicitor treatment. Collect at least 3-4 leaves from each plant considering that, depending on leaf size, 2-3 discs can be obtained. In the case of OG treatment four additional discs are required as a negative control (see below).
    1. Grow Arabidopsis plants in a growth chamber until pre-bolting stage (4-5 weeks) at optimum condition (22 °C, 70% relative humidity, and a photoperiod of 12/12 h light/dark).
    2. Select and harvest appropriate leaves (third to fifth from the top) from at least two different plants, by cutting them from the petiole with a surgical blade. Gently wash harvested leaves with sterile distilled water (SDW).
      Note: Appropriate leaves are defined as leaves from plants that i) do not have different height and age, due to non-synchronous germination; ii) are not wounded; iii) do not show any disease symptoms caused by the presence of soil fungi and/or bacteria.
    3. To produce leaf discs, place the leaf above the bur block with the midrib in the correspondence of one of the holes. Punch the leaf with the cork borer positioned exactly above the hole (see Figure 1A-B).
    4. Collect and recover leaf discs for 2 h in a Petri dish containing abundant SDW (25 ml) in dark condition, by covering the plate with aluminum foil. Replace the SDW every 30 min (see Figure 1C).
      Note: At this point of the procedure the leaf discs do not need to be completely immersed in water. However, it is important that they are well in contact with the aqueous medium to prevent dehydration and to remove secondary metabolites produced upon wounding.
    5. Gently place, by using a flat tweezer, one leaf disc per well in a 96-well luminometer plate each containing 200 µl SDW (see Figure 1D).
    6. Cover the plate with aluminum foil and incubate in the growth chamber (see step A1 for specifications) overnight.


      Figure 1. Preparation of plant material. Arabidopsis leaf A is punched at the level of the midrib by using a bur block and a cork borer B. The resulting leaf discs are rinsed with 25 ml of SDW in Petri dish for 2 h C, replacing the SDW every 30 min. The leaf discs are then placed in a 96-well luminometer plate, 1 disc/1 well containing 200 µl SDW D.

  2. Detection of flg22- or elf18-triggered H2O2 production
    1. Prepare stock solutions (500x) both for luminol (LSS) and horseradish peroxidase (HPSS) in SDW. Aliquots of both LSS an HPSS can be stored at -20 °C for further uses.
    2. Prepare a luminol/peroxidase working solution (1.25x each) in SDW; consider that you will need 200 µl of this solution for each well in the analysis.
      Example: For 12 wells mix 6 µl LSS with 6 µl HPSS up to 2,400 µl with SDW. After use, discard the solution.
    3. Prepare 1 mM flg22 or elf18 stock solution (FSS or ESS) in SDW. Stock solution can be aliquoted and stored at -20 °C for further uses.
    4. Prepare a 5x working solution containing flg22 or elf18 (FWS or EWS) in SDW; consider that you will need 50 µl per well and 1 ml of dead volume to charge the injector. The elicitor final concentration may be comprised between 0.1 µM and 1 µM.
      Example: For 12 wells mix 0.8 µl FSS or ESS up to 1600 µl with SDW to obtain 0.5 µM FWS or EWS. After use discard the solution.
    5. Gently replace the 200 µl of water with an equal volume of luminol/peroxidase working solution in each well containing leaf disc.
      Critical step: Avoid leaf discs injuries; make sure to completely submerge the leaf discs in the luminol/peroxidase solution prior elicitor injection.
      Note: Since leaf discs have hydrophobic surfaces gently use a pipette tip, to fully immerse both sides of the leaf disc in the solution.
    6. Manually place the plate in the GloMax®-Multi+ Detection System (see Figure 2). Charge one of the injectors with the 5x elicitor solution and aliquot 50 µl per well.
    7. Perform the luminescence detection assay for at least 40 min with a signal integration time of 1 sec.
      Note: The indicated time only refers to the reading of the plate, which is done in stationary stage and in automated way by the detection system (for the manufacturer’s instructions as well as for a video about how to use the GloMax®-Multi+ Detection System refer to the link in the legend of Figure 2).
    8. Express luminescence in relative light units (RLU) (Figure 3A-B).
      Note: RLU represents raw data.

  3. Detection of OG-triggered H2O2 production
    Note: Compared to flg22 and elf18, OGs are larger negatively charged molecules. These characteristics may affect their diffusion and entry into the plant tissue generating inhomogeneous responses between treated leaf discs. To standardize the response it is necessary to vacuum infiltrate OGs into the leaf discs. Any ROS production caused by the vacuum infiltration step is assessed by the leaf discs infiltrated with water, as a control.
    1. Prepare a luminol/peroxidase working solution (5x each); considering that you will need 50 µl per well and 1 ml of dead volume to charge the injector.
      Example: For 12 wells mix 16 µl LSS with 16 µl HPSS and SDW up to 1,600 µl.
    2. Prepare a 1.25x working solution containing OGs (OWS) in SDW; consider that you will need 200 µl per well. The final concentration of the OG solution may be comprised between 200 or 300 µg/ml.
      Example: For 12 wells dissolve 0.6 mg of OGs in 1,600 µl SDW to obtain 250 µg/ml OWS. After use, discard the solution.
    3. Gently and rapidly replace the 200 µl of water with an equal volume of OG-containing solution in each well containing leaf disc. Use 4 discs as negative control by replacing the water with the same SDW used for the OG suspension.
      Note: It is not recommended to analyze more than 2 genotypes per plate, since the time required to manually replace the water with the OG solution may be too long and elicitation may occur in the firstly treated discs. In this regard it is recommended to alternate wells containing wild-type and mutant (if in analysis) discs during water replacing with the OG suspension.
      Critical step: Make sure to completely submerge leaf discs in the OG solution (see step B5) prior vacuum infiltration.
    4. Immediately vacuum infiltrate the leaf discs with the OG solution or SDW for 2 min, by using a desiccator connected to a vacuum pump.
      Note: Make sure to charge the injector with the luminol/peroxidase working solution (5x each) before or during the vacuum infiltration.
    5. Rapidly manually place the plate in the GloMax®-Multi+ Detection System. Inject 50 µl of the luminol/peroxidase working solution per well.
    6. Perform the luminescence detection assay for at least 40 min with a signal integration time of 1 sec.
    7. Express luminescence in RLU (Figure 3C).
      Note: See step B7.

Representative data


Figure 2. GloMax®-Multi+ Detection System with dual injectors. For a video about how to use the GloMax click on the following link: https://ita.promega.com/resources/multimedia/instruments/features-glomax-multi/. For the manufacturer’s manual click on the following link: https://www.promega.com/~/media/files/resources/protocols/technical manuals/0/glomax-multi detection system protocol.pdf.


Figure 3. ROS production in elicitors-treated Arabidopsis leaf discs. The H2O2 production was measured in Col-0 ecotype with the luminol-based assay after treatments with flg22 (100 nM) A), elf18 (100 nM) B), OGs (200 µg/ml) C). Results are average ± SE [n = 12 for elicitor treatments; n = 4 for water treatment (see Figure 3C)].

Recipes

  1. 500x luminol stock solution (LSS)
    Dissolve 15 mg of luminol in 1 ml of DMSO, by vortexing for 10-20 sec until the solution becomes uniformly light green. After use, aliquots can be stored at -20 °C up to 3 months.
    Note: Solutions are extremely sensitive to light.
  2. 500x horseradish peroxidase stock solution (HPSS)
    Dissolve 10 mg of peroxidase in 1 ml of water, by vortexing for 10-20 sec until the solution becomes uniformly brown. After use, aliquots can be stored at -20 °C up to 3 months.
  3. 1 mM flg22 stock solution (FSS - flg22 Molecular Weight: 2272.52)
    Dissolve 2.27 mg of flg22 in 1 ml of SDW, by vortexing for 10-20 sec until the solution becomes transparent.
    After use, aliquots can be stored at -20 °C for 4-6 months.
    Prepare the flg22 5x working solution (FWS) by diluting the FSS with SDW.
    After use, discard the solution.
  4. 1 mM elf18 stock solution (ESS - elf18 Molecular Weight: 2069.37)
    Dissolve 2.07 mg of elf18 in 1 ml of SDW, by vortexing for 10-20 sec until the solution becomes transparent.
    After use, aliquot and store at -20 °C for 4-6 months.
    Prepare the elf18 5x working solution (EWS) by diluting the ESF with SDW.
    After use, discard the solution.

References

  1. Bellincampi, D., Dipierro, N., Salvi, G., Cervone, F. and De Lorenzo, G. (2000). Extracellular H(2)O(2) induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiol 122(4): 1379-1385.

简介

在拟南芥中,在诱导剂感知之后诱导的非常早期的免疫相关反应之一是氧化爆发,即包括超氧阴离子和过氧化氢的活性氧(ROS)产生(H 2 O 2 O 2)。 ROS产生在宽范围的生物和非生物应激反应中起不同的作用,包括气孔的闭合和细胞扩增的调节。特别地,诱导剂诱导的H 2 O 2 O 2主要由膜定位的NAD(P)H氧化酶呼吸道刺激氧化酶同源物D和F产生。在该方案中,我们描述了简单和可再现的基于鲁米诺/过氧化物酶的测定,以检测和评价在拟南芥叶盘中产生的H 2 O 2亚型的免疫相关累积(OGs),鞭毛蛋白(flg22)或延伸因子 - 热不稳定的(EF-Tu-elf18)处理。该方法基于检测在H 2 O 2 O 2存在下在辣根过氧化物酶(HRP) - 催化的鲁米诺分子氧化后产生的激发发光氨分子发出的发光, sub>。发光的水平以及持续时间与由引发的叶盘产生的H 2 O 2 O 2的量成比例。

材料和试剂

  1. 培养皿(90×15mm)
  2. 96孔光度计板(Thermo Fisher Scientific,目录号:136101)
  3. 铝箔
  4. 每个诱导剂治疗至少两个拟南芥(例如生态型哥伦比亚-0)植物
  5. 鲁米诺(Sigma-Aldrich,目录号:123072)
  6. 来自辣根的过氧化物酶(Sigma-Aldrich,目录号:P8125)
  7. 无菌蒸馏水
  8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:41640)
  9. Flg22(QRLSTGSRINSAKDDAAGLQIA)和elf18(ac-SKEKFERTKPHVNVGTIG)-EZBiolab(http://www.ezbiolab.com/)
  10. 如先前所述(Bellincampi等人,2000)制备的平均聚合度为10至16的OGs。
  11. 500x鲁米诺储备液(LSS)(参见配方)
  12. 500x辣根过氧化物酶储备液(HPSS)(见配方)
  13. 1 mM flg22储备溶液(见配方)
  14. 1 mM elf18储备溶液(见配方)

设备

  1. 拟南芥生长室
  2. 外科刀片
  3. 软木钻孔器(0.125cm 2面积)(Sigma-Aldrich,目录号:Z165220)
  4. 塑料块
  5. 平镊子
  6. GloMax ? -Multi +检测系统(Promega公司)
  7. Microsart e.jet实验室真空泵(Sartorius AG,??目录号:16612)
  8. 塑料或玻璃干燥器

程序

  1. 植物材料的制备
    注意:每个诱导剂治疗使用至少12片叶片。从每株植物收集至少3-4叶,考虑到,根据叶的大小,可以获得2-3张光盘。在OG处理的情况下,需要四个附加的盘作为阴性对照(见下文)。
    1. 在生长室中生长拟南芥植物,直到预先固定阶段(4-5 ?周),在最佳条件(22℃,70%相对湿度,和a 12/12小时光/暗的光周期)
    2. 选择和收获 适当的叶子(从顶部第三到第五)从至少两个 不同的植物,通过从手术切除叶柄 刀。用无菌蒸馏水(SDW)轻轻洗涤收获的叶子。
      注意:适当的叶片被定义为来自植物的叶子 不具有不同的高度和年龄,由于非同步发芽; ii)没有受伤; iii)不显示由所引起的任何疾病症状 ?土壤真菌和/或细菌的存在。
    3. 为了生产叶盘, 把叶子放在上面的bur块与midrib在通信 ?的一个孔。打开叶子与软木钻孔器定位 正好在孔的上方(见图1A-B)。
    4. 收集和恢复 叶盘在含有丰富SDW(25ml)的培养皿中培养2小时 黑暗条件,通过用铝箔覆盖板。更换 SDW每30分钟(见图1C)。
      注意:在这一点上 叶片不需要完全浸入水中。 ?然而,重要的是它们与水相良好接触 ?介质以防止脱水和除去次级代谢物 受伤后产生。
    5. 轻轻放置,用平镊子, 在96孔光度计板中每孔含有一个叶片 200μlSDW(见图1D)。
    6. 用铝箔覆盖板,并在生长室中孵育(参见步骤A1的规格)过夜。


      图1.植物材料的制备。拟南芥叶A 在中间的水平通过使用块塞和软木钻斧B. 将所得叶盘用培养皿中的25ml SDW漂洗2小时 C,每30分钟更换SDW。然后将叶圆片放入 96孔光度计板,含有200μlSDW D的1个盘/1个孔。

  2. 检测flg22-或elf18-触发的H 2 2 O 2亚型生产物
    1. 在SDW中制备鲁米诺(LSS)和辣根过氧化物酶(HPSS)的储备溶液(500x)。 两种LSS和HPSS的等分试样可以储存在-20℃用于进一步使用
    2. 在SDW中制备鲁米诺/过氧化物酶工作溶液(各1.25x) 考虑你将需要200微升的这个解决方案为每个井 ?分析。
      例如:对于12个孔,使用SDW将6μlLSS与6μlHPSS混合至2,400μl。使用后,丢弃溶液。
    3. 在SDW中制备1 mM flg22或elf18储备溶液(FSS或ESS)。原液可以等分并在-20℃下保存 ?进一步使用。
    4. 准备一个5x工作溶液含flg22或 ?elf18(FWS或EWS);考虑你将需要每孔50μl 和1ml死体积以对注射器充气。诱导者决赛 浓度可以在0.1μM和1μM之间。
      示例:对于12 孔混合0.8μlFSS或ESS,最高达1600μl,使用SDW获得0.5μMFWS 或者EWS。使用后丢弃溶液。
    5. 轻轻更换200 μl的水与等体积的鲁米诺/过氧化物酶工作溶液 在每个含有叶盘的孔中。
      关键步骤:避免叶片 伤害确保将叶片完全浸没在中 鲁米诺/过氧化物酶溶液。
      注意:自 叶盘有疏水表面轻轻地使用移液器吸头,以完全 将叶盘的两侧浸入溶液中。
    6. 手动 将板置于GloMax -Multi + Detection System(参见图2)。 用5x引发剂溶液和等分试样50加入一个注射器 ?μl/孔。
    7. 执行发光检测分析至少40分钟,信号积分时间为1秒。
      注意:指示的时间仅指板的读数,即 ?在固定阶段和通过检测以自动化方式进行 系统(对于制造商的说明以及视频的 ?如何使用GloMax ? -Multi + Detection System参考中的链接 ?图2的图例)。
    8. 以相对光单位(RLU)表示发光(图3A-B)。
      注意:RLU表示原始数据。

  3. 检测OG-触发的H sub 2 O 2子产生
    注意:与flg22和elf18相比,OGs是较大的带负电荷的分子。这些特征可能影响它们扩散和进入植物组织,在处理的叶片之间产生不均匀的反应。为了标准化响应,有必要将OGs真空渗入叶盘中。作为对照,通过用水浸润的叶盘评价由真空渗透步骤引起的任何ROS产生。
    1. 准备鲁米诺/过氧化物酶工作溶液(每次5x);考虑 你将需要50微升每孔和1毫升死体积充电 注射器。
      例如:对于12个孔,混合16μl具有16μlHPSS的LSS和最多1,600μl的SDW。
    2. 在SDW中制备含有OGs(OWS)的1.25x工作溶液; 考虑你将需要每孔200μl。最终浓度 OG溶液可以包含在200或300μg/ml之间。
      实施例:对于12个孔,在1,600μlSDW中溶解0.6mg OG以获得250μg/ml OWS。使用后,舍弃解决方案。
    3. 轻轻快速更换等体积的200μl水 的含有叶片的每个孔中的含OG的溶液。使用4张光盘 ?作为阴性对照,用用于的相同SDW替换水 OG悬浮液。
      注意:不建议分析 ?2基因型每板,因为手动更换所需的时间 水与OG溶液可能太长,并且可能发生引起 首先处理的盘。在这方面,建议交替 ?含有野生型和突变体(如果在分析中)盘的孔 水代替OG悬浮液。
      关键步骤:在真空渗透之前,确保将叶圆片完全浸没在OG溶液中(参见步骤B5)。
    4. 立即用OG溶液真空渗入叶盘 ?SDW 2分钟,通过使用连接到真空泵的干燥器。
      注意:确保用鲁米诺/过氧化物酶对注射器充电 工作溶液(每次5次),在真空渗透之前或期间。
    5. 快速手动将板放在GloMax ? -Multi + Detection中 系统。注射50微升鲁米诺/过氧化物酶工作溶液 好。
    6. 执行发光检测测定至少40分钟,信号积分时间为1秒。
    7. 在RLU中的快速发光(图3C)。
      注意:请参阅步骤B7。

代表数据


图2. GloMax ? -Multi + Detection System with dual injectors。有关如何使用GloMax点击以下链接: https://ita.promega.com/资源/多媒体/仪器/功能 - glomax-multi /。对于制造商的手册,点击以下链接: https://www.promega.com/~/media/files/resources/protocols/technical手册/0/glomax-multi检测系统协议.pdf。

A)

B)

C)

图3.在诱导子处理的拟南芥叶片中的ROS产生。测量H 2 O 2 O 2亚基产生。 (100nM)A),elf18(100nM)B),OGs(200μg/ml)C)处理后,用基于发光氨的测定测定Col-0生态型。结果是平均值±SE [对于诱导剂治疗n = 12; n = 4用于水处理(参见图3C)]。

食谱

  1. 500x鲁米诺储备液(LSS)
    通过涡旋10-20秒溶解15mg鲁米诺在1ml的DMSO中,直到溶液变得均匀的浅绿色。使用后,等分试样可以储存在-20°C至3个月。
    注意:解决方案对光非常敏感。
  2. 500x辣根过氧化物酶储备液(HPSS)
    通过涡旋10-20秒溶解10毫克过氧化物酶在1毫升水中,直到溶液变得均匀的棕色。使用后,等分试样可以储存在-20°C至3个月。
  3. 1mM flg22储备液(FSS-flg22分子量:2272.52)
    通过涡旋10-20秒溶解2.27mg flg22在1ml SDW中直到溶液变得透明。
    使用后,可将等分试样在-20℃下储存4-6个月。
    通过用SDW稀释FSS制备flg22 5x工作溶液(FWS)。
    使用后,丢弃溶液。
  4. 1mM elf18储备溶液(ESS-elf18分子量:2069.37) 通过涡旋10-20秒,溶解2.07mg的elf18在1ml的SDW中直到溶液变得透明。
    使用后,等分并在-20°C下储存4-6个月。
    通过用SDW稀释ESF制备elf18 5x工作溶液(EWS)。
    使用后,丢弃溶液。

参考文献

  1. Bellincampi,D.,Dipierro,N.,Salvi,G.,Cervone,F.and De Lorenzo,G。(2000)。 由寡聚半乳糖醛酸苷诱导的细胞外H(2)O(2)不参与生长素的抑制 调节的rolB基因在烟草叶外植体中的表达。植物生理学122(4):1379-1385。
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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. Bisceglia, N. G., Gravino, M. and Savatin, D. V. (2015). Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves. Bio-protocol 5(24): e1685. DOI: 10.21769/BioProtoc.1685.
  2. Bellincampi, D., Dipierro, N., Salvi, G., Cervone, F. and De Lorenzo, G. (2000). Extracellular H(2)O(2) induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiol 122(4): 1379-1385.
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