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Localisation and Quantification of Reactive Oxygen Species and Nitric Oxide in Arabidopsis Roots in Response to Fungal Infection
拟南芥根部响应真菌感染的活性氧和一氧化氮的定位和定量测定

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Abstract

Nitric oxide and reactive oxygen species have emerged as important signalling molecules in plants. The half-lives of NO and ROS are very short therefore rapid and precise measurements are required for the understanding biological roles of these redox active species. Various organelles and compartments generate NO and ROS thus it is important to determine precise location of these free radicals in order to understand their signalling roles. Diaminofluorescen (DAF) and fluorescent 2', 7'-dichlorofluorescein (DCF) dyes are employed to determine NO and ROS localisation. The advantage of this approach is that the dyes diffuse precisely to NO and ROS producing sites and generate fluorescence which can be detected by fluorescence- or confocal laser scanning microscopes. However, this technique has its disadvantages; particularly the specificity of the fluorescence signals needs to be established. Therefore, the use scavenger of NO such as cPTIO and ROS such as ascorbate is required to confirm the specificity of the fluorescence signal and ideally, confirmation of data obtained using other methods due to advantage and disadvantage associated with each method (Gupta and Igamberdiev, 2013). Here we describe a method to detect NO and ROS production from Arabidopsis roots in response to infection by Trichoderma, Fusarium using DAF, gas phase Griess reagent assay and DCF fluorescence methods.

Materials and Reagents

  1. Plant materials: 1-3 weeks old Arabidopsis thaliana sterile seedlings grown in vitro conditions
  2. 20 mM HEPES (pH 7.2) (Sigma-Aldrich, catalog number: H3375 )
  3. MS medium with vitamins (Duchefa Biochemie, catalog number: M0222 )
  4. Sodium hypochlorite (NaOCl) (Sigma-Aldrich, catalog number: 425044 )
  5. DAF-2DA (Enzo Life Sciences, catalog number: ALX-620-056-M001 )
  6. PDA medium (Potato dextrose agar)
  7. DCF-2DA fluorescent dye (Life Technologies, InvitrogenTM, catalog number: D-399 )
  8. Carboxy-PTIO potassium salt (Sigma-Aldrich, catalog number: C221 )
  9. Sulphaniliamide (Sigma-Aldrich, catalog number: S9251 )
  10. N-(1-naphthyl) ethylenediamine (NED) (Sigma-Aldrich, catalog number: 222488 )
  11. Potato dextrose agar (PDA) (Difco)

Equipment

  1. Slides
  2. Coverslips
  3. Leica fluorescent microscope
  4. Sterilised forceps
  5. Sterile pipette tips
  6. Micro centrifuge tubes
  7. Micropore tape (VWR International, catalog number: 115-8172 )
  8. Petri dishes

Software

  1. Image J software (version 1.45) Wayne Rasband (NIH)

Procedure

  1. Localisation of NO and ROS by DAF and DCF fluorescence
    1. Surface sterilize seeds of Arabidopsis thaliana (L.) Heynh. (Col-0) by incubating seeds in 10% NaOCl and 0.1% Tween-20 detergent for 10 min and washed three times with autoclaved distilled water.
    2. Transfer the sterilised seeds horizontally on Murashige Skoog (MS medium) 1.5% agar plates, close the plates with micropore tape and culture for 12 days under long day conditions .
    3. Grow Trichoderma asperelloides (T. asperelloides) T203 and Fusarium oxysporum (F. oxysporum) f. sp. lentis on potato dextrose agar (PDA; 39 g/L) plates for 10 days at 28 °C.
    4. Harvest T203 conidia by gentle scraping the petridish in 5 ml sterile water, consisting of both mycelium and spores, and add the solution (that contain mycelium+spores) to a 2-ml microcentrifuge tube in which the plant intact root system has to be placed (shown in Figure 1).
    5. The following treatments are recommended:
      1. Untreated plants (control).
      2. Incubate with T. asperelloides for various time intervals as described by Gupta et al. (2014).
      3. Incubate F. oxysporum-for 10 min to 4 h depending on experiment.
      4. Incubate plants simultaneously with T. asperelloides sand F. oxysporum.
    6. Once the fungal incubation is finished remove plant from microcentrifuge and wash three times with HEPES buffer.
    7. Incubate the whole plant with 10 µM DAF-2DA or DCF-2DA (2 ml volume) for 10 min in dark (Figure 1).
    8. Wash excess of DAF or DCF-2DA dye three times with HEPES buffer.
    9. Observe roots under bright field mode and capture picture.
    10. The observe fluorescence emission using a 505- to 530-nm band-excitation filter coupled with a 515-nm long-emission filter.
      Quantify images using Image J software by selecting area of interest and go to menu under Image J and click for analyse and then click submenu to measure.

  2. Determination of NO by gas phase griess reagent assay
    1. Place 1 g of root material in a 10 ml small conical flask that contain 4 ml of HEPES buffer (Figure 1).
    2. Take a loop-full of inoculum, that contains both mycelium and spores, and add to a conical flask.
    3. The following treatments are recommended.
      1. Untreated plants (control).
      2. Incubate with T. asperelloides for various intervals.
      3. Incubate F. oxysporum-for various Intervals Figure 1.
      4. Incubate plants simultaneously with T. asperelloides and F. oxysporum.
    4. Flush the solution in conical flask with NO free air (It is a safe process) (shown in Figure 1B).
    5. Trap the emitted air (bubbling) containing NO into 2 ml solution containing 1 ml of 1% w/v sulphanilamide and 0.02% w/v N-(1)-(naphthyl) ethylene-diaminedihydrochloride (Greiss reagent) (shown in Figure 1B).
    6. Read the absorbance at 540 nm in spectrophotometer.
    7. Make a standard curve with 0.1 to 5 µM nitrite.


      Figure1. A. NO and ROS detection by DAF and DCF2DA fluorescence; Roots were incubated in the solution containing fungus+spores and then transferred to DAF or DCF solution and monitored fluorescence B. Detection of NO released into gas phase detected by Griess reagent assay. 1 g of roots were placed in conical flask containing 4 ml of HEPES (pH 7.6) and flushed with NO free air and then NO generated from roots in response to fungal infection was trapped in griess reagent.

Acknowledgments

This work was supported by Max Planck Society (K. J. Gupta; Y. Brotman) and Marie Curie Intra European Fellowship for Carrier Development within the 7th European Community Framework Programme (K. J. Gupta) and the BBSRC-DEFRA-HGCA SCORE LINK grant (L. A. J. Mur).

References

  1. Gupta, K. J., Mur, L. A. and Brotman, Y. (2014). Trichoderma asperelloides suppresses nitric oxide generation elicited by Fusarium oxysporum in Arabidopsis roots. Mol Plant Microbe Interact 27(4): 307-314.
  2. Gupta, K. J. and Igamberdiev, A. U. (2013). Recommendations of using at least two different methods for measuring NO. Fron Plant Sci 4.

简介

一氧化氮和活性氧在植物中作为重要的信号分子出现。 NO和ROS的半衰期非常短,因此需要快速和精确的测量来理解这些氧化还原活性物质的生物学作用。各种细胞器和隔室产生NO和ROS,因此重要的是确定这些自由基的精确位置,以了解他们的信号传导作用。使用二氨基荧光素(DAF)和荧光2',7'-二氯荧光素(DCF)染料来确定NO和ROS定位。这种方法的优点是染料精确扩散到NO和ROS产生位点,并产生可以通过荧光或共聚焦激光扫描显微镜检测的荧光。然而,这种技术有其缺点;特别是需要建立荧光信号的特异性。因此,需要诸如cPTIO和抗坏血酸的ROS的使用清除剂来确认荧光信号的特异性,并且理想地,由于与每种方法相关的优点和缺点,理想地确认使用其它方法获得的数据(Gupta和Igamberdiev,2013 )。在这里我们描述了一种方法来检测响应于木霉属,镰刀菌属使用DAF,气相Griess试剂测定和DCF荧光方法的感染的拟南芥根的NO和ROS产生。

材料和试剂

  1. 植物材料:1-3周龄体外生长的拟南芥无菌幼苗条件
  2. 20mM HEPES(pH7.2)(Sigma-Aldrich,目录号:H3375)
  3. MS培养基(Duchefa Biochemie,目录号:M0222)
  4. 次氯酸钠(NaOCl)(Sigma-Aldrich,目录号:425044)
  5. DAF-2DA(Enzo Life Sciences,目录号:ALX-620-056-M001)
  6. PDA培养基(马铃薯葡萄糖琼脂)
  7. DCF-2DA荧光染料(Life Technologies,Invitrogen TM ,目录号:D-399)
  8. 羧基-PTIO钾盐(Sigma-Aldrich,目录号:C221)
  9. 硫酰胺(Sigma-Aldrich,目录号:S9251)
  10. (1-萘基)乙二胺(NED)(Sigma-Aldrich,目录号:222488)。
  11. 马铃薯葡萄糖琼脂(PDA)(Difco)

设备

  1. 幻灯片
  2. 盖舌
  3. 徕卡荧光显微镜
  4. 灭菌镊子
  5. 无菌移液器吸头
  6. 微量离心管
  7. 微孔胶带(VWR International,目录号:115-8172)
  8. 培养皿

软件

  1. Image J软件(版本1.45)Wayne Rasband(NIH)

程序

  1. NO和ROS的本地化   DAF和DCF荧光
    1. 表面消毒拟南芥的种子(L.)Heynh。 (Col-0) 在10%NaOCl和0.1%Tween-20洗涤剂中孵育种子10分钟,   用高压蒸馏水洗涤三次
    2. 转让 灭菌种子在Murashige Skoog(MS培养基)上水平1.5% 琼脂平板,用微孔胶带封闭平板并培养12小时 天。
    3. 生长木霉 (< em> T。asperelloides)T203和尖孢镰孢(Fusarium oxysporum)( F 尖孢镰孢)f。 sp。 在PDA马铃薯葡萄糖琼脂(PDA; 39g/L)平板上 在28℃下培养10天
    4. 通过温和刮擦收获T203分生孢子 在5毫升无菌水中的培养皿,由菌丝体和 孢子,并将溶液(含有菌丝体+孢子)添加到2-ml 微量离心管中必须有植物完好的根系 放置(如图1所示)。
    5. 建议进行以下治疗:
      1. 未处理植物(对照)。
      2. 与 T孵育。 对于各种时间间隔的非对映异构体,如Gupta等人 (2014)。
      3. 根据实验孵育尖孢镰孢 - 10分钟至4小时。
      4. 与胚胎同时孵育。 asperelloides sand F。 oxysporum。
    6. 一旦真菌孵育完成,从微量离心机中取出植物并用HEPES缓冲液洗涤三次
    7. 在黑暗中用10μMDAF-2DA或DCF-2DA(2ml体积)孵育整个植物10分钟(图1)。
    8. 用HEPES缓冲液洗涤过量的DAF或DCF-2DA染料三次
    9. 在明视场模式下观察根,并捕获图片。
    10. 观察荧光发射使用505至530nm 带有515 nm长发射滤光片的激发滤光片 量化   图像使用Image J软件通过选择感兴趣的区域并转到 菜单下的图像J,然后单击以进行分析,然后单击子菜单 测量。

  2. 通过气相试剂测定法测定NO
    1. 将1g根材料放入含有4ml HEPES缓冲液的10ml小锥形瓶中(图1)。
    2. 取一圈充满接种物,其中含有菌丝体和孢子,并加入到锥形瓶中
    3. 建议进行以下治疗。
      1. 未处理植物(对照)。
      2. 用不同的间隔孵育T. asperelloides 。
      3. 孵育 F。 oxysporum - 对于各种时间间隔图1.
      4. 与胚胎同时孵育。 非对映异构体和 F。 oxysporum 。
    4. 在无空气的情况下在锥形瓶中冲洗溶液(这是一个安全的过程)(如图1B所示)
    5. 将排放的含有NO的空气(鼓泡)捕获到2ml溶液中 含有1ml 1%w/v磺胺和0.02%w/v N-(1) - (萘基) 乙二胺二盐酸盐(Greiss试剂)(如图1B所示)
    6. 在分光光度计中读取540 nm处的吸光度
    7. 用0.1到5μM的亚硝酸盐制成标准曲线。


      Figure1。 A.通过DAF和DCF2DA荧光检测NO和ROS; 根 在含有真菌+孢子的溶液中温育,然后转移 到DAF或DCF溶液和监测的荧光B. 释放到气相中,通过Griess试剂检测。 1g根 置于含有4ml HEPES(pH7.6)和的三角烧瓶中 用无NO空气冲洗,然后从根产生的NO响应   真菌感染被捕获在试剂中。

致谢

这项工作得到了马克斯·普朗克学会(KJ Gupta; Y。布罗特曼)和Marie Curie Intra欧洲团队在第七届欧洲共同体框架计划(KJ Gupta)和BBSRC-DEFRA-HGCA SCORE LINK赠款(LAJ Mur) 。

参考文献

  1. Gupta,K.J.,Mur,L.A。和Brotman,Y。(2014)。 Trichoderma asperelloides抑制尖孢镰刀菌引起的一氧化氮产生 拟南芥根。 Mol Plant Microbe Interact 27(4):307-314。
  2. Gupta,K.J。和Igamberdiev,A.U。(2013)。 建议使用至少两种不同的方法来衡量NO。 Fron Plant Sci 4。
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引用:Gupta, K. J., Brotman, Y. and Mur, L. A. (2014). Localisation and Quantification of Reactive Oxygen Species and Nitric Oxide in Arabidopsis Roots in Response to Fungal Infection. Bio-protocol 4(19): e1259. DOI: 10.21769/BioProtoc.1259.
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