搜索

DAB Staining and Visualization of Hydrogen Peroxide in Wheat Leaves
利用DAB染色原位检测小麦叶片中的过氧化氢   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

The production of hydrogen peroxide (H2O2) has been recognized as an important feature of plant cells that undergo programmed cell death (PCD) during host-pathogen interaction. Thordal-Christensen et al. (1997) first described a method using chemical 3,3-diaminobenzidine (DAB) to detect the presence and distribution of H2O2 in barley leaves challenged by the powdery mildew fungus (Thordal-Christensen et al., 1997). Since then, this method has been adapted to many other plant species for in situ detection of H2O2. Here, we describe a modified protocol to stain and visualize H2O2 production in wheat leaves during infection by the necrotrophic fungus, Stagonospora nodrum or infiltration by the necrotrophic effectors produced by the fungus. The short version of this method has been reported in (Liu et al. 2012).

Materials and Reagents

  1. 3,3-diaminobenzidine (Sigma-Aldrich, catalog number: D8001 )
  2. 0.1 N HCl
  3. 95% ethanol (bulked)
  4. Glacial acetic acid (EMD Millipore, catalog number: UN2789 )
  5. L-(+)-Lactic acid (Sigma-Aldrich, catalog number: L1750 )
  6. Glycerol (Life Technologies, Invitrogen™, catalog number: 15514-011 )
  7. Aluminum foil
  8. Round plastic screens with the diameter closed to that of 15 ml tube (handmade, help the wheat leaf segments immerse in the staining solution). These plastic screens were cut from a big piece of plastic mesh that can be purchased from a local hardware store or online store (for example, Industrial Netting)
  9. Wheat leaves at three leaf stage (~2 weeks old grown under the greenhouse conditions)

Equipment

  1. Polyethylene 50 ml conical centrifuge tube (BD Biosciences, Falcon®, catalog number: C2745 )
  2. Polyethylene 15 ml conical centrifuge tube (BD Biosciences, Falcon®, catalog number: C2750 )
  3. Glass petri dish 100 mm with lid (Sigma-Aldrich, catalog number: CLS3160102 )
  4. Balance
  5. pH meter
  6. Shaker
  7. Vacuum
  8. Microscope
  9. Dessicator

Procedure

  1. Preparation of fresh DAB staining solution
    1. Measure ~50 ml distilled water and add 0.1 N HCl (1-2 drops) till pH reaches 3.6.
    2. Pour 10 ml of the above solution into each 15 ml tube (the number of tubes needed is dependent on the number of treatments).
    3. Weigh 10 mg 3, 3-diaminobenzidine and add to each tube.
    4. Close the cap, wrap the whole tube with Aluminum foil, and vigorously shake at 37 °C in an orbital shaker for at least 1 h (leaf samples can be collected during this time period).
    5. Check the solution to see if most of the DAB is dissolved. The solution should be colorless or slight pink and should be used fresh for staining.

  2. Staining of wheat leaves
    1. Collect wheat leaves at the designated time points after inoculation with the pathogen or infiltration with toxins. The fungal inoculation and toxin infiltration in this case were done at three leaf stage.
    2. Cut infiltrated or inoculated areas of the leaves into 3 cm long segments and immerse them in the DAB solution with a plastic screen (no more than 8 leaf segments in one 15 ml tube).
    3. Place all tubes with the caps removed in a vacuum dessicator and vacuum for 30 min at room temperature. It’s not necessary to keep the tubes in the dark at this step.
    4. Release vacuum and place all tubes in a dark cabinet with the caps closed and incubate overnight.
    5. The necrotic areas caused by the fungus or the toxin should be dark brown in color.

  3. Clearing of leaves for examination (all procedures should be carried out in a fume hood)
    1. Gently rinse DAB solution off the stained leaf segments using distilled water and dry slightly on paper towels.
    2. Place the leaf segments on paper towels saturated with a fixative solution (ethanol/acetic acid 3:1, V/V) in a petri dish. A little amount of extra solution left in petri dish is allowed.
    3. Incubate the leaf segments at room temperature in a fume hood for at least 24 h (it may require additional time to clear all the chlorophyll from the leaf tissue).
    4. Transfer the cleared leaf segments to paper towels saturated with water and leave for 30 min to remove any fixative residues.
    5. Transfer leaf segments on paper towels saturated with a lactoglycerol solution (lactic acid/glycerol/H2O 1:1:1, v/v/v). A little amount of extra solution left in petri dish is allowed.
    6. Leaf segments can be subjected to examination after a couple of hour incubation at room temperature or stored on paper towel saturated with the same lactoglycerol solution for several weeks.
    7. For examination, carefully mount leaf segments on a glass slide and observe under the microscope to determine the cellular location of DAB. Photographs can be made using a white background.

Acknowledgments

This protocol was adapted from a method published in Liu et al. (2012), which was originally based on the descriptions by Thordal-Christensen et al. (1997). Development and implementation of this protocol was funded by USDA-NIFA AFRI Microbial Biology Program - Competitive grant # 2010-65108-20543, and by USDA-ARS CRIS Project #5442-22000-048-0D.

References

  1. Liu, Z., Z. Zhang, J. D. Faris, R. P. Oliver, R. Syme, M. C. McDonald, B. A. McDonald, P. S. Solomon, S. Lu, W. L. Shelver, S. Xu and T. L. Friesen (2012). The cysteine rich necrotrophic effector SnTox1 produced by Stagonos pora nodorum triggers susceptibility of wheat lines harboring Snn1. PLoS Pathog 8(1): e1002467.
  2. Thordal-Christensen, H., Z. Zhang, Y. Wei and D. B. Collinge (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. Plant J 11(6): 1187-1194.

简介

过氧化氢(H 2 O 2 O 2)的产生已被认为是在宿主 - 病原体相互作用期间经历程序性细胞死亡(PCD)的植物细胞的重要特征。 Thordal-Christensen等人(1997)首先描述了使用化学3,3-二氨基联苯胺(DAB)检测H 2 O 2基团的存在和分布的方法, (Thordal-Christensen等人,1997)攻击的大麦叶中的抗性。 从那时起,该方法已经适应于许多其它植物物种用于原位检测H 2 O 2 O 2。 在这里,我们描述了在感染期间通过坏死性真菌,角叉状瘤或由产生的坏死营养效应物的浸润来染色和可视化小麦叶中H 2 O 2 O 2产生的修饰的方案 真菌。 该方法的简短版本已经在(Liu等人2012年)中报道。

材料和试剂

  1. 3,3-二氨基联苯胺(Sigma-Aldrich,目录号:D8001)
  2. 0.1 N HCl
  3. 95%乙醇(膨化)
  4. 冰乙酸(EMD Millipore,目录号:UN2789)
  5. L - (+) - 乳酸(Sigma-Aldrich,目录号:L1750)
  6. 甘油(Life Technologies,Invitrogen TM,目录号:15514-011)
  7. 铝箔
  8. 圆形塑料筛网,其直径接近15ml管(手工制作,帮助小麦叶节浸入染色溶液)。 这些塑料屏幕是从一块大块塑料网上切下来的,可以从当地的五金商店或在线商店购买(例如,工业网)。
  9. 小麦叶在三叶期(〜2周龄,在温室条件下生长)

设备

  1. 聚乙烯50ml圆锥形离心管(BD Biosciences,Falcon ,目录号:C2745)
  2. 聚乙烯15ml锥形离心管(BD Biosciences,Falcon ,目录号:C2750)
  3. 带有盖的玻璃培养皿(Sigma-Aldrich,目录号:CLS3160102)
  4. 余额
  5. pH计
  6. 振动器
  7. 真空
  8. 显微镜
  9. 干燥剂

程序

  1. 制备新鲜DAB染色溶液
    1. 测量〜50ml蒸馏水并加入0.1N HCl(1-2滴),直至pH达到3.6
    2. 将10ml上述溶液倒入每个15ml管中(所需的管数取决于处理次数)。
    3. 称量10mg 3-二氨基联苯胺并加入每个试管中
    4. 盖上盖子,用铝箔包裹整个管子,并在轨道振荡器中在37℃下剧烈摇动至少1小时(在此期间可以收集叶子样品)。
    5. 检查溶液以查看大部分DAB是否溶解。 溶液应无色或浅粉红色,应用于新鲜染色

  2. 小麦叶子的染色
    1. 在接种病原体或用毒素浸润后的指定时间点收集小麦叶。 在这种情况下,在三叶处进行真菌接种和毒素浸润 阶段
    2. 将叶的渗透或接种区域切成3cm长的区段,并将其浸入具有塑料筛(在一个15ml管中不超过8个叶节段)的DAB溶液中。
    3. 将在真空干燥器中移除的所有管和在室温下真空30分钟。 在这一步不需要保持管在黑暗中。
    4. 释放真空,将所有管子放在黑暗的橱柜中,盖子关闭并孵育过夜
    5. 由真菌或毒素引起的坏死区域应该是深棕色的

  3. 清除叶子进行检查(所有程序应在通风橱中进行)
    1. 使用蒸馏水轻轻冲洗DAB溶液离开染色的叶片段,并稍微用纸巾擦干
    2. 将叶片放在用固定液(乙醇/乙酸3:1,V/V)饱和的纸巾上,在培养皿中。允许在培养皿中留有少量额外的溶液。
    3. 在室温下在通风橱中孵育叶片段至少24小时(可能需要额外的时间从叶组织中清除所有叶绿素)。
    4. 将清除的叶片段转移到用水饱和的纸巾上,并离开30分钟以去除任何固定残留物
    5. 在用乳甘油溶液(乳酸/甘油/H 2 O 1:1:1,v/v/v)饱和的纸巾上转移叶片段。允许在培养皿中留有少量额外的溶液。
    6. 叶片可以在室温下孵育几小时后进行检查,或者储存在用相同乳糖甘油溶液饱和的纸巾上数周。
    7. 为了检查,将叶片段小心地安装在载玻片上并在显微镜下观察以确定DAB的细胞位置。 照片可以使用白色背景进行。

致谢

该方案改编自Liu等人(2012)中公开的方法,其最初基于Thordal-Christensen等人(1997)的描述。 本协议的制定和实施由USDA-NIFA AFRI微生物生物学计划 - 竞争性补助金#2010-65108-20543和USDA-ARS CRIS项目#5442-22000-048-0D资助。

参考文献

  1. Liu,Z.,Z.Zhang,J.D.Faris,R.P.Oliver,R.Syme,M.C.McDonald, B. A. McDonald,P. S. Solomon,S. Lu,W. L. Shelver,S. Xu和T. L. Friesen(2012)。   半胱氨酸富集的坏死营养效应物SnTox1由Stagonos pora产生 nodorum触发对携带Snn1的小麦品系的易感性。

    8(1):e1002467。

  2. Thordal-Christensen,H.,Z.Zhang,Y.We和D.B.Colllinge(1997)。 H sub 2 O 2的亚细胞定位 。 在大麦 - 白粉病相互作用期间,在乳头中的H 2 O 2积累和过敏反应。植物J 1(6) :1187-1194。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 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. Liu, Z. and Friesen, T. (2012). DAB Staining and Visualization of Hydrogen Peroxide in Wheat Leaves. Bio-protocol 2(24): e309. DOI: 10.21769/BioProtoc.309.
  2. Liu, Z., Z. Zhang, J. D. Faris, R. P. Oliver, R. Syme, M. C. McDonald, B. A. McDonald, P. S. Solomon, S. Lu, W. L. Shelver, S. Xu and T. L. Friesen (2012). The cysteine rich necrotrophic effector SnTox1 produced by Stagonos pora nodorum triggers susceptibility of wheat lines harboring Snn1. PLoS Pathog 8(1): e1002467.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。