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Detection and Measurement of ROS in Tobacco Leaves
烟草叶中活性氧(ROS)的检测与测定

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Abstract

Leaf metabolism produces hydrogen peroxide (H2O2) at high rates, high level H2O2 accumulation can cause oxidative stress. This protocol describes a method for determining H2O2 concentration in tobacco leaves. In this method all extractions were performed with HClO4, neutralized, and pretreated with ascorbate oxidase to eliminate ascorbate interferences. H2O2 content was determined using a colorimetric assay spiked with an internal control. Interfering peroxides were determined in parallel using a negative control treated with catalase and subsequently subtracted.

Materials and Reagents

  1. Tobacco leaves (Nicotiana tabaccum, Wisconsin 38)
  2. HClO4
  3. Polyvinylpolypyrrolidone (PVP)
  4. K2CO3 in 0.3 M phosphate buffer (pH 5.6)
  5. Ascorbate oxidase (Sigma-Aldrich, catalog number: A0157 )
  6. Phosphate buffer (pH 6.5)
  7. Catalase (Sigma-Aldrich, catalog number: C3155 )
  8. H2O2
  9. Horseradish peroxidase (Sigma-Aldrich, catalog number: 77332 )
  10. 3-(dimethylamino) benzoic acid (DMAB)
  11. 3-methyl-2-benzothiazoline hydazone (MBTH)
  12.  Liquid nitrogen
  13. 0.1 M phosphate buffer (pH 6.5)

Equipment

  1. DU-800 Spectrophotometer (Beckman Coulter)

Procedure

  1. The seeds of tobacco plants were allowed to germinate on MS medium, and then the plants were transferred to soil and grown for two weeks in a growth chamber at 25 ± 1 °C with PPFD of 100 μmol/m2/s, a relative humidity of 75–80%, and a photoperiod of 12/12 h light/dark. For H2O2 content determination, tobacco leaves (50 mg) were ground with mortar and pestle to a fine powder in liquid nitrogen and the powder was extracted in 2 ml 1 M HClO4 including 5% insoluble PVP.
  2. The homogenate was centrifuged at 12,000 x g for 10 min and the supernatant was neutralized with several microlitre 5 M K2CO3 to pH 5.6. The homogenate was centrifuged at 12,000 x g for 1 min to remove KClO4.
  3. To eliminate the interference of ascorbate, the supernatant was incubated for 10 min with 1 U/ml ascorbate oxidase at room temperature to oxidize ascorbate.
  4. Preparing reaction mixture: the reaction mixture consisted of 0.05 M phosphate buffer (pH 6.5); 3.3 mM DMAB; 0.07 mM MBTH and 50 ng/ml horseradish peroxidase. The reaction was initiated by addition of an aliquot (50 μl) of the sample.

    Reaction mixture (1 ml)
    Stock
    Volume
    Final conc.
    0.1 M phosphate buffer (pH 6.5)
    500 μl
    0.05 M
    33 mM DMAB
    100 μl
    3.3 mM
    0.7 mM MBTH
    100 μl
    0.07 mM
    horseradish peroxidase (0.1 U/μl)
    1 μl
    0.1 U
    sample
    50 μl

    ddH2O

    to 1,000 μl
       
    The absorbance change at 590 nm (ΔOD1) per minute was monitored at 25 °C for 1-5 min. The value represents the total peroxides including H2O2 in the sample.
  5. To eliminate the interference of other peroxides, an aliquot of the supernatant pre-incubated with ascorbate oxidase as mentioned in step 3 was then incubated for 10 min with 1 U/m catalase at room temperature to catalyze H2O2 decomposition. The interference of other peroxides was then determined using the same procedure as described in step 4 (ΔOD2).
  6. The absorbance change at 590 nm caused by H2O2 in sample was calculated as ΔOD1-ΔOD2.
  7. H2O2 content in the sample was quantified by reference to an internal standard: in each determination in step 4, a parallel aliquot was assayed with addition of 2 nmol H2O2 to the reaction mixture, and the absorbance change at 590 nm (ΔOD3) was monitored at 25 °C.
  8. The final H2O2 concentration was calculated as: (ΔOD1-ΔOD2)/ (ΔOD3-ΔOD1) × 2 nmol/50 μl × total volume of the extraction/total fresh weight of the tobacco leaves.

Recipes

  1. 0.1 M phosphate buffer (pH 6.5)
    pH
    1 M K2HPO4 (ml)
    1 M KH2PO4 (ml)
    6.5
    30.4
    69.6

Acknowledgments

This protocol was adapted from Ding et al. (2009). This study was supported by the National Natural Science Foundation of China (30725024) and the State Key Basic Research and Development Plan of China (2009CB118503) to L.C.

References

  1. Ding, S. H., Lu, Q. T., Zhang, Y., Yang, Z. P., Wen, X. G., Zhang, L. X., Lu, C. M. (2009). Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state. Plant Mol Biol 69(5): 577–592.
  2. Queval, G., Hager, J., Gakière, B., Noctor, G. (2008). Why are literature data for H2O2 contents so variable? A discussion of potential difficulties in the quantitative assay of leaf extracts. J Exp Bot 59(2): 135–146.
  3. Veljovic-Jovanovic, S., Noctor, G., Foyer, C. H. (2002). Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate. Plant Physiol Biochem 40(6-8): 501–507.

简介

叶代谢以高速率产生过氧化氢(H 2 O 2 O 2),高水平的H 2 O 2 O 2积累 可引起氧化应激。 该方案描述了用于测定烟草叶中H 2 O 2 O 2浓度的方法。 在该方法中,所有提取用HClO 4进行,中和,并用抗坏血酸氧化酶预处理以消除抗坏血酸盐干扰。 使用掺入内部对照的比色测定法测定H 2 O 2含量。 使用用过氧化氢酶处理的阴性对照平行测定干扰过氧化物,随后扣除。

材料和试剂

  1. 烟叶( Nicotiana tabaccum ,Wisconsin 38)
  2. HClO 4
  3. 聚乙烯吡咯烷酮(PVP)
  4. 在0.3M磷酸盐缓冲液(pH 5.6)中的K 2 CO 3 3 /
  5. 抗坏血酸氧化酶(Sigma-Aldrich,目录号:A0157)
  6. 磷酸盐缓冲液(pH 6.5)
  7. 过氧化氢酶(Sigma-Aldrich,目录号:C3155)
  8. H 2 2
  9. 辣根过氧化物酶(Sigma-Aldrich,目录号:77332)
  10. 3-(二甲基氨基)苯甲酸(DMAB)
  11. 3-甲基-2-苯并噻唑啉hydazone(MBTH)
  12.  液氮
  13. 0.1M磷酸盐缓冲液(pH6.5)

设备

  1. DU-800分光光度计(Beckman Coulter)

程序

  1. 使烟草植物的种子在MS培养基上发芽,然后将植物转移到土壤中,并在25±1℃下在生长室中生长两周,PPFD为100μmol/m 2相对湿度为75-80%,光周期为12/12h光/暗。对于H 2 O 2含量测定,将烟叶(50mg)用研钵和杵在液氮中研磨成细粉末,并将粉末在2ml 1包括5%不溶性PVP。
  2. 将匀浆在12,000×g离心10分钟,并用几微升5M K 2 CO 3至pH 5.6中和上清液。将匀浆在12,000×g离心1分钟以除去KClO 4。
  3. 为了消除抗坏血酸的干扰,将上清液与1U/ml抗坏血酸氧化酶在室温下温育10分钟以氧化抗坏血酸。
  4. 制备反应混合物:反应混合物由0.05M磷酸盐缓冲液(pH 6.5)组成; 3.3mM DMAB; 0.07mM MBTH和50ng/ml辣根过氧化物酶。通过加入等分试样(50μl)的样品开始反应。

    反应混合物(1ml)
    股票

    最终浓度
    0.1M磷酸盐缓冲液(pH6.5) 500微升
    0.05 M/m
    33mM DMAB
    100微升
    3.3 mM
    0.7mM MBTH
    100微升
    0.07 mM
    辣根过氧化物酶(0.1U /μl) 1微升
    0.1 U
    样品
    50微升

    ddH sub 2 O

    到1000微升
       
    在25℃下监测每分钟590nm处的吸光度变化(ΔOD1)1-5分钟。该值表示样品中的总过氧化物,包括H 2 O 2 O 2。
  5. 为了消除其它过氧化物的干扰,然后将如在步骤3中提及的用抗坏血酸氧化酶预温育的上清液的等分试样与1U/m过氧化氢酶在室温下温育10分钟以催化H 2 - O 2分解。然后使用与步骤4(ΔOD2)中所述相同的程序测定其它过氧化物的干扰
  6. 将样品中由H 2 O 2引起的590nm处的吸光度变化计算为ΔOD1-ΔOD2。
  7. 通过参考内部标准来定量样品中的H 2 O 2 O 2含量:在步骤4中的每次测定中,加入2nmol H 2 O 2测定平行等分试样,并在25℃下监测590nm处的吸光度变化(ΔOD3)。
  8. 最终的H 2 O 2 O 2浓度计算为:(ΔOD1-ΔOD2)/(ΔOD3-ΔOD1)×2nmol /50μl×提取的总体积/总计烟叶的鲜重

食谱

  1. 0.1M磷酸盐缓冲液(pH6.5)
    pH
    1 M K 2 HPO 4(ml)
    1 M KH sub 2 PO 4(ml)
    6.5
    30.4
    69.6

致谢

该方案改编自Ding等人(2009)。本研究得到了中国国家自然科学基金(30725024)和国家重点基础研究发展规划(2009CB118503)的支持。

参考文献

  1. Ding,S.H.,Lu,Q.T.,Zhang,Y.,Yang,Z.P.,Wen,X.G.,Zhang,L.X.,Lu,C.M。(2009)。 对具有降低的谷胱甘肽还原酶活性的转基因烟草植物中氧化应激的增强的敏感性导致抗坏血酸池和抗坏血酸盐氧化还原状态。 Plant Mol Biol 69(5):577-592
  2. Queval,G.,Hager,J.,Gakière,B.,Noctor,G。(2008)。 叶过氧化氢浓度通常被高估了吗? 组织酚和抗坏血酸的伪影干扰的潜在影响。 植物生理生物化学(Plant Physiol Biochem)40(6-8):501-507。
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引用:Ding, S. and Lu, C. (2013). Detection and Measurement of ROS in Tobacco Leaves. Bio-protocol 3(5): e407. DOI: 10.21769/BioProtoc.407.
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Wing Lee
Rothamsted Research
Hello,
I am trying to prepare the 33 mM DMAB solution called for in this protocol. As DMAB is not very soluble in water, do you dissolve the powder in ethanol first and then make it up to the appropriate molarity using water? If so, what final percentage of ethanol do you use?
Thank you
7/18/2013 8:46:39 AM Reply