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Detection and Measurement of ROS in Tobacco Leaves    

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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)


  1. DU-800 Spectrophotometer (Beckman Coulter)


  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)
    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
    50 μl


    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.


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


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.


  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.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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
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
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