Detection and Measurement of ROS in Tobacco Leaves

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.