Quantification of Superoxide and Hydrogen Peroxide in Leaves

Part of the Methods in Molecular Biology book series (MIMB, volume 1166)


Reactive oxygen species (ROS) are produced in plants under both non-stressful and stressful conditions. Various histochemical staining methods have been developed and are widely used to visualize ROS accumulation sites. In contrast to qualitative analysis, quantification of ROS has been time- and labor consuming. As a consequence, the number of samples, which could be analyzed in parallel, has been limited. To overcome this problem, we introduce an improved semiquantitative method, in which ROS levels are quantified after histochemical staining in plant organs with the digital image analysis package ImageJ.

Key words

ROS Chloroplast H2O2 staining Superoxide staining Digital imaging Quantification 


  1. 1.
    Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599PubMedCrossRefGoogle Scholar
  2. 2.
    Heidervand L, Amiri LM (2010) What happens in plant molecular responses to cold stress? Acta Physiol Plant 32:419–431CrossRefGoogle Scholar
  3. 3.
    Juszczak I, Rudnik R, Pietzenuk B, Baier M (2012) Natural genetic variation in the expression regulation of the chloroplast antioxidant system among Arabidopsis thaliana accessions. Physiol Plant 146:53–70PubMedCrossRefGoogle Scholar
  4. 4.
    Torres MA, Dangl JL, Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci U S A 99:517–522PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F (2002) The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 53:1367–1376PubMedCrossRefGoogle Scholar
  6. 6.
    Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:851–863PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Mehler AH (1951) Studies on reactivities of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents. Acta Biochem Biophys 33:65–77Google Scholar
  8. 8.
    Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410PubMedCrossRefGoogle Scholar
  9. 9.
    Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  10. 10.
    Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498PubMedCrossRefGoogle Scholar
  11. 11.
    Baier M, Dietz KJ (1997) The plant 2-Cys peroxiredoxin BAS1 is a nuclear-encoded chloroplast protein: its expressional regulation, phylogenetic origin, and implications for its specific physiological function in plants. Plant J 12:179–190PubMedCrossRefGoogle Scholar
  12. 12.
    Baier M, Pitsch NT, Mellenthin M, Guo W (2010) Regulation of genes encoding antioxidant enzymes in comparison to regulation of the extra-plastidic antioxidant defense system. In: Anjum NA, Chan MT, Umar S (eds) Ascorbate-glutathione pathway and stress tolerance in plants. Springer, Dordrecht, Heidelberg, London, New YorkGoogle Scholar
  13. 13.
    Foyer CH, Shigeoka S (2011) Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol 155:93–100PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Vanacker H, Carver TL, Foyer CH (2000) Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hyper-sensitive response in the barley-powdery mildew interaction. Plant Physiol 123:1289–1300PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Kawai-Yamada M, Ohori Y, Uchimiya H (2004) Dissection of Arabidopsis Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-induced cell death. Plant Cell 16:21–32PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Bournonville CF, Diaz-Ricci JC (2010) Quantitative determination of superoxide in plant leaves using a modified NBT staining method. Phytochem Anal 22:268–271CrossRefGoogle Scholar
  17. 17.
    Hideg E, Barta C, Kalai T, Vass I, Hideg K, Asada K (2002) Detection of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photoinhibition or UV radiation. Plant Cell Physiol 43:1154–1164PubMedCrossRefGoogle Scholar
  18. 18.
    Fryer MJ, Oxborough K, Mullineaux PM, Baker NR (2002) Imaging of photo-oxidative stress responses in leaves. J Exp Bot 53:1249–1254PubMedCrossRefGoogle Scholar
  19. 19.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Plant Physiology, Dahlem Center of Plant SciencesFreie Universität BerlinBerlinGermany

Personalised recommendations