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A simple colorimetric method for determination of hydrogen peroxide in plant tissues

Abstract

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 µmol g−1 FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.

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References

  • Aebi H (1974) Methods of enzymatic analysis. Academic Press, New York, pp 673–684

    Google Scholar 

  • Aroca R, Irigoyen JJ, Sànchez-Díaz M (2003) Drought enhances maize chilling tolerance. II. Photosynthetic traits and protective mechanisms against oxidative stress. Physiol Plant 117:540–549

    PubMed  CAS  Article  Google Scholar 

  • Asada K (1999) The water–water cycle in chloroplast: scavenging of active oxygens and dissipation of excess photons. Ann Rev Plant Physiol Plant Mol Biol 50:601–639

    PubMed  CAS  Article  Google Scholar 

  • Fadzillah NM, Gill V, Finch RP, Burdon RH (1996) Chilling, oxidative stress and antioxidant responses in shoot cultures of rice. Planta 199:552–556

    CAS  Article  Google Scholar 

  • Foyer CH, López-Delgado H, Dat J, Scott IM (1997) Hydrogen peroxide and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol Plant 100:241–254

    CAS  Article  Google Scholar 

  • Genfa Z, Dasgupta PK (1992) Hematin as a peroxidase substitute in hydrogen peroxide determinations. Anal Chem 64:517–522

    PubMed  CAS  Article  Google Scholar 

  • Hu X, Bidney DL, Yalpani N, Duvick JP, Crasta O, Folkerts O, Lu G (2003) Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiol 133:170–181

    PubMed  CAS  Article  Google Scholar 

  • Huang M, Guo Z (2005) Responses of antioxidative system to chilling stress in two rice cultivars differing in sensitivity. Biol Plant 49:81–84

    CAS  Article  Google Scholar 

  • Jiang M, Zhang J (2003) Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings. Plant Cell Environ 26:929–939

    PubMed  CAS  Article  Google Scholar 

  • Law MY, Charles SA, Halliwell B (1983) Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. Biochem J 210:899–903

    PubMed  CAS  Google Scholar 

  • Li J, Lu B, Xu LL (2000) An improved method for the determination of hydrogen peroxide in leaves. Prog Biochem Biophys 27:548–551

    CAS  Google Scholar 

  • Moller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive species. Ann Rev Plant Physiol Plant Mol Biol 52:561–591

    CAS  Article  Google Scholar 

  • Neill S, Desikan R, Hancock J (2002) Hydrogen peroxide signaling. Curr Opin Plant Biol 5:386–395

    Article  Google Scholar 

  • Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279

    CAS  Article  Google Scholar 

  • Noctor G, Veljoric-Jovanovic SD, Riscoll S, Novitskaya L, Foyer CH (2002) Drought and oxidative load in wheat leaves. A predominant role for photorespiration? Ann Bot 89:841–850

    PubMed  CAS  Article  Google Scholar 

  • Orozco-Cárdenas ML, Narveáz-Vásquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13:179–191

    PubMed  Article  Google Scholar 

  • Patterson BD, Macrae EA, Ferguson IB (1984) Estimation of hydrogen peroxide in plant extracts using titanium (IV). Anal Biochem 139:487–492

    PubMed  CAS  Article  Google Scholar 

  • Pérez FJ, Rubio S (2006) An improved chemiluminescence method for hydrogen peroxide determination in plant tissues. Plant Growth Regul 48:89–95

    Article  CAS  Google Scholar 

  • Prasad TK, Anderson MD, Stewart CR (1994) Acclimation, hydrogen peroxide, and abscisic acid protect mitochondria against irreversible chilling injury in maize seedlings. Plant Physiol 105:619–627

    PubMed  CAS  Google Scholar 

  • Srivalli B, Sharma G, Khanna-Chopra R (2003) Antioxidative defense system in an upland rice cultivar subjected to increasing intensity of water stress followed by recovery. Physiol Plant 119:503–512

    CAS  Article  Google Scholar 

  • Trinder P (1969) Determination of glucose in blood using glucose with an alternative oxygen an alternative oxygen acceptor. Ann Clin Biochem 6:24

    CAS  Google Scholar 

  • Vaidyanathan H, Sivakumar P, Chakrabarty R, Thomas G (2003) Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)—differential response in salt-tolerant and sensitive varieties. Plant Sci 165:1411–1418

    CAS  Article  Google Scholar 

  • Warm E, Laties CG (1982) Quantification of hydrogen peroxide in plant extracts by the chemiluminiscense reaction with luminol. Phytochemistry 21:827–831

    CAS  Article  Google Scholar 

  • Wohlgemuth H, Mittelstrass K, Kschieschan S, Bender J, Weigel HJ, Overmyer K, Kangasjărvi J, Sandermann H, Langerbartels C (2002) Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone. Plant Cell Environ 25:717–726

    CAS  Article  Google Scholar 

  • Zhou B, Guo Z, Liu Z (2005) Effects of abscisic acid on antioxidant systems of Stylosanthes guianensis (Aublet) Sw. under chilling stress. Crop Sci 45: 599–605

    CAS  Article  Google Scholar 

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Acknowledgements

The project was funded by grants from Guangdong Science and Technology Projects (2003C201018), Guangdong Provincial Natural Science Foundation (04105978, 5300846), and National Natural Science Foundation (30571283).

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Correspondence to Zhenfei Guo.

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Zhou, B., Wang, J., Guo, Z. et al. A simple colorimetric method for determination of hydrogen peroxide in plant tissues. Plant Growth Regul 49, 113–118 (2006). https://doi.org/10.1007/s10725-006-9000-2

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  • DOI: https://doi.org/10.1007/s10725-006-9000-2

Keywords

  • 4-aminoantipyrine
  • Hydrogen peroxide
  • Peroxidase
  • Plant tissue