Detection of Peroxynitrite in Plants Exposed to Bacterial Infection

  • Diana Bellin
  • Massimo Delledonne
  • Elodie Vandelle
Part of the Methods in Molecular Biology book series (MIMB, volume 1424)


Peroxynitrite is a highly reactive derivative of nitric oxide (NO) which is gaining attention in the plant biology community because it may play a role in NO signaling during biotic stress. Peroxynitrite can react with many different biomolecules, but its ability to nitrate the tyrosine residues of proteins is particularly important because this may regulate defense signaling in response to pathogens. The analysis of peroxynitrite levels in the context of its proposed defense role requires an accurate and specific detection method. Here, we describe a photometric assay using the fluorescent dye Hong Kong Green 2 as a specific and quantitative probe for peroxynitrite in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the preparation of plant samples, the assay procedure, the measurement of peroxynitrite-specific fluorescence, and data presentation.

Key words

Nitric oxide Peroxynitrite Pseudomonas syringae Hong Kong Green 2 Signaling Superoxide Nitrosative stress 


  1. 1.
    Delledonne M, Xia RA, Dixon RA, Lamb C (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394:585–588CrossRefPubMedGoogle Scholar
  2. 2.
    Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci U S A 98:13454–13459CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Szabó C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680CrossRefPubMedGoogle Scholar
  4. 4.
    Gow AJ, Duran D, Malcolmc S, Ischiropoulosa H (1996) Effects of peroxynitrite-induced protein modifications on tyrosine phosphorylation and degradation. FEBS Lett 385:63–66CrossRefPubMedGoogle Scholar
  5. 5.
    Liaudet L, Vassalli G, Pacher P (2009) Role of peroxynitrite in the redox regulation of cell signal transduction pathways. Front Biosci 14:4809–4814CrossRefGoogle Scholar
  6. 6.
    Romero-Puertas MC, Laxa M, Mattè A, Zaninotto F, Finkemeier I, Jones AM, Perazzolli M, Vandelle E, Dietz KJ, Delledonne M (2007) S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration. Plant Cell 19:4120–4130CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Oury TD, Tatro L, Ghio AJ, Piantadosi CA (1995) Nitration of tyrosine by hydrogen peroxide and nitrite. Free Radic Res 23:537–547CrossRefPubMedGoogle Scholar
  8. 8.
    Sakamoto A, Sakurao SH, Fukunaga K, Matsubara T, Ueda-Hashimoto M, Tsukamoto S, Takahashi M, Morikawa H (2004) Three distinct Arabidopsis hemoglobins exhibit peroxidase-like activity and differentially mediate nitrite-dependent protein nitration. FEBS Lett 572:27–32CrossRefPubMedGoogle Scholar
  9. 9.
    Saito S, Yamamoto-Katou A, Yoshioka H, Doke N, Kawakita K (2006) Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells. Plant Cell Physiol 47:689–697CrossRefPubMedGoogle Scholar
  10. 10.
    Sun ZN, Wang HL, Liu FQ, Chen Y, Tam PK, Yang D (2009) BODIPY-based fluorescent probe for peroxynitrite detection and imaging in living cells. Org Lett 11:1887–1890CrossRefPubMedGoogle Scholar
  11. 11.
    Gaupels F, Spiazzi-Vandelle E, Yang D, Delledonne M (2011) Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response. Nitric Oxide 25:222–228CrossRefPubMedGoogle Scholar
  12. 12.
    Peng T, Wong N-K, Chen X, Chan Y-K, Hoi-Hang Ho D, Sun Z, Hu JJ, Shen J, El-Nezami H, Yang D (2014) Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues. J Am Chem Soc 136:11728–11734CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Diana Bellin
    • 1
  • Massimo Delledonne
    • 1
  • Elodie Vandelle
    • 1
  1. 1.Dipartimento di BiotecnologieUniversità degli Studi di VeronaVeronaItaly

Personalised recommendations