Skip to main content
SpringerLink
Log in

Measurement of Nitric Oxide (NO) Generation Rate by Chloroplasts Employing Electron Spin Resonance (ESR)

  • Protocol
  • First Online:
Book cover Plant Nitric Oxide

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1424))

Abstract

Chloroplasts are among the more active organelles involved in free energy transduction in plants (photophosphorylation). Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was measured as an endogenous product assessed by electron paramagnetic resonance (ESR) spin-trapping technique. ESR spectroscopy is a methodology employed to detect species with unpaired electrons (paramagnetic). This technology has been successfully applied to different plant tissues and subcellular compartments to asses both, NO content and generation. The spin trap MGD-Fe2+ is extensively employed to efficiently detect NO. Here, we describe a simple methodology to asses NO generation rate by isolated chloroplasts in the presence of either l-Arginine or nitrite (NO2 −) as substrates, since these compounds are required for enzymatic activities considered as the possible sources of NO generation in plants.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 39.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 54.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 54.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rosen GM (1985) Use of spin trap in biological systems. Free Radic Biol Med 1:345–375

    Article  CAS  Google Scholar 

  2. Moncada S, Palmer RMJ, Higgs EA (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109–142

    CAS  PubMed  Google Scholar 

  3. Stamler JS, Singel DJ, Loscalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258:1898–1902

    Article  CAS  PubMed  Google Scholar 

  4. Saran M, Michel C, Bors W (1990) Reaction of NO with O2. Implications for the action of endothelium-derived relaxing factor (EDRF). Free Radic Res Commun 10:221–222

    Article  CAS  PubMed  Google Scholar 

  5. Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434–456

    Article  CAS  PubMed  Google Scholar 

  6. Davis KL, Martin E, Turko IV et al (2001) Novel effects of nitric oxide. Annu Rev Pharmacol Toxicol 41:203–236

    Article  CAS  PubMed  Google Scholar 

  7. Rubbo H, Parthasarathy S, Barnes S et al (1995) Nitric oxide inhibition of lipoxygenase-dependent liposome and low density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen containing oxidized lipid derivatives. Arch Biochem Biophys 324:15–25

    Article  CAS  PubMed  Google Scholar 

  8. Gisone P, Dubner D, Pérez MR et al (2004) The role of nitric oxide in the radiation-induced effects in the developing brain. In Vivo 18:281–292

    CAS  PubMed  Google Scholar 

  9. Grisham MB, Johnson GG, Lancaster JR Jr (1996) Quantitation of nitrate and nitrite in extracellular fluids. Methods Enzymol 268:237–246

    Article  CAS  PubMed  Google Scholar 

  10. Miles AM, Wink DA, Cook JC et al (1996) Determination of nitric oxide using fluorescence spectroscopy. Methods Enzymol 268:105–120

    Article  CAS  PubMed  Google Scholar 

  11. Ventakaraman S, Martin SM, Schafer FQ et al (2000) Detailed methods for the quantification of nitric oxide in aqueous solutions using either an oxygen monitor or EPR. Free Radic Biol Med 29:580–585

    Article  Google Scholar 

  12. Jackson SK, Hancock JT, James PE (2007) Biological free radicals and biomedical applications of EPR spectroscopy. Electron Paramag Res 20:157–191

    Google Scholar 

  13. Caro A, Puntarulo S (1999) Nitric oxide generation by soybean embryonic axes. Possible effect on mitochondrial function. Free Radic Res 31:S205–S212

    Article  CAS  PubMed  Google Scholar 

  14. Jasid S, Galatro A, Villordo JJ et al (2009) Role of nitric oxide in soybean cotyledon senescence. Plant Sci 176:662–668

    Article  CAS  Google Scholar 

  15. Galatro A, Puntarulo S, Guiamet JJ et al (2013) Chloroplast functionality has a positive effect on nitric oxide level in soybean cotyledons. Plant Physiol Biochem 66:26–33

    Article  CAS  PubMed  Google Scholar 

  16. Jasid S, Simontacchi M, Puntarulo S (2008) Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes. J Exp Bot 59:3953–3962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Galatro A, Simontacchi M, Puntarulo S (2004) Effect of nitric oxide exposure on antioxidant capacity of soybean leaves. Current Topics Plant Biol 5:69–79

    CAS  Google Scholar 

  18. Jasid S, Simontacchi M, Bartoli CG et al (2006) Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins. Plant Physiol 142:1246–1255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. del Rio LA (2011) Peroxisomes as a cellular source of reactive nitrogen species signal molecules. Arch Biochem Biophys 506:1–11

    Article  PubMed  Google Scholar 

  20. Foissner I, Wendehenne D, Langebartels C et al (2000) In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. Plant J 23:817–824

    Article  CAS  PubMed  Google Scholar 

  21. Gould KS, Lamotte O, Klinguer A et al (2003) Nitric oxide production in tobacco leaf cells: a generalized stress response? Plant Cell Environ 26:1851–1862

    Article  CAS  Google Scholar 

  22. Arnaud N, Murgia I, Boucherez J et al (2006) An iron induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression. J Biol Chem 281:23579–23588

    Article  CAS  PubMed  Google Scholar 

  23. Tewari RK, Prommer J, Watanabe M (2013) Endogenous nitric oxide generation in protoplast chloroplasts. Plant Cell Rep 32:31–44

    Article  CAS  PubMed  Google Scholar 

  24. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  25. Edwards GE, Lilley RMC, Craig S et al (1979) Isolation of intact and functional chloroplasts from mesophyll and bundle sheath protoplasts of C4 plant Panicum miliaceum. Plant Physiol 63:821–827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schwitzguébel JP, Siegenthaler PA (1984) Purification of peroxisomes and mitochondria from spinach leaf by Percoll-density gradient centrifugation. Plant Physiol 75:670–674

    Article  PubMed  PubMed Central  Google Scholar 

  27. Quy LV, Foyer C, Champigny ML (1991) Effect of light and NO3 - on wheat leaf phosphoenolpyruvate carboxylase activity. Plant Physiol 97:1476–1482

    Article  Google Scholar 

  28. Bartoli CG, Gómez F, Martínez DE et al (2004) Mitochondria are the main target for oxidative damage in leaves of wheat (Triticum aestivum L.). J Exp Bot 55:1663–1669

    Article  CAS  PubMed  Google Scholar 

  29. Puntarulo S, Jasid S, Boveris AD et al (2009) Electron Paramagnetic Resonance as a tool to study nitric oxide generation in plants. In: Hayat S, Mori M, Pichtel J, Ahmad A (eds) Nitric oxide in plant physiology. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 17–30

    Chapter  Google Scholar 

Download references

Acknowledgments

This study was supported by grants from the UBA, ANPCyT and CONICET. S.P. and A.G. are career investigators from CONICET.

Author information

Authors and Affiliations

  1. Physical Chemistry-Institute of Biochemistry and Molecular Medicine (IBIMOL), School of Pharmacy and Biochemistry, University of Buenos Aires-CONICET, Junín, 956, C1113AAD, Buenos Aires, Argentina

    Andrea Galatro & Susana Puntarulo

Authors
  1. Andrea Galatro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susana Puntarulo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susana Puntarulo .

Editor information

Editors and Affiliations

  1. National Institute of Plant Genome Resea, New Delhi, India

    Kapuganti Jagadis Gupta

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Galatro, A., Puntarulo, S. (2016). Measurement of Nitric Oxide (NO) Generation Rate by Chloroplasts Employing Electron Spin Resonance (ESR). In: Gupta, K. (eds) Plant Nitric Oxide. Methods in Molecular Biology, vol 1424. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3600-7_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3600-7_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3598-7

  • Online ISBN: 978-1-4939-3600-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation