In vivo Imaging of Nitric Oxide and Reactive Oxygen Species Using Laser Scanning Confocal Microscopy

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 913)

Abstract

Both nitric oxide (NO) and reactive oxygen species (ROS) are versatile molecules that mediate a variety of cellular responses in plants. In this chapter, methods for imaging NO and ROS using laser scanning confocal microscopy (LSCM) are presented. Arabidopsis roots, dyed with DAF-FM or H2DCF, are observed using the Leica TCS-SP2 LSCM. NO or ROS production are imaged and their kinetic changes monitored with the laser excitation and emission wavelengths at 488 nm and between 500 and 530 nm, respectively. In addition, Leica software is employed to visualize and calculate the fluorescence intensity data.

Key words

Nitric oxide Reactive oxygen species Laser scanning confocal microscopy 

References

  1. 1.
    Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526PubMedCrossRefGoogle Scholar
  2. 2.
    Prast H, Philippu A (2001) Nitric oxide as modulator of neuronal function. Prog Neurobiol 64:51–68PubMedCrossRefGoogle Scholar
  3. 3.
    Esplugues JV (2002) NO as a signaling molecule in the nervous system. Br J Pharmacol 135:1079–1095PubMedCrossRefGoogle Scholar
  4. 4.
    Culotta E, Koshland DE Jr (1992) NO news is good news. Science 258:1862–1865PubMedCrossRefGoogle Scholar
  5. 5.
    Snyder SH (1992) Nitric oxide: first in a new class of neurotransmitters. Science 257:494–496PubMedCrossRefGoogle Scholar
  6. 6.
    Vincent SR (1992) Nitric oxide and arginine-evoked insulin secretion. Science 258:1376–1378PubMedCrossRefGoogle Scholar
  7. 7.
    Durner J, Klessig DF (1999) Nitric oxide as a signal in plants. Curr Opin Plant Biol 2:369–374PubMedCrossRefGoogle Scholar
  8. 8.
    Wendehenne D, Pugin A, Klessig DF, Durner J (2001) Nitric oxide: comparative synthesis and signaling in animals and plant cells. Trends Plant Sci 6:177–183PubMedCrossRefGoogle Scholar
  9. 9.
    Crawford NM, Guo FQ (2005) New insights into nitric oxide metabolism and regulatory functions. Trends Plant Sci 10:195–200PubMedCrossRefGoogle Scholar
  10. 10.
    Lamattina L, García-Mata C, Graziano M, Pagnussat G (2003) Nitric oxide: the versatility of an extensive signal molecule. Annu Rev Plant Biol 54:109–136PubMedCrossRefGoogle Scholar
  11. 11.
    Besson-Bard A, Pugin A, Wendehenne D (2008) New insights into nitric oxide signaling in plants. Annu Rev Plant Biol 59:21–39PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang H, Shen WB, Zhang W, Xu LL (2005) A rapid response of β-amylase to nitric oxide but not gibberellin in wheat seeds during the early stage of germination. Planta 220:708–716PubMedCrossRefGoogle Scholar
  13. 13.
    Courtois C, Besson A, Dahan J, Bourque S, Dobrowolska G, Pugin A, Wendehenne D (2008) Nitric oxide signaling in plants: interplays with Ca2+ and protein kinases. J Exp Bot 59:155–163PubMedCrossRefGoogle Scholar
  14. 14.
    Hetrick EM, Schoenfisch MH (2009) Analytical chemistry of nitric oxide. Annu Rev Anal Chem 2:409–433CrossRefGoogle Scholar
  15. 15.
    Zhao MG, Tian QY, Zhang WH (2007) Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in arabidopsis. Plant Physiol 144:206–217PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao MG, Chen L, Zhang LL, Zhang WH (2009) Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151:755–767PubMedCrossRefGoogle Scholar
  17. 17.
    Asai S, Ohta K, Yoshida H (2008) MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana. Plant Cell 20:1390–1406PubMedCrossRefGoogle Scholar
  18. 18.
    Xie YJ, Ling TF, Han Y, Liu KL, Zheng QS, Huang LQ, Yuan XX, He Z, Hu B, Fang L, Shen ZG, Yang Q, Shen WB (2008) Carbon monoxide enhances salt tolerance by nitric oxide-mediated maintenance of ion homeostasis and up-regulation of antioxidant defense in wheat seedling roots. Plant Cell Environ 31:1864–1881PubMedCrossRefGoogle Scholar
  19. 19.
    Lozano-Juste J, León J (2010) Enhanced abscisic acid-mediated responses in nia1nia2noa1-2 triple mutant impaired in NIA/NR- and AtNOA1-dependent nitric oxide biosynthesis in Arabidopsis. Plant Physiol 152:891–903PubMedCrossRefGoogle Scholar
  20. 20.
    Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Nagano T (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70:2446–2453PubMedCrossRefGoogle Scholar
  21. 21.
    Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T (1999) Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed Engl 38:3209–3212PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.College of Life SciencesNanjing Agricultural UniversityNanjingChina

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