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Extracellular and Intracellular NO Detection in Plants by Diaminofluoresceins

  • Neidiquele Maria Silveira
  • Eduardo Caruso Machado
  • Rafael Vasconcelos Ribeiro
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1990)

Abstract

Many assays focus on determining NO content within plant tissues to assess the actual concentration that impacts on cellular processes. Diaminofluorescein fluorescent dyes (DAFs) have been very widely used by plant scientists to reveal likely sites of NO production inside and outside cells. In general, DAFs dyes react with N2O3, a byproduct of NO oxidation, resulting in fluorescence. It is initially available in the form of diacetate (DAF-2DA), which allowed the ready absorption by the cells. The diacetate group is removed by cell esterases leaving the membrane impermeable to DAF-2 and available for N2O3 nitration to generate the highly fluorescent triazole (DAF-2T). Here, we describe two methods for detection of NO by fluorescence, one for NO extracellular detection by DAF-2 and the other one for NO intracellular detection, in this case using DAF-2DA.

Key words

Fluorescence emission Leaf Root NO production 

Notes

Acknowledgments

N.M.S. acknowledges the São Paulo Research Foundation (Fapesp, Brazil) (Grant no. 2012/19167-0 and 2015/21546-7), and R.V.R. and E.C.M. acknowledge the fellowship granted by the National Council for Scientific and Technological Development (CNPq, Brazil). The authors also acknowledge the Centre for Research in Biosciences at University of the West of England (UWE).

References

  1. 1.
    Sthör C, Stremlau S (2006) Formation and possible roles of nitric oxide in plant roots. J Exp Bot 57:463–470CrossRefGoogle Scholar
  2. 2.
    Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70:2446–2454CrossRefGoogle Scholar
  3. 3.
    Yao D, Vlessidis AG, Evmiridis NP (2004) Determination of nitric oxide in biological samples. Microchim Acta 147:1–20CrossRefGoogle Scholar
  4. 4.
    Mur LAJ, Mandon J, Cristescu SM, Harren FJM, Prats E (2011) Methods of nitric oxide detection in plants: a commentary. Plant Sci 181:509–519CrossRefGoogle Scholar
  5. 5.
    Nagano T (1999) Practical methods for detection of nitric oxide. Luminescence 14:283–290CrossRefGoogle Scholar
  6. 6.
    Halbach O (2003) Nitric oxide imaging in living neuronal tissues using fluorescent probes. Nitric Oxide 9:217–228CrossRefGoogle Scholar
  7. 7.
    Arita NO, Cohen MF, Tokuda G, Yamasaki H (2006) Fluorometric detection of nitric oxide with diaminofluoresceins (DAFs): applications and limitations for plant NO research. In: Lamattina L, Polacco JC (eds) Nitric oxide in plant growth, development and stress physiology. Springer, Berlin, pp 269–280Google Scholar
  8. 8.
    Ruemer S, Krischke M, Fekete A, Lesch M, Mueller MJ, Kaiser WM (2016) Methods to detect nitric oxide in plants: are DAFs really measuring NO? Methods Mol Biol 1424:57–68CrossRefGoogle Scholar
  9. 9.
    Desikan R, Griffiths R, Hancock JT, Neill S (2002) A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid induced stomatal closure in Arabidopsis thaliana. Proc Natl Acad Sci U S A 99:16314–16318CrossRefGoogle Scholar
  10. 10.
    Silveira NM, Hancock JT, Frungillo L, Siasou E, Marcos FCC, Salgado I, Machado EC, Ribeiro RV (2017) Evidence towards the involvement of nitric oxide in drought tolerance of sugarcane. Plant Physiol Biochem 115:354–359CrossRefGoogle Scholar
  11. 11.
    Bright J, Hiscock SJ, James PE, Hancock JT (2009) Pollen generates nitric oxide and nitrite: a possible link to pollen-induced allergic responses. Plant Physiol Biochem 47:49–55CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Neidiquele Maria Silveira
    • 1
  • Eduardo Caruso Machado
    • 1
  • Rafael Vasconcelos Ribeiro
    • 2
  1. 1.Laboratory of Plant Physiology “Coaracy M. Franco”, Center R&D in Ecophysiology and BiophysicsAgronomic Institute (IAC)CampinasBrazil
  2. 2.Laboratory of Crop Physiology, Department of Plant Biology, Institute of BiologyUniversity of Campinas (UNICAMP)CampinasBrazil

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