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.
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References
Sthör C, Stremlau S (2006) Formation and possible roles of nitric oxide in plant roots. J Exp Bot 57:463–470
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 70:2446–2454
Yao D, Vlessidis AG, Evmiridis NP (2004) Determination of nitric oxide in biological samples. Microchim Acta 147:1–20
Mur LAJ, Mandon J, Cristescu SM, Harren FJM, Prats E (2011) Methods of nitric oxide detection in plants: a commentary. Plant Sci 181:509–519
Nagano T (1999) Practical methods for detection of nitric oxide. Luminescence 14:283–290
Halbach O (2003) Nitric oxide imaging in living neuronal tissues using fluorescent probes. Nitric Oxide 9:217–228
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 269–280
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 1424:57–68
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 99:16314–16318
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 115:354–359
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 47:49–55
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).
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Silveira, N.M., Machado, E.C., Ribeiro, R.V. (2019). Extracellular and Intracellular NO Detection in Plants by Diaminofluoresceins. In: Hancock, J., Conway, M. (eds) Redox-Mediated Signal Transduction. Methods in Molecular Biology, vol 1990. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9463-2_9
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DOI: https://doi.org/10.1007/978-1-4939-9463-2_9
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