Extracellular and Intracellular NO Detection in Plants by Diaminofluoresceins
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 wordsFluorescence emission Leaf Root NO production
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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