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.