Quantitative Estimation of Nitric Oxide in situ (Griess Assay)

Freshly treated root tissues were crushed in phosphate-buffered saline in an ice bath and centrifuged at 4°C for 30 min, and the supernatant was used for further analyses. Since nitrite is the only stable component that can convert into NO, 1 U nitrate reductase (NR, Sigma) was added to the suspension which was then incubated for 30 min; 100 μl of supernatant from each set was then dispensed into a plate reader in triplicate, and an equal volume of Griess reagent [1% sulfanilamide in 5% phosphoric acid; and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride; Sigma-Aldrich] was added, before incubation for 15 min. The color reaction was assayed using a Bio-Rad iMark^TM microplate reader at 550 nm (Saha et al., 2004) and expressed as μg g^–1 fresh weight.