Abstract
Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.
Keywords: Nitrate reductase, in vitro NR activity assay, Nitrite concentration
Background
Nitrogen is crucial macronutrient required by plants and is mainly absorbed in the form of nitrate. Nitrate reductase is the first enzyme of the nitrogen assimilation in higher plants. Homodimers of plant nitrate reductase catalyze the NAD(P)H-dependent reduction of nitrate to nitrite as follows:NO3- + NADH + H+ → NO2- + NAD+ + H2OMethods to measure NR activity may be a powerful tool to investigate biological factors influencing NR activity (Park et al., 2011). Nitrogen assimilation affects the contents of amino acid in plant, thus regulating NR activity could be used for increasing quality of some crop (Croy and Hageman, 1970; Dalling and Loyn, 1977; Ruan et al., 1998).In this protocol, increased nitrite concentrations during limited time in optimized buffer condition are acquired as comparable values. Nitrite concentration is measured by its absorbance at 540 nm through Griess assay. Briefly, nitrite forms a diazonium salt with sulfanilic acid, then N-(1-naphthyl) ethylenediamine dihydrochloride is formed colored azo compound. It is possible to compare the values to determine how samples have different NR activity. Furthermore, the values could be converted to exact increased nitrite concentration through a simple process.
Materials and Reagents
Equipment
Procedure
Data analysis
Values acquired from this protocol are quantity of nitrite molecules. Thus, calculate the NR activity as follows: ΔA = Asample - Ablank Each sample is assayed in triplicate and ΔA value is blank subtracted from the average of triplicates. ΔA value means the increased concentration of nitrite which is produced during 2 h incubation time (see Step C2) and it could be used as a comparable scale of NR activity. For someone who wants to acquire the exact increased nitrite concentration, calculate the concentration change using the standard curve. The ratio of increasing nitrite content of the sample (nmol NO2- g-1 FW h-1) using the formula as follows: [change of nitrite concentration (μM)] x [extracted volume (ml)]/[fresh weight (g)]/[reaction time (h)]
Recipes
Acknowledgments
This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ01327601), Rural Development Administration, Republic of Korea. This protocol was adapted and modified as described previously (Samuelson and Larsson, 1993; Yu et al., 1998, Hachiya and Okamoto, 2016). The authors declare that they have no conflict of interest.
References
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