Characterization of in Vivo Nitrate Reductase Activity in Triticum Vulgare Seedlings

Vinodh Ratnala¹, Dsvgk Kaladhar², B. Seshagiri³ and N. Sarada Mani4

¹Department of Biotechnology, MAS Lab, NRC for Sorghum, Rajendranagar, Hyderabad India.

²Department of Bioinformatics, GITAM Institute of Science, GITAM University, Visakhapatnam India.

³Department of Biochemistry, M.V.R College of Post-Graduate Studies, Visakhapatnam India.

⁴Department of Botany, Andhra University, Visakhapatnam India.

ABSTRACT: Nitrate assimilation plays an important role in plant growth and bio-productivity. The first step of this process is the reduction of nitrate to nitrite in the cytosol. Investigations are undertaken to study the optimum conditions for assaying this enzyme in seedlings of Triticum vulgare. The in vivo NR activity in seven-day-old green seedlings increased linearly with the mass of the leaf tissue in the infiltration (assay) medium (5 ml total volume) up to 400 mg tissue. The optimum concentration of substrate, nitrate, in the external assay medium was 100 mM, the optimum assay pH was 7.5, with a decrease in enzyme activity in both acidic and alkaline ranges. The optimum assay temperature was 30°C with greatest inhibition of enzyme activity at 50°C (67%) at the end of 1 h exposure of leaf segments in the assay medium. Among the low molecular weight organic solvents added to the assay medium to enhance enzyme activity, 1% (v/v) n-butanol was the most effective causing 420% increase in enzyme activity. Where as 1%(v/v) acetone was the least potent organic solvent. 0.1% Triton X-100, a surfactant as well as a detergent, caused a 3.7 fold enhancement in NR activity over control.

KEYWORDS: In vivo characterization; Nitrate Reductase(NR) activity; Triticum vulgare

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