Otimização do método para a análise da enzima redutase do nitrato na cultura do milho

Abstract

In Brazil, corn occupies great importance and prominence among the products of the agricultural sector due to its versatile use and has prominence among grasses for the response with greater intensity to nitrogen fertilization. Nitrogen is absorbed mainly in the form of nitrate (NO3-) and this anion is first reduced to nitrite (NO2-) by the enzyme Nitrate Reductase (NR). Determining nitrate reductase activity (aRN) becomes important because plants with high enzyme activity have a higher ability to assimilate nitrogen and respond to nitrogen fertilization. Mulder, Boxma and van Veen (1959) were the first to publish the in vivo nr assay in leaf discs of olericultural plants, however, each species has different anatomical and physiological characteristics, so it is necessary to establish appropriate and particular analytical conditions for each type of plant. The general objective of this study was to standardize and characterize the optimal conditions for determining the activity of nitrate reductase enzyme (aRN) in fresh corn leaves. The BM 3051 hybrid was cultivated at Fazenda Escola Boa Esperança located in Barra do Garças - MT. To estimate aRN, the simplified in vivo method of Mulder, Boxma and van Veen (1959) was used. Diagnostic leaves of corn were collected and incubated in potassium nitrate solution at 0.25 M for two hours at 35°C. Subsequently, 1 mL of this solution was transferred to volumetric balloon and sulphanyl acid, alphanaphthylamine and sodium acetate buffer were added. After solution rested, small amount was transferred to a cuvette and read in spectrophotometer set to 540nm. The optimization of the method consisted of sequential experiments, in which different conditions of preparation the incubation medium were studied, varying: type of cut of the fresh leaf sample, leaf tissue mass used, incubation time of the samples, potassium nitrate concentration (KNO3) in the buffer solution and incubation temperature. In each experiment, one factor was modified and, for the next one, the highest aRN value observed in the previous experiment was based. The proposed changes to the current enzymatic method to be optimized in corn crop consist of cutting the samples of diagnostic leaves in the whole disk format, using 0.2 g of leaf tissue mass that should be incubated in solution with 0.16 M KNO3 in phosphate buffer pH 7.4, for 1 hour and 30 minutes, temperature that can range from 30°C to 35°C. The conditions for quantifying nitrate reductase enzyme activity in corn leaves were standardized.