Otimização da análise da atividade da redutase do nitrato e sua caracterização em folhas de cana-de-açúcar

Abstract

Resumo -O objetivo deste trabalho foi padronizar e caracterizar as condições para determinação da atividade da redutase do nitrato em tecido foliar de cana-de-açúcar, com uso do método in vivo. Amostras foliares foram coletadas de uma lavoura de primeira soqueira da cultivar IACSP 933046, com idade de seis meses. Foram estudadas diferentes condições de preparo das amostras foliares e do meio de incubação. O material que possibilitou a maior atividade da redutase do nitrato foi obtido pela amostragem de 25 discos de 1 cm de diâmetro, coletados às 13h, do centro da folha do tipo +1 sem nervura. O meio de incubação otimizado para a determinação da atividade dessa enzima em folhas de cana-de-açúcar deve ser composto por: 2,5 mL de KNO 3 300 mmol L -1 ; 2,5 mL de tampão fosfato 285 mmol L -1 pH 7,3; 1,0 mL de Tween 20 a 0,6% (v/v); e 4,0 mL de água deionizada. A maior atividade da redutase do nitrato é obtida pela incubação das amostras por 90 min, a 32°C, no escuro; é observada em plantas jovens formadas pela brotação da soqueira; e alcança o valor mínimo na fase de maturação das plantas.Termos para indexação: Saccharum, atividade enzimática, estádio fenológico, método in vivo, nitrogênio, ritmo diurno. Optimization of nitrate reductase activity analysis and its characterization in sugarcane leavesAbstract -The objective of this work was to standardize and characterize the conditions for determining the nitrate reductase activity in sugarcane leaf tissue, using in vivo method. Leaf samples were collected from a first ratoon crop of the IACSP 933046 cultivar, with six months of age. Different preparation conditions of leaf samples and incubation media were studied. The material that allowed the highest nitrate reductase activity was obtained by the sampling of 25 discs of 1 cm in diameter, collected at 13h from the center of the +1 leaf type without center rib. The incubation medium optimized to determine nitrate reductase activity in sugarcane leaves must be comprised of: 2.5 mL KNO 3 300 mmol L -1 ; 2.5 mL phosphate buffer 285 mmol L -1 pH 7.3; 1.0 mL Tween 20 0.6% (v/v); and 4.0 mL deionized water. The highest nitrate reductase activity is obtained by incubating samples for 90 min, at 32°C, in the dark; is observed in young plants formed by stump sprouting; and reaches a minimum value at the plant maturity phase.Index terms: Saccharum, enzymatic activity, phenological stage, in vivo method, nitrogen, diurnal rhythm. IntroduçãoA cana-de-açúcar (Saccharum officinarum L.) é considerada a principal matéria prima para a produção de biocombustíveis renováveis no mundo (Miller, 2010); e, portanto, é grande o interesse por conhecimento relacionado à cultura. Muitos fenômenos ligados à aquisição de N pelas plantas de cana-de-açúcar ainda não estão bem esclarecidos, e práticas, como adubação, irrigação e correção do solo para o cultivo, precisam ser melhor estudadas.A atividade da enzima redutase do nitrato (RN) [EC 1.6.6.1] pode ser utilizada como ferramenta complementar para interpretações de resultados experimentais. Div...

Characterization of nitrate reductase activity (NR) in foliar and radicular tissues of Physalis angulata L.: diurnal variations and protocol optimization

“…The enzyme activity in the leaf was observed when exogenous nitrate was supplied, being 50mM the adopted concentration for subsequent analyses, since there were no differences between the higher concentrations tested ( Figure 3C). This stabilization tendency was observed by other authors to sugarcane, Brachiaria and pineapple leaves (Santos et al, 2014;Cazetta and Villela, 2004;Nievola and Mercier, 2001). The initial increase in aNR in response to nitrate addition to the medium has been linked to a de novo enzyme synthesis as well as its activation (Solomonson and Barber, 1990).…”

“…However, these effects varies according to cellular and anatomical characteristics of plants of different species. In sugarcane leaves, the reduction of aNR was observed with the increase of n-propanol concentration (Santos et al, 2014). Regarding the radicular tissue of Citrus sinensis L. Osbeck, there was no difference between concentrations, but for foliar tissues a reduction was occurred starting at 3% (Dovis et al, 2014).…”

“…Nievola and Mercier (2001) also observed this performance in pineapple leaves, which presented greater aNR in the presence of light. Santos et al (2014) observed maximum values of aNR at 01:00pm for sugarcane, indicating that the global solar radiation may be the climatic factor that most influences the enzyme activity. However, for coffee plants it was observed a gradual decline throughout the light period, followed by an increase of activity in the first hours of the night (Queiroz et al, 1993).…”

“…However, this method has the disadvantage of requiring adaptations in assay conditions, according to the nature of the vegetal tissue (Jaworski, 1971). Also, another important factor is the knowledge of the diurnal rhythm of NR, which has already been observed in some plants (Nievola and Mercier, 2001;Santos et al, 2014;Oliveira et al, 2005;Queiroz et al, 1993), indicating the increase of NR activity concomitant to luminous intensity. It is extremely important to determine the ideal schedule for performing the assay, coinciding with the period of NR maximum activity (Nievola and Mercier, 2001).…”

“…It is extremely important to determine the ideal schedule for performing the assay, coinciding with the period of NR maximum activity (Nievola and Mercier, 2001). Each species has its anatomical and physiological particularities, so it is necessary to determine appropriate conditions to each species (Santos et al, 2014). Passos (2013) evaluated the NR activity in plants of Physalis peruviana L. and Physalis pubescens L. under different doses of nitrogen fertilizing.…”