Determination of nitrate reductase activity (NR) was based on the in vivo assay method, which was standardised for leaves and roots of pineapple cultivated in vitro. The highest activity was obtained when tissues were incubated in 0.1 M phosphate buffer, 100 mM KNO3 and 3% of n-propanol. The optimal pH ranged from 6.5 to 7.5. The incubation period was 60 min at 30 °C. This standardisation was important for the diurnal rhythm analysis of nitrate reductase activity in pineapple because the conditions of in vivo assay for this enzyme vary significantly among different plant species. The diurnal rhythm analysis showed that in leaves, the highest activity occurred during the day. The NR activity in the roots during the dark period was similar to that observed during the light period. The NR activity in roots was always higher than in leaves, indicating that roots are an important site for nitrate reduction when pineapple is cultivated in vitro. The nitrate accumulation in leaves during the diurnal cycle showed that it is present in higher concentrations in the light period. Therefore a positive correlation between nitrate concentration and NR activity could be established. In roots, however, the highest nitrate concentrations were observed in the absence of light. In this case, it is possible that other factors, besides nitrate, contributed to a higher enzymatic activity during the light period.
Nitrate reductase; pineapple; diurnal rhythm; bromeliad; Bromeliaceae
