Optimization of the nitrate reductase activity assay for citrus trees

The aim of this study was to assess factors that can affect the in vivo and in vitro assays of the nitrate reductase activity (NRA) in sweet orange trees and then standardize conditions for tissue sampling and analysis. Seven-month-old plants grown in pots were used, of which, mature and healthy leaves between the third and the fifth position from the branch apex, stems, and roots were assessed. One half of each leaf was used for in vivo tests, and the other half was used for in vitro tests. In addition to varied incubation time and temperature, in vivo KNO3 and n-propanol concentrations and in vitro KNO3 and NADH concentrations were evaluated. The optimum conditions for the in vivo NRA assay in leaves were: 200 mmol L-1 KNO3 and 1 % n-propanol at 40 degrees C for 20 min. The highest leaf NRA occurred at 11:00 h for the in vivo assay and at 13:00 h for the in vitro assay, with both analysis showing similar results. Overestimation of the in vitro NRA occurred as compared to the in vivo analysis when accessed early morning and late afternoon. Branches bearing fruits show reduced NRA in young mature leaves, whereas sprouting significantly increases NRA in correspondent leaves. For the root assays, the optimized conditions for the NRA estimation were the same as for leaves. Although roots and stems (bark) have shown some NRA, it was six times lower than leaf NRA. Our data indicate that NO3- reduction occurs mostly in leaves and there is a significant effect of daytime and leaf position in relation to fruit or sprouts on NRA of citrus trees.