15N Studies on the In Vivo Assay of Nitrate Reductase in Leaves: Occurrence of Underestimation of the Activity Due to Dark Assimilation of Nitrate and Nitrite

摘要

The reduction of nitrate and nitrite in leaf disks from seven di- and two monocotyledonous species under in vivo nitrate reductase assay conditions was studied using 15N-labeled substrates. Significant reduction of both nitrate and nitrite into ammonia and amino acids was detected under aerobic conditions (in an atmosphere of air): in some cases, the amount of nitrate-N reduced to ammonia and amino acids was more than that remaining as nitrite. Anaerobic incubation (under an atmosphere of N2gas) enhanced the accumulation of nitrite, but the subsequent reduction to the basic nitrogen compounds was 40 to 180of the aerobic rates. The present examination indicates that in vivo assays of nitrate reductase under aerobic conditions may give greatly underestimated results due to nitrite reduction and that exclusion of oxygen from the in vivo assay mixture is desirable in terms of the quantity of nitrite formed.n-Propanal treatment increased nitrite accumulation under aerobic but not under anaerobic conditions, and depressed the incorporation of nitrate-N into basic fractions under both conditions. Therefore, addition ofn-propanol may be desirable for assays under aerobic conditions. No significant difference in the reduction of nitrate supplied as sodium and potassium salts was observed on the nitrite formation and on the incorporation of nitrate-N into basic fractions.15N experiments on dark assimilation of nitrate, nitrite and ammonia into amino acids in wheat leaves showed that these three nitrogen sources were assimilated through the same route and that the glutamine synthetase/glutamate synthase pathway was the major route. With anaerobic treatment, the incorporation of nitrogen into alanine and serine remained at relatively high, but the incorporation into aspartate and asparagine was much lower than in the cases of aerobic treatment.