Critical evaluation of the in vivo nitrate reductase assay for detection of two nitrate pools in wheat leaves

The usefulness of the nitrate‐free in vivo nitrate reductase assay for the study of nitrate pools in wheat leaves was investigated. Leaf sections from 7‐day‐old wheat seedlings, exposed 24 h before harvest to 1.5, 3.0 or 5.0 mM KNO3 were used. After 2 to 4 h of incubation nitrite production ceased, reaching a plateau. The time required to reach the plateau and the level of the plateau increased with increasing endogenous nitrate content. At nitrite plateau the amount of nitrate left in the tissue was independent of the original nitrate content in the tissue. Addition of nitrate at plateau caused resumed nitrite production. It is concluded that nitrate was the limiting factor in nitrite production. Oxygen inhibited nitrate reduction and stimulated further assimilation of nitrite. A considerable initial leakage of nitrate from tissue to the assay medium, followed by a slower continuous leakage, was observed throughout the incubation. N2‐flushing or inclusion of Triton X‐100 in the assay medium increased nitrite production by making more nitrate available for reduction. These treatments also increased the leakage of nitrate. At plateau levels the amount of nitrate left in the tissue was dependent on the oxygen tension in the assay medium. Under low oxygen tension nearly all nitrate in the tissue was available for reduction. Nitrite production at plateau is not a useful index for a metabolic nitrate pool and nitrate left in the tissue is not a useful index for a nitrate storage pool because both parameters are highly dependent on the oxygen tension in the assay medium. Further, in view of the considerable leakage, the nitrate‐free in vivo nitrate reductase assay cannot be used to detect two separate nitrate pools in wheat leaves.