Nitrate Reductase in the Field Pea (Pisutn arvense L.)

The extraction and assay of a soluble, NADH-requiring nitrate reductase is described. Induction of the enzyme by nitrate is demonstrated in the roots and shoot of young seedlings and in the leaves of older, nodulated plants grown without inorganic nitrogen. Induction occurs most readily in actively growing tissues. Studies with sterile cultures of excised roots confirm the presence of an endogenous enzyme system within the root. Assays of the enzyme in vitro are combined with analyses of free nitrate and organic compounds of nitrogen in the bleeding sap. Either the root or shoot may act as the main centre for reduction of incoming nitrate. The extent to which these organs function is apparently influenced by environmental factors, particularly the level of nitrate in the rooting medium. The diurnal rhythm of export of nitrogenous substances from the root is correlated with observations on daily fluctuations in the level of extractable enzyme in root and leaves. The activity of the enzyme is studied in different ages of leaf of plants grown on a constant supply of nitrate. Assays suggest that the enzyme is most active just as a leaf is fully expanded. Thereafter enzyme activity falls sharply, although small amounts of active enzyme may be recovered until a leaf becomes senescent.