Enzymic Assimilation of Nitrate in Tomato Plants. II. Reduction of Nitrite to Ammonia.

It is generally assumed at present that nitrate is reduced to the level of amino-nitrogen or ammonia, via nitrite (20, 21, 32). Support for this assumption comes from the demonstration of a widespread occurrence of nitrate reductase in higher plants (27). However, as was pointed out previously (27), a final decision as to the validity of this assumption awaits the characterization of an enzyme system capable of reducing nitrite to ammonia. Nason, Abraham, and Averbach (22) in a brief report stated that nitrite was reduced to ammonia, in the presence of either DPNH or TPNH, and Mn++, by a partially purified soybean leaf extract. However, the results showed that significant quantities of ammonia were formed even in the absence of added nitrite in spite of the fact that the enzyme preparation should have been free of nitrate and nitrite due to the purification carried out. Vaidyanathan and Street (29) reported cell-free enzyme extracts of excised tomato roots which caused a disappearance of nitrite in the presence of added DPNH and Mn+ +, but only 2 % of the nitrite lost could be recovered as ammonia. Roussos and Nason (23) reported a highly purified enzyme from soybean leaves which would oxidize DPNH and TPNH in the presence of nitrite or hydroxylamine. However, nitrite did not disappear and the product of hydroxylamine disappearance could not be identified. Huzisige and Satoh (13) have reported an enzyme from spinach leaves which will cause a disappearance of nitrite in the presence of illuminated chloroplasts without the addition of an electron carrier. The product of the reaction was not identified. The reduction of nitrite was inhibited by the addition of TPN. Hageman, Cresswell, and Hewitt (10) were able to demonstrate that ammonia was the product of nitrite reduction by a marrow leaf enzyme preparation, but their system was dependent on the use of reduced benzyl viologen as an electron donor. Neither DPNH nor TPNH could serve as electron donor for this enzymic reduction in the absence of benzyl viologen. The present investigation was undertaken for the purpose of clarifying these conflicting reports in order to establish the role of nitrate reductase in nitrate assimilation in higher plants. During the course of the investigation additional information on the nature