Relation Between CO2 Evolution and in situ Reduction of Nitrate in Wheat Leaves

In wheat leaf discs the evolution of 14CO2 from exogenously supplied 14C-labelled citric acid cycle intermediates was stimulated during the in situ anaerobic reduction of nitrate in the dark. Under these conditions, however, [1,4-14C]succinate was not metabolized. Similarly, when leaves were allowed to assimilate 14CO2 in the dark, thus producing endogenously labelled organic acids, the subsequent evolution of 14CO2 from discs prepared from these leaves was strongly dependent on nitrate reduction. A 1 : 1 stoichiometry between nitrite production and CO2 evolution was recorded during this in situ reduction of nitrate. The in situ reduction of nitrate was inhibited by malonate and D-malate and this effect was reversed by fumarate, probably by generating L-malate within the mitochondria. Mitochondrial NAD-malic enzyme (decarboxylating) (EC 1.1.1.38) was similarly inhibited competitively by malonate and D-malate, but not by succinate. These results indicate that the citric acid cycle dehydrogenases which generate CO2 supply NADH for nitrate reduction in wheat leaves. It is likely that, under anaerobic conditions, nitrate acts as an alternative oxidant to O2 for the NADH generated by the citric acid cycle dehydrogenases resulting in simultaneous evolution of CO2. This ensures that the citric acid cycle operates at the required rate for nitrate assimilation.