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

Abstract

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.