Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [(15)N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O(2) inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.Oxygen control of nitrate reduction was not observed in the light, since protoplasts assimilated [(15)N]nitrate to amino-N under light aerobic conditions. It has been proposed that the increase of the ATP-to-ADP ratio in the light inhibits dark respiration and allows nitrate reduction to occur under aerobic conditions. To test this hypothesis, protoplast N and C assimilation was assayed in the presence of nigericin, an uncoupler of photophosphorylation. The dark to light increase of the protoplast energy charge was not observed in the presence of nigericin, and CO(2) fixation was completely inhibited by the uncoupler. In contrast, rates of in vivo nitrate reduction (N(2) and air) and nitrite reduction were relatively unaffected by nigericin, and light-driven nitrate assimilation was inhibited by only 20%. Nigericin had no effect on the dark-to-light increase of protoplast NADH and NADPH levels. It is proposed that the light-induced increase of cytoplasmic-reducing equivalents suppresses the competition between nitrate reduction and dark respiration and allows nitrate reduction to occur under aerobic conditions. Dark-to-light changes of the ATP-to-ADP ratio apparently are not critical to the regulation of nitrate reduction.