Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O(2) evolution (mean of 13 preparations, 17.5 mumole per mg chlorophyll per hour, sd 3.64). The optimum concentration of nitrite was 0.5 mm; 0.12 mm nitrite supported V(max)/2. The reaction was accompanied by the consumption of nitrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. In short experiments (less than 10 minutes) the O(2) to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O(2) evolution with chloroplasts from plants grown without added nitrate but such chloroplasts could assimilate ammonia at about the usual rate. The results are consistent with the reduction of nitrite to ammonia involving nitrate-induced nitrite reductase and a reductant generated by the chloroplast electron transport chain.In the presence of ADP, pyrophosphate, and MgCl(2) the O(2) to nitrite ratio was typically 0.5 to 0.6 and the recovery of nitrite-N as ammonia about 60%. Under these conditions, alpha-ketoglutarate increased the O(2) to nitrite ratio (0.9-1.35) and the recovery of nitrite-N as ammonia decreased to 27%. These data and the results of nitrite plus ammonia addition experiments (with and without alpha-ketoglutarate) are attributed to incorporation of nitrite-N into glutamate via the chloroplast enzymes nitrite reductase, glutamine synthetase, and glutamate synthetase.