Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

Abstract

Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O₂ evolution (mean of 13 preparations, 17.5 μmole 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 O₂ to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O₂ 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 MgCl₂ the O₂ to nitrite ratio was typically 0.5 to 0.6 and the recovery of nitrite-N as ammonia about 60%. Under these conditions, α-ketoglutarate increased the O₂ 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 α-ketoglutarate) are attributed to incorporation of nitrite-N into glutamate via the chloroplast enzymes nitrite reductase, glutamine synthetase, and glutamate synthetase.