Nitrite assimilation and amino nitrogen synthesis in isolated spinach chloroplasts

Abstract

The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed CO(2) at rates of approximately 100 mumoles hr(-1) mg chlorophyll(-1).The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 mumoles hr(-1) mg chlorophyll(-1). The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of alpha-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of amino-nitrogen: NO(2) (-) ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when (alpha)-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants.