Initial Organic Products of Assimilation of [13N]Ammonium and [13N]Nitrate by Tobacco Cells Cultured on Different Sources of Nitrogen

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Abstract
Glutamine is the first major organic product of assimilation of 13NH4+ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of 13NO3− by tobacco cells cultured on nitrate. The percentage of organic 13N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. 13NO3−, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of 13N in glutamine, and a concomitant increase in the percentage of 13N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of 13N from 13NH4+ into glutamine more extensively than it inhibits the incorporation of 13N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when 13NH4+ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of 13NH4+ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of 13N from 13NO3− into glutamate more strongly than it inhibits the incorporation of 13N into glutamine, suggesting that the assimilation of 13NH4+ derived from 13NO3− may be mediated solely by the glutamine synthetase-glutamate synthase pathway.