INORGANIC NITROGEN-METABOLISM IN EMBRYOGENIC WHITE SPRUCE CULTURES - A NITROGEN-14 15 NMR-STUDY/

Inorganic nitrogen metabolism in embryogenic white spruce cultures, wa s investigated by N-14/N-15 NMR spectroscopy, amino acid analysis, and nitrate determinations. Cultures were exposed to N-15-isotopically-la belled nitrate and ammonium in various combinations, and their uptake, storage, reduction and incorporation into metabolites was followed by N-14 and N-15 NMR. The isotopic-labelling experiments indicated that nitrate pools turned over slowly (T-1/2 = 3.1 days), and that ammonium was taken up and incorporated into metabolites faster than nitrate. N MR perfusion experiments with (NH4+)-N-15 showed that the amide nitrog en of Gln was the first resonance labelled followed by alpha-amino nit rogen and Ala. Use of the glutamine synthetase inhibitor methionine su lfoximine indicated that the glutamine synthetase/glutamate synthase p athway is the major route of nitrogen assimilation in these tissues. T race amounts of Arg were found in early filamentous embryos, and in th eir embryonal masses only. Amino acid analysis showed that developing embryos contained significant amounts of Glu, Gln and Arg; metabolites that were also substantially labelled with N-15 during long-term expo sure. Hence, during white spruce somatic embryo development there is a switch in amino acid metabolism, involving increases in Glu, Gln and Arg and a decrease in Ala between the early filamentous and precotyled onary stages of embryo development.