Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds

In vivo 15N nuclear magnetic resonance (NMR) as well as 15N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH4+ is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into α-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of 15NH4+ into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and γ-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH4+ can only be assimilated through the GS/GOGAT pathway in P. glauca buds.