A 14N and 15N Nuclear Magnetic Resonance Study of Nitrogen Metabolism in Shoot-Forming Cultures of White Spruce (Picea glauca) Buds

Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other alphaNH(2) groups), Pro, Ala, and the side chain groups in Gln, Arg, Orn, and gamma-aminobutyric acid could be detected in in vivo(15)N NMR spectra. Peaks for alpha-amino groups, Pro, NO(3) (-) and NH(4) (+) could also be identified in (14)N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that (15)N-labeled NH(4) (+) and NO(3) (-) are first incorporated into the amide group of Gln and then in the alphaNH(2) pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH(4) (+) is much faster than that of NO(3) (-). Consequently after 10 days of growth more than 70% of the newly synthesized internal free amino acid pool derives its nitrogen from NH(4) (+) rather than NO(3) (-). If NH(4) (+) is omitted from the medium, no NO(3) (-) is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH(4) (+) and NO(3) (-) are both required for the induction of nitrate- and nitrite reductase.