Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999