Nitrate use by tobacco cells in response to N-stress and ammonium nutrition

Summary Characterization of NO ₃ ^– use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO ₃ ^– uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO ₃ ^– and NH ₄ ⁺ (40 mM NO ₃ ^– plus 20 mM NH ₄ ⁺ , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO ₃ ^– only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO ₃ ^– . The NO ₃ ^– influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO ₃ ^– influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO ₃ ^– and NH ₄ ⁺ had lower NO ₃ ^– reductase (NR) activity and higher cell NO ₃ ^– levels than those of cells grown with NO ₃ ^– only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO ₃ ^– was depleted. The level of cell NO ₃ ^– plays a critical role in the expression of the NO ₃ ^– uptake and reduction processes. The transitions in the expression of NO ₃ ^– uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.Abbreviations DTT DL-dithiothreitol - EDTA ethylenediamine tetraacetate - FW fresh weight - MS media Murashige & Skoog media - NADH ß-nicotinamide adenine dinucleotide reduced form - PMSF phenylmethyl-sulfonyl fluoride