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

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.