A Nitrate Reductase-less Variant Isolated from Suspension Cultures of Datura innoxia (Mill.)

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Abstract
A comparative study was carried out of the growth of 2 lines of D. inoxia (Mill.) cells, designated DI-6 and NR1, their resistance to chlorate, and their ability to assimilate nitrate in sterile culture. The NR1 cell line was isolated from DI-6 cultures by first growing the latter in a nitrate-based medium for 5 days and then transferring the cells to a medium containing 2 g/l of casein hydrolysate as the sole N source and 49 millimolar KClO3 for a 6 wk incubation period. Cells which survived the chlorate treatment then were transferred to casein hydrolysate medium and have been cultured in the absence of chlorate for more than 18 mo. (NR1). DI-6 cells can grow in a nitrate-based medium, whereas NR1 cells can take up nitrate but cannot use it as a N source. The inability of NR1 to assimilate nitrate appears to be due to the lack of an active nitrate reductase in these cells. Through the use of a variety of electron donors and acceptors, the lack of nitrate reductase activity in NR1 cells was due to the absence of, or a defect in, that component of the enzyme which mediates the reduction of nitrate to nitrite. In other experiments, DI-6 and NR1 were grown on a solid medium containing casein hydrolysate (2 g/l) as the sole N source. Under these culture conditions, neither cell line contained an active nitrate reductase. The growth on this medium was compared to that on the same medium containing chlorate at concentrations from 0-100 mmol. DI-6 culture growth was inhibited by 70% at a chlorate concentration of 30 micromolar, whereas growth of NR1 was stimulated by more than 60% on the same medium and by 100% at a chlorate concentration of 30 mmol. In the presence of 100 mmol chlorate, the growth of both cell lines was completely inhibited. This clear difference between the response of DI-6 and NR1 cells to chlorate even in the absence of nitrate lends support to the observations by others that chlorate inhibits cells by a mechanism other than, or in addition to, its nitrate reductase-catalyzed conversion to chlorite. Nitrite reductase was induced by nitrate in NR1 cells as well as in DI-6. This observation is a further confirmation of the fact that nitrate, not nitrite, is the true inducer of the nitrate assimilatory pathway in higher plants.