Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

Abstract

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaCIO3. This sensitivity depended on the age of the cultures that were plated. Chlorateresistant colonies isolated from 5-to 7-day cultures retained their resistance through many generations of growth in medium lacking NaCIO3; they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4'-methoxymethyltrioxsalen, increased the proportion that was resistant to NaCIO3. However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.In tobacco, Muller and Grafe (11) isolated nine strains on the basis ofchlorate resistance. Seven ofthese strains lacked detectable nitrate reductase and did not grow on medium containing nitrate as the sole nitrogen source. Two other strains had very little nitrate reductase and grew very poorly on nitrate medium. It seems that the presence of nitrate reductase, which presumably reduces chlorate to chlorite, is necessary for the full toxic effect of chlorate to be expressed.If strains of plant cells that possess both nitrate reductase and chlorate resistance were located, they might provide clues to further steps in the mechanism by which chlorate kills normal cells. Cove (3) discovered a group of chlorate-resistant mutants of A. nidulans that could utilize NO3; at least three separate genes were involved, but these mutants were very rare. One group of chlorate-resistant mutants of N. muscorum (17) retained nitrate reductase activity (inferred, not measured) and lacked nitrogenase activity. Here we report that when strains of Rosa damascena cultured cells are selected for chlorate resistance, only a minor fraction lacks the ability to grow on medium containing nitrate as the sole nitrogen source and probably lacks the ability to reduce chlorate to chlorite; the major fraction retains the ability to grow on nitrate medium and possesses the ability to transform chlorate to toxic products. MATERIALS AND METHODSThe lethal effect of NaClO3 on higher plants has long been recognized, but the mechanism of chlorate toxicity rema...

Growth and Water Relations of Cultured Tomato Cells after Adjustment to Low External Water Potentials

“…17). A number of reports have shown growth cycle-related changes in various characteristics of higher plant cells including plating efficiency (20), nutrient uptake (11), accumulation ofnitrogen (25), ethylene production (15) …”

Isolation and characterization of nitrate reductase-deficient cell lines from a haploid culture of peony.

“…This fact supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. However, in n CR strains from diploid Rosa damascene cultured cells, approximately 85% of them grew on N03-medium, although the rest, approximately 15%, could not grow on the same medium (Murphy and Imbrie 1981). Other factor leading to chlorate resistance may therefore have functioned in the case of these Rosa cultured cells.…”

Chlorate‐resistant rose cells: influx, efflux and reduction of [36Cl]‐chlorate

“…Sodiutn chlorate has proved to be a useful compoutid for selecting resistance mutants of bacteria (Puig et al 1967), fungi (Cove 1976), algae (Singh andSonie 1977, Sosa et al 1978) and higher plants (Oostindier-Braaksma and Feenstra 1973, Mtiller and Grafe 1978, King and Khanna 1980, Murphy and Imbrie 1981, Buchanan and Wray 1982, M,arton et al 1982, Nelson et al 1983. In many cases, the mutants resistant to high external concentrations of ClOj^ lack nitrate reductase (NR, EC 1.6.6.2) and thereby the ability to grow on nitrate as sole N source.…”

“…This is consistent with the idea, first popularized by Aberg (1948), that CIO7 is reduced in vivo to ClOj^ in non-resistant organisms and that the CIO,' is the true toxic compound. Some experiments, have been reported in which ClOj^-resistant mutants of higher plants possess NR (Oostindier-Braaksma and Feenstra 1973, Murphy and Imbrie 1981, Buchanan and Wray 1982, Marton et al 1982. The basis of the resistance of these mutants is un-known.…”