Regulation of sulfate uptake in carrot cells: Properties of a hypercontrolled variant

Abstract

Sulfate uptake in haploid carrot cultures can be experimentally controlled by the sulfur source provided for growth. The rate of sulfate uptake is low in cells grown on cystine or sulfate and high in sulfur-starved cells. A selenate-resistant variant cell line has been isolated from a haploid carrot line. The variant shows hypersuppression of sulfate uptake by cystine and essentially normal control by the other treatments. While both lines efflux intracellular sulfate in the presence ofexternal sulfate, the rate of efflux from the variant is 4-6 times higher at comparable levels of initial -intracellular sulfate.-Further, properties of the efflux and uptake in both lines suggest that they are mediated by the same system. We propose that the variant possesses an altered uptake-efflux system -that is more readily reversed and more subject to control by some metabolite derived from cystine.Sulfate uptake has been studied in a variety of organisms, and permease-deficient mutants have been selected by resistance to sulfate analogs in Escherichia coli (1), yeast (2), Aspergillus (3), and Neurospora (4). In Aspergillus, a mutant showing hypersuppression of sulfate uptake by methionine has been isolated and was found to be allelic to other permease-less mutants (5). In general, the regulation of these permease activities has been attributed to repression by some reduced sulfur compound; however, there is also evidence for inhibition of uptake by some early intermediate in reduction (2).Tobacco cells (6) and potato tuber discs (7) show suppression of sulfate uptake if incubated with sulfur-containing amino acids. Using tobacco cells, Smith (8,9) found that sulfate uptake was suppressed by growth on cyst(e)ine or sulfate and, on the basis of correlative evidence, suggested that uptake was controlled by feedback inhibition by the intracellular sulfate pool.In this paper, we describe a variant carrot cell line that has altered control of sulfate uptake by cystine. The results show that this line has an altered uptake-efflux system and is impaired in sulfate reduction. The relationships among these phenotypes are discussed. MATERIALS AND METHODSCulture Methods and Variant Isolation. The parental cell line has been described (10), and growth in suspension culture was monitored turbidimetrically (11). Media used in this work were based on Gamborg's B5 medium (12), modified by replacing (NH4)2504 with NH4NO3 and other sulfates with equimolar chlorides. This medium, referred to as -S medium, was used unsupplemented or supplemented with cystine (Eastman). Suspension cultures of both lines were grown in 100 ml of medium in 500-ml flasks with continuous shaking at 120 rpm at 250C in the light. Plants were produced from both lines on B5 medium devoid of2,4-dichlorophenoxyacetic acid and callus was reinitiated from root tips on B5 medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg/liter. Chromosome counts were done as described (10).The variant was isolated from the parental line, HA, by plating cells on -S medi...

Characterization of a Selenocystine-Resistant Carrot Cell Line

“…In carrot cells, growth on cystine suppresses the development of sulfate uptake activity (5). In a previous paper, we described a sulfate uptake variant which is hypersuppressed by cystine (5). We concluded that the cell line possessed an altered sulfate permease more sensitive than the wild type to suppression by the same metabolite derived from cystine (5).…”

“…In a previous paper, we described a sulfate uptake variant which is hypersuppressed by cystine (5). We concluded that the cell line possessed an altered sulfate permease more sensitive than the wild type to suppression by the same metabolite derived from cystine (5). However, the nature of the metabolite is unknown.…”

“…The uptake and metabolite pools were examined as previously described (5). Briefly, cells grown on the sulfur source indicated were washed three times in incubation medium (1 mM MgCl2; 1 mm CaCl2; 1 mm Mes, pH 5.0; glucose 2% w/v).…”

“…Chromosome counts were performed as previously described (13). The tissue culture and media were as before (5). Plants were obtained by somatic embryogenes on B5 (6) without 2,4-D. Resistance of plants was tested by placing small, 2-cm long, plants on B5-2,4-D supplemented with 0.1 mM selenocystine.…”

“…Thus, we studied the effect of cystine, rather than cysteine, on carrot culture growth. In carrot cells, growth on cystine suppresses the development of sulfate uptake activity (5). In a previous paper, we described a sulfate uptake variant which is hypersuppressed by cystine (5).…”