Interaction of sulfate and glutathione transport in cultured tobacco cells

Abstract

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. Km (photoheterotrophic cells: 16.0±2.0 μM; heterotrophic cells: 11.8±1.8 μM) and Vmax (photoheterotrophic cells: 323±50 nmol·min(-1)·g(-1) dry weight; heterotrophic cells: 233±3 nmol·min(-1)·g(-1) dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.