Recovery of sulfate transport into heterotrophic tobacco cells from inhibition by reduced glutathione

Abstract

In heterotrophic tobacco cells (Nicotiana tabacum L. cv. Samsun) inhibition of sulfate transport by reduced glutathione (GSH) is a reversible process. When GSH was removed from the culture medium subsequent to a 10‐h treatment with 1 mM GSH, sulfate transport began to recover after a lag period of ca 4 h and reached the transport rates of controls without GSH within another 3–4 h. Recovery was prevented when inhibitors of protein synthesis, i.e. cycloheximide or puromycin, were added to the medium upon removal of GSH, even if low concentrations (cycloheximide 1 μM; puromycin 250 μM) were applied. At these low concentrations the rate of synthesis of sulfate transport entities was maintained at the rate of degradation in the absence of GSH. The post‐transcriptional polyadenylation inhibitor cordycepin and the transcription inhibitor α‐amanitin only slightly reduced recovery of sulfate transport from inhibition by GSH. Apparently, protein synthesis is required for this recovery, suggesting that inhibition of synthesis of sulfate carrier entities is the mechanism of action of GSH on sulfate transport in heterotrophic tobacco cells. An initial rate of net increase in sulfate transport during recovery from inhibition of GSH of 3.6±0.2 U h−1 was calculated [1 U=1 nmol sulfate (g DW)−1 min−1]. This rate of increase is small compared with the rate of decrease in sulfate transport at maximum inhibition by cycloheximide (110±3 U h−1). However, with increasing time of exposure without GSH, the net increase in sulfate transport was enhanced to a maximum rate of 96±3 U h−1, measured 5–7 h after GSH had been removed from the media. Apparently, the rate of synthesis of sulfate transport entities in heterotrophic tobacco cells is about twice its rate of degradation.