Effect of short-term dark incubation with sulfate, chloride and selenate on the glutathione content of spinach leaf discs

A 24 h incubation of leaf discs of spinach ( Spinacia oleracea L. cv. Estivato) in darkness with 50 and 100 m M sulfate resulted in a two- to three-fold increase in the level of glutathione (GSH), a compound which may serve as storage of excess reduced sulfur in the plant. The accumulated GSH was a small fraction (around 1%) of the sulfate taken up in the spinach leaf discs. Incubation of spinach leaf discs with 50 and 100 m M chloride resulted in only a 30% increase of the water-soluble non-protein-SH; the uptake of electrolytes was comparable to that observed with sulfate. This indicated that the increase of the GSH level upon incubation with sulfate was rather specific and not due to salinity. Incubation with 50 m M Na₂SO₄ did not affect water-soluble protein-SH content after 24 h. Addition of 1 and 10 m M selenate, an inhibitor of sulfate reduction, strongly reduced sulfate-induced GSH accumulation in spinach leaf discs, both in light and darkness. It was concluded that the sulfate-induced SH accumulation was due to a substantial de novo reduction of sulfate in darkness and subsequent incorporation of the reduced sulfur into GSH. The role of the sulfate concentration at the reaction site of ATP sulfurylase in the regulation of sulfur assimilation in the plant is discussed with respect to the low affinity of the enzyme for sulfate.