Reactive oxygen species-induced release of intracellular ascorbate in plant cell-suspension cultures and evidence for pulsing of net release rate

P>Apoplastic ascorbate has been proposed to confer resistance to oxidative stresses, e.g. ozone. We investigated reactive oxygen species (ROS)-induced secretion and catabolism of ascorbate.

Late-growth-phase cultured cells of rose and Arabidopsis were preloaded with [14C]ascorbate. Radiolabelled metabolites and secretion products were analysed by high-voltage electrophoresis.

In both species, exogenous 1 mM hydrogen peroxide (H2O2) rapidly stimulated [14C]ascorbate and [14C]dehydroascorbate accumulation in the medium (apoplast). Net 14C export was most rapid within 100 s of washing, and often showed superimposed pulses, of c. 10-s duration, whose amplitude was greater after H2O2 treatment. Oxidative stress did not cause indiscriminate metabolite leakage from the cells. H2O2 caused c. 20-40% of the intracellular [14C]ascorbate to be irreversibly catabolized to [14C]oxalyl-threonate and related products; however, the great majority of the extracellular radioactivity remained as [14C]ascorbate and [14C]dehydroascorbate. Much of the apoplastic dehydroascorbate was probably reabsorbed by the cells and reduced back to ascorbate.

The data show that exported ascorbate can serve an apoplastic antioxidant role in these late-growth-phase cells without being irreversibly lost, whereas in early-growth-phase cells most extracellular ascorbate is irreversibly degraded. In conclusion, cultured plant cells can respond actively to oxidative stress by reversibly exporting ascorbate into the apoplast.