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

SUMMARY: *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 [(14)C]ascorbate. Radiolabelled metabolites and secretion products were analysed by high-voltage electrophoresis. *In both species, exogenous 1 mM hydrogen peroxide (H(2)O(2)) rapidly stimulated [(14)C]ascorbate and [(14)C]dehydroascorbate accumulation in the medium (apoplast). Net (14)C export was most rapid within 100 s of washing, and often showed superimposed pulses, of c. 10-s duration, whose amplitude was greater after H(2)O(2) treatment. Oxidative stress did not cause indiscriminate metabolite leakage from the cells. H(2)O(2) caused c. 20-40% of the intracellular [(14)C]ascorbate to be irreversibly catabolized to [(14)C]oxalyl-threonate and related products; however, the great majority of the extracellular radioactivity remained as [(14)C]ascorbate and [(14)C]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.