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Keywords:

  • apoplast;
  • ascorbate;
  • cell-suspension cultures;
  • dehydroascorbate;
  • hydrogen peroxide (H2O2);
  • oxidative stress;
  • pulsing of secretion;
  • reactive oxygen species (ROS)

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 [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.