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

Abstract

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.

Ascorbic acid metabolism and functions: A comparison of plants and mammals

“…Plant cell cultures rapidly oxidase ascorbate and this is most likely mediated by the apoplastic ascorbate oxidase ( Section 8 ). Cultured cells exposed to H 2 O 2 release a pulse of ascorbate, which oxidises in the culture medium and is then re-absorbed indicating a possibility that oxidative stress activates an ascorbate efflux system to protect the apoplast [116] . Recently, electrophysiological measurements have provided evidence for ascorbate efflux via an anion channel which is activated by gluconic acid and blocked by anthracene-9-carboxylic acid [117] .…”

Plant signalling in acute ozone exposure

“…Extracellular ROS accumulation stimulates ascorbate supply to the apoplast. Treatment of Arabidopsis suspension cells with hydrogen peroxide resulted in fast release of ascorbate to the extracellular space (Parsons & Fry ). Active transport of ascorbate across the plasma membrane suggests the presence of specific ascorbate transporters; however, their identity has remained unknown.…”

The Impact of Global Change Factors on Redox Signaling Underpinning Stress Tolerance

“…Arabidopsis mutants that are deficient in ascorbate (vtc) are characterized by high sensitivity to O 3 (Conklin et al, 2000) as well as constitutive up-regulation of SA-dependent defense responses and an enhanced basal resistance to biotrophic pathogens (Pastori et al, 2003;Pavet et al, 2005;Mukherjee et al, 2010). Plants respond to O 3 and other apoplastic oxidants by rapidly exporting ascorbate into the apoplast (Parsons and Fry, 2010). The products of ascorbate breakdown such as oxalate are considered to contribute to a network of signals in the apoplast that transfer information concerning the redox state of the extracellular environment to the nucleus.…”