Levels of Active Oxygen Species Are Controlled by Ascorbic Acid and Anthocyanin in Arabidopsis

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O2 •-) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of ∼300−500 SOD units/g of fresh weight. The ESR pattern indicated that most (40−50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by γ-irradiation. The radical scavenging activity in irradiated plants increased ∼10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3, tt5, ttg1, and vtc1) was examined after γ-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower. Keywords: Active oxygen species; ascorbic acid; anthocyanin; electron spin resonance