Accelerated light-induced necrosis in an Arabidopsis thaliana triple mutant deficient in vitamin C and stromal and thylakoid chloroplast ascorbate peroxidases

Ascorbate (vitamin C) and ascorbate peroxidases (APXs) are part of a multi-layered defense system that protects the cell from damage by reactive oxygen species (ROS). In this study, we investigated the combined functional significance of the soluble stromal ascorbate peroxidase (SAPX) and the thylakoid bound isoform (TAPX) in chloroplasts of Arabidopsis thaliana. In addition, an APX-like protein is present in the thylakoid lumen. We generated homology models for these three chloroplast APXs, and showed that the lumenal APX-like protein is not a bona fide ascorbate peroxidase. A null mutant in this lumenal APX behaved as wild-type under different light regimes. Single and double T-DNA-tagged null mutants in SAPX and TAPX were obtained and were each crossed with vtc2, partially deficient in ascorbate accumulation (Conklin et al, 2000). The single mutants (sapx, vtc2 and tapx), double mutants (sapx-vtc2, tapx-vtc2, and tapx-sapx), and the triple mutant (tapx-sapx-vtc2) were all viable on soil. We determined levels of α-tocopherol, ascorbate and glutathione, as well as growth parameters, photosynthetic activity and H2O2 accumulation in the mutants. All vtc2 genotypes accumulated more glutathione, most likely to compensate for the 80% loss of ascorbate. Anthocyanin accumulation increased under light stress conditions, but was strongly reduced in the vtc2 background. Surprisingly, single, double and triple mutants did not show significant differences in light sensitivity when constantly grown under moderate or high light intensities. However, when mutants were shifted from moderate to high light (120 to 1000 μmol photons m-2sec-1), fully expanded leaves in the triple mutant rapidly showed necrotic lesions in the mesophyll but not in the vasculature. Necrosis in the triple mutant was concomitant with accumulation of H2O2 in the cell. tapx-vtc2 and sapx-vtc2 also showed signs of necrosis, but significantly less than the triple mutant. Importantly, tapx-sapx did not show any light stress symptoms, or H2O2 accumulation upon shift to high light, suggesting that other peroxidase activities are sufficient to detoxify H2O2 when cellular ascorbate concentration is not limiting.