Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress.

The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen (¹O₂) and activating ¹O₂-mediated cell death. Thylakoids of aba1 produced twice as much ¹O₂ as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. ¹O₂ was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of ¹O₂ generation in aba1. Up-regulation of the ¹O₂ responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct ¹O₂ cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of ¹O₂-mediated cell death. In conclusion, aba1 may serve as an alternative model to other ¹O₂-overproducing mutants of Arabidopsis for investigating ¹O₂-mediated cell death.

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