Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana

Absorption of excess excitation energy induces overproduction of singlet oxygen (¹O₂) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates located in the chloroplast. Over-accumulation of the chlorophyll biosynthetic intermediate protochlorophyllide by the exogenous application of 5-aminolevulinic acid (ALA), the precursor of tetrapyrrole, induced singlet oxygen production in the plastidic membranes. Over-expression of protochlorophyllide oxidoreductase C (PORC) in Arabidopsis thaliana resulted in efficient light-induced photo-transformation of protochlorophyllide to chlorophyllide that limited the accumulation of protochlorophyllide. Consequently, the ¹O₂ generation decreased in the PORC overexpressors (PORCx) and their cell death was minimal. Conversely, porC-2 over-accumulated protochlorophyllide in response to ALA treatment and generated higher amounts of ¹O₂ in light and had highest cell death as monitored by Evans blue staining. The protoplasts isolated from PORCx plants, when treated with ALA, generated minimal amounts of ¹O₂ as revealed by singlet oxygen sensor green (SOSG) fluorescence emission from chloroplasts. Conversely, the protoplasts of porC-2 mutants under identical conditions generated the maximum SOSG fluorescence in their chloroplasts and cytosol surrounding the chloroplasts most likely due to the leakage from the organelle. The membrane blebbing, a hallmark of programmed cell death, was clearly visible in WT and porC-2 protoplasts. Similarly, the nick end labelling (TUNEL) assay revealed nicks in the DNA. The TUNEL-positive nuclei after 30 min of light exposure were highest in porC-2 and lowest in PORCx protoplasts. The results demonstrate that higher amounts of singlet oxygen produced in the chloroplasts play an important role in programmed cell death.