Reactive oxygen species in programmed death of pea guard cells

Hydrogen peroxide potentiates CN⁻-induced apoptosis of guard cells recorded as destruction of cell nuclei in the epidermis from pea leaves. A still stronger effect was exerted by the addition of H₂O₂ and NADH, which are the substrates of the plant cell wall peroxidase producing [graphic removed] coupled to the oxidation of NADH. The CN⁻-or (CN⁻ + H₂O₂)-induced destruction of guard cell nuclei was completely removed by nitroblue tetrazolium (NBT) oxidizing [graphic removed] and preventing there-by the subsequent generation of H₂O₂. The reduced NBT was deposited in the cells as formazan crystals. Cyanide-induced apoptosis was diminished by mannitol and ethanol, which are OH· traps. The dyes Rose Bengal (RB) and tetramethylrhodamine ethyl ester (TMRE) photosensitizing singlet oxygen production suppressed the CN⁻-induced destruction of the cell nuclei in the light. This suppression was removed by exogenous NADH, which reacts with ¹O₂ yielding [graphic removed] . Incubation of leaf slices with RB in the light lowered the photosynthetic O₂ evolution rate and induced the permeability of guard cells for propidium iodide, which cannot pass across intact membranes. Inhibition of photosynthetic O₂ evolution by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or bromoxynil prevented CN⁻-induced apoptosis of guard cells in the light but not in the dark. RB in combination with exogenous NADH caused H₂O₂ production that was sensitive to NBT and estimated from dichlorofluorescein (DCF) fluorescence. Data on NBT reduction and DCF and TMRE fluorescence obtained using a confocal microscope and data on the NADH-dependent H₂O₂ production are indicative of generation of reactive oxygen species in the chloroplasts, mitochondria, and nuclear region of guard cells as well as with participation of apoplastic peroxidase. Cyanide inhibited generation of reactive oxygen species in mitochondria and induced their generation in chloroplasts. The results show that H₂O₂, OH·, and [graphic removed] resources utilized for H₂O₂ production are involved in apoptosis of guard cells. It is likely that singlet oxygen generated by RB in the light, judging from the permeability of the plasmatic membrane for propidium iodide, makes Photosystem II of chloroplasts inoperative and induces necrosis of the guard cells.