* To whom correspondence should be addressed.
Received January 30, 2008; Revision received April 21, 2008
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 O2-· 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 O2-· and preventing thereby 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 O2-·. 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 O2-· 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.
KEY WORDS: programmed cell death, reactive oxygen species, chloroplasts, mitochondria, cell nucleus, apoplastic peroxidase, Pisum sativum L.