Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

Chitosan, CN(-), or H(2)O(2) caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H(2)O(2), or chitosan + H(2)O(2) on EC. H(2)O(2) formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN(-) and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN(-)-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN(-)-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H(2)O(2) on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H(2)O(2) as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Pea leaf epidermis incubated with cyanide displayed ultrastructural changes in guard cells that are typical of apoptosis. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and lincomycin, an inhibitor of protein synthesis in chloroplasts and mitochondria, produced different effects on the dynamics of programmed death of guard cells. According to light microscopy data, cycloheximide reinforced and lincomycin suppressed the CN(-)-induced destruction of cell nuclei. Lincomycin lowered the effect of cycloheximide in the light and prevented it in the dark. According to electron microscopy data, the most pronounced effects of cycloheximide in the presence of cyanide were autophagy and a lack of apoptotic condensation of nuclear chromatin, the prevention of chloroplast envelope rupturing and its invagination inside the stroma, and the appearance of particular compartments with granular inclusions in mitochondria. Lincomycin inhibited the CN(-)-induced ultrastructural changes in guard cell nuclei. The data show that programmed death of guard cells may have a combined scenario involving both apoptosis and autophagy and may depend on the action of both cytoplasm synthesized and chloroplast and mitochondrion synthesized proteins.

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN--induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (alpha-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN--induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN--induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN--induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Qo site of the chloroplast cytochrome b6f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.

Treatment with cyanide of epidermal peels isolated from pea leaves resulted in destruction of nuclei in the guard cells of stomata, which is visible with a light microscope. The process was accelerated by illumination. Electron microscopy revealed significant CN--induced changes in the ultrastructure of guard cells, which increased with time. Margination of chromatin, which is one of the first signs of apoptosis, was observed in the guard cells even after 1 h incubation of the isolated epidermis with CN-. Subsequent chromatin condensation, swelling of the endoplasmic reticulum with formation of large tanks covered with ribosomes, changes in the structure of dictyosomes, and a slight swelling of mitochondria were observed after 3 h of the epidermis incubation with CN-. After 6 h of incubation with CN-, the bulk volume of the guard cells was filled with vacuoles, the cytoplasm occupied the thin marginal layer, the nucleus was in the center similarly to the control experiment, but it was polylobal, extended in narrow cytoplasmic bands, and, despite the loss of the nuclear envelope integrity, appeared to be a self-dependent structure. In the envelope-free open regions of the nucleus, mitochondria and chloroplasts directly contacted with chromatin. Much like the cell nucleus, chloroplasts lost the integrity of the membrane, but did not swell and retained the stroma and integrity of the thylakoid system. An antioxidant di-tert-butyl-4-hydroxytoluene prevented ultrastructural changes in the cells observed after 6 h of incubation with CN-. Thus, the CN--induced death of the guard cells of stomata occurs through the mechanism of apoptosis.