Mitochondria constitute a major source of reactive oxygen species and have been proposed to integrate the cellular responses to stress. In animals, it was shown that mitochondria can trigger apoptosis from diverse stimuli through the opening of MTP, which allows the release of the apoptosis-inducing factor and translocation of cytochrome c into the cytosol. Here, we analyzed the role of the mitochondria in the generation of oxidative burst and induction of programmed cell death in response to brief or continuous oxidative stress in Arabidopsis cells. Oxidative stress increased mitochondrial electron transport, resulting in amplification of H 2 O 2 production, depletion of ATP, and cell death. The increased generation of H 2 O 2 also caused the opening of the MTP and the release of cytochrome c from mitochondria. The release of cytochrome c and cell death were prevented by a serine/cysteine protease inhibitor, Pefablock. However, addition of inhibitor only partially inhibited the H 2 O 2 amplification and the MTP opening, suggesting that protease activation is a necessary step in the cell death pathway after mitochondrial damage.Generation of reactive oxygen species (ROS) in plants has been implicated in biotic and abiotic stresses. A biphasic oxidative burst is triggered in the plant cells following recognition of invading avirulent pathogens. Despite extensive research on the source of ROS, the subcellular location and the mechanism of ROS generation has not been unequivocally clarified (Bolwell, 1999). Many different mechanisms have been shown to be involved in pathogeninduced ROS production, including peroxidases and diverse oxidases (oxalate oxidase, amine oxidase, and NADPH oxidase; Pugin et al., 1997;Wojtaszek, 1997;Rea et al., 1998). An intracellular and an apoplastic source of ROS production were detected during elicitation of tobacco (Nicotiana tabacum) epidermal cells with cryptogein from Phytophthora cryptogea (Allan and Fluhr, 1997). The connection between pathogen response and respiration is further substantiated by the recent finding that salicylic acid, an important component of the pathogen response mechanism, inhibits ATP formation and uptake of respiratory O 2 in nonphotosynthetic tobacco cells (Xie and Chen, 1999). Mitochondria is a major source of ROS formation, and it is possible that this organelle could participate in the oxidative burst in plants. During respiration, molecular oxygen may undergo a univalent reduction at the sites of ROS generation in complexes I and III of the respiratory chain, forming superoxide, which subsequently dismutates to hydrogen peroxide (Braidot et al., 1999).We have shown previously that a brief treatment with H 2 O 2 induced a programmed cell death (PCD) in suspension-cultured soybean (Glycine max) cells (Levine et al., 1994(Levine et al., , 1996. Recently, Desikan and coworkers (1998) showed that a similar response is activated in suspension-cultured Arabidopsis cells. In those experiments, PCD induction by H 2 O 2 in the soybean and Arabidopsis cell cu...

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.

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