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...

Adaptation to stress requires removal of existing molecules from various cellular compartments and replacing them with new ones. The transport of materials to and from the specific compartments involved in the recycling and deposition of macromolecules is carried out by an intracellular vesicle trafficking system. Here, we report the isolation of a vesicle trafficking-regulating gene, AtRabG3e (formerly AtRab7), from Arabidopsis. The gene was induced during programmed cell death after treatment of intact leaves with superoxide and salicylic acid or infection with necrogenic pathogens. Transgenic plants that expressed the AtRabG3e gene under the constitutive 35S promoter from cauliflower mosaic virus exhibited accelerated endocytosis in roots, leaves, and protoplasts. The transgenic plants accumulated sodium in the vacuoles and had higher amounts of sodium in the shoots. The transgenic plants also showed increased tolerance to salt and osmotic stresses and reduced accumulation of reactive oxygen species during salt stress. These results imply that vesicle trafficking plays an important role in plant adaptation to stress, beyond the housekeeping function in intracellular vesicle trafficking.Plants are constantly exposed to changes in the environment that results in development of stress, compelling them to adjust to the new conditions. The perturbations in the surrounding environmental conditions often cause an oxidative stress. A mild oxidative stress usually induces antioxidant defenses, whereas a severe stress causes rapid necrosis. Intermediate levels of reactive oxygen species (ROS) often trigger a programmed cell death (PCD) cascade, which eliminates the compromised cells (Datt et al., 2003). The induction and execution of PCD are tightly controlled processes and can be modulated by signaling molecules, such as jasmonic acid, salicylic acid (SA), and ethylene (Lam et al., 1999).Little information exists on the role of intracellular vesicle trafficking in resistance to environmental stresses. Endocytosis has been viewed traditionally as a constitutive housekeeping function in both animal and plant cells. Recently, however, a syntaxinrelated protein, NtSyr1, which is one of the central components of the vesicle trafficking machinery in eukaryotes, was implicated in abscisic acid-mediated responses in tobacco (Nicotiana tabacum; Leyman et al., 1999). In yeast (Saccharomyces cerevisiae), we found that vesicle trafficking between cytosol and the plasma membrane was inhibited by oxidative stress. Overexpression of an Arabidopsis synaptobrevin homolog partially restored the traffic and provided tolerance to lethal concentrations of hydrogen peroxide (H 2 O 2 ; Levine et al., 2001). A link between regulation of endocytosis by GDI:Rab5 complex and the p38-dependent stress response was described recently in animal cells (Cavalli et al., 2001).We have shown previously that a low concentration of superoxide in the presence of nontoxic concentration of SA-induced PCD in intact Arabidopsis leaves (Mazel and Levine, 2001)...

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

Salt-tolerant Pokkali rice plants accumulate higher polyamines (PAs) such as spermidine (Spd) and spermine (Spm) in response to salinity stress, while the sensitive cultivarM-1-48 is unable to maintain high titres of these PAs under similar conditions. The effects of the triamine Spd and the tetramine Spm on physiological and biochemical changes in 12-day-old rice seedlings were investigated during salinity stress to determine whether they could protect the sensitive plants from stress effects. At physiological concentrations Spd and Spm significantly prevented the leakage of electrolytes and amino acids from roots and shoots induced by salinity stress. To different degrees they also prevented chlorophyll loss, inhibition of photochemical reactions of photosynthesis as well as downregulation of chloroplast-encoded genes like psbA, psbB, psbE and rbcL, indicating a positive correlation between salt tolerance and accumulation of higher PAs in rice. The inhibitory effect of salinity stress and its reversal by exogenous PAs were more pronounced in the salt-sensitiveM-1-48 plants than in the tolerant Pokkali plants.

Botrytis cinerea is a necrotrophic fungus that infects over 200 plant species. Previous studies showed that host cells collapse in advance of the hyphae, suggesting secretion of toxins or elicitors. We have partially characterized elicitor activity from intercellular fluid extracted from Arabidopsis thaliana leaves infected with B. cinerea. Treatment of intact leaves or cell cultures with either intercellular fluid from infected leaves or medium from inoculated A. thaliana cell culture induced generation of reactive oxygen species, resulting in reduced photosynthesis, electrolyte leakage, and necrotic lesions that resembled the hypersensitive response (HR). The necrosis was inhibited by diphenyleneiodonium, a specific inhibitor of NADPH oxidase, and by chelating free iron, suggesting the involvement of hydroxyl radicals. The necrosis was also suppressed in dnd1 mutants that are compromised in HR. In contrast, increased cell death was observed in acd2 mutants, indicating the involvement of the host defense signaling pathways. Treatment with the intercellular fluid from infected leaves also induced transcription of pathogenesis-related (PR) genes, such as PR-1, PR-5, HSR203J, and of senescence-associated gene SAG-13. Moreover, rapid transcription of the ethylene-dependent AtEBP gene was detected, indicating induction of ethylene production. The inter-cellular fluid from infected A. thaliana induced cell death in other plants, in line with the lack of B. cinerea specificity. In summary, the intercellular fluid mimicked a range of molecular and physiological host responses that are observed during infection with a live fungus. Moreover, it accelerated the B. cinerea infection, suggesting that the elicitor may act as a pathogenicity factor in the progression of gray mold disease.