To gain some insight into the mechanisms by which plant cells die as a result of abiotic stress, we exposed tobacco (Nicotiana tabacum) Bright-Yellow 2 cells to heat shock and investigated cell survival as a function of time after heat shock induction. Heat treatment at 55°C triggered processes leading to programmed cell death (PCD) that was complete after 72 h. In the early phase, cells undergoing PCD showed an immediate burst in hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 ⅐ -) anion production. Consistently, death was prevented by the antioxidants ascorbate (ASC) and superoxide dismutase (SOD). Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, also prevented cell death, but with a lower efficiency. Induction of PCD resulted in gradual oxidation of endogenous ASC; this was accompanied by a decrease in both the amount and the specific activity of the cytosolic ASC peroxidase (cAPX). A reduction in cAPX gene expression was also found in the late PCD phase. Moreover, changes of cAPX kinetic properties were found in PCD cells. Production of ROS in PCD cells was accompanied by early inhibition of glucose (Glc) oxidation, with a strong impairment of mitochondrial function as shown by an increase in cellular NAD(P)H fluorescence, and by failure of mitochondria isolated from cells undergoing PCD to generate membrane potential and to oxidize succinate in a manner controlled by ADP. Thus, we propose that in the early phase of tobacco Bright-Yellow 2 cell PCD, ROS production occurs, perhaps because of damage of the cell antioxidant system, with impairment of the mitochondrial oxidative phosphorylation.In plants, programmed cell death (PCD) is responsible for removal of redundant, misplaced, or damaged cells, and, thus, contributes significantly to both development and maintenance of these multicellular organisms. Activation of PCD in plants takes place during a variety of processes including differentiation of tracheary elements (Fukuda, 2000; Yu et al., 2002) and female gametophytes (Wu and Cheun, 2000), the development of cereal endosperm and aleurone cells (Young and Gallie, 2000; Fath et al., 2002), responses to external stimuli such as the hypersensitive reaction against pathogen attacks (Lamb and Dixon, 1997; Beers and McDowell, 2001) and severe abiotic stresses (Koukalovà et al., 1997; Weaver et al., 1998; Rao and Davis, 2001). At present, however, how signaling pathways lead plant cells toward death via apoptosis and how death occurs are far from being elucidated. Mammalian and plant PCD processes share several morphological and biochemical features, including cytoplasm shrinkage, nuclear condensation, DNA laddering, expression of caspase-like proteolytic activity, and release of cytochrome c from mitochondria (Balk et al., 1999; Sun et al., 1999; Kim et al., 2003). Nonetheless, in distinction from mammals (for instance, see Atlante et al., 2003aAtlante et al., , 2003b, how plant PCD takes place remains somewhat obscure. In this context, the role of the react...

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2-to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.

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Although mitochondria have been the object of intensive study over many decades, some aspects of their metabolism remain to be fully elucidated, including the L L-lactate metabolism. We review here the novel insights arisen from investigations on [7]. Our present position, based on our own work and that of others reviewed here is that the evidence for mitochondrial metabolism of L L-LAC, due to the existence of both L L-LAC carrier-mediated transport processes and of the mitochondrial L L-lactate dehydrogenase (mL-LDH) is now compelling. SpermatozoaThe first evidence in favour of the presence of a putative mL-LDH was from Clausen who found that about 40% of total activity in sperm cells was present in a particulate fraction showing high succinate dehydrogenase activity [8].The existence of the mL-LDH which can reduce pyridine nucleotide in the mitochondrial matrix was demonstrated in hypotonically treated rabbit epididymal spermatozoa by means of oxygen uptake and fluorimetric studies [9]. It was also shown that the mL-LDH functions actively in these cells. In sperm cells the cytosolic L L-lactate dehydrogenase (cL-LDH) and mL-LDH appear to be the same isoenzyme unique to those cells: L-LDH-X. The dual localization of the enzyme enables mammalian spermatozoa to exchange cytosolic and mitochondrial reducing equivalents by means of a L L-LAC/ pyruvate (PYR) shuttle, as suggested by Blanco et al. [10] and reconstituted later [11]. LiverSince liver possesses the enzymatic machinery for gluconeogenesis (GNG) and since L L-LAC is a major substrate for GNG, liver play a major role in L L-LAC metabolism. Indeed, metabolism of L L-LAC has traditionally been considered solely as a function of the cL-LDH in spite of the fact that the L L-LAC oxidation by mitochondria and the existence of an mL-LDH in the inner compartments of isolated rat liver mitochondria (RLM) has been widely reported [12][13][14][15]. In a recent detailed investigation de Bari et al. [16] showed that externally added L L-LAC can be oxidized in the matrix by an mL-LDH, with reduction of intramitochondrial NAD(P) +

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.