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...

not received MitochondriaLaser Transmembrane proton gradient Membrane potential A TP synthesis Oxygen uptake

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as`glutamate neurotoxicity' (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K ؉ ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K ؉ is measured fluorimetrically by using safranine. A detailed investigation of K ؉ uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K ؉ uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K ؉ ATP channel in plants (PmitoK ATP ). Interestingly, Mg 2؉ and glyburide, which can inhibit mammalian K ؉ channel, have no effect on PmitoK ATP . In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoK ATP activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K ؉ uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation.One of most outstanding problems in mitochondria bioenergetics concerns the mitochondrial permeability to metabolites, organic compounds, including vitamins, their derived cofactors, and metal ions. The mitochondrial inner membrane contains metabolite carriers (for review, see Refs. 1 and 2), responsible for shuttling substrates between matrix and cytosol and for catabolism dependent on matrix enzymes, as well as vitamin and cofactor translocators (for review, see Refs. 3 and 4). Moreover, the inner membrane also contains the cation carriers and channels that regulate cell and mitochondrial physiology. In particular, as regards K ϩ ion, in mammalian mitochondria, the transport properties are such that net potassium flux across the mitochondrial membrane determines mitochondrial volume (Refs. 5 and 6 and references therein). It has been shown that K ϩ uptake is mediated by diffusion leak, driven by the high electric membrane potential maintained by redox-driven electrophoretic proton ejection, and that regulated K ϩ efflux is mediated by the inner membrane K ϩ /H ϩ antiporter (see Ref. 7). There is also evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K ϩ uptake into mammalian (5-12) and yeast (13, 14) mitochondria. As far as plant mitochondria are concerned, even though mitochondrial structure and function are expected to be strictly dependent on K ϩ transport across the mitochondrial membrane, the knowledge of K ϩ permeability is not established at present. Indeed, the presence of a powerful K ϩ /H ϩ antiporter, which partially collapses ⌬pH, thereby increasing ⌬⌿, 1 has been shown (Refs. 15 and 16 and ...

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1⌬ mutant strain was constructed. Coupled mitochondria isolated from flx1⌬ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1⌬ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation (for reviews see Refs. 1 and 2).As far as mammalian mitochondria are concerned, we have demonstrated that in rat liver the main source of intramitochondrial flavin cofactors is riboflavin (Rf) 1 taken up from the cytosol. FAD synthesis occurs inside the organelle from imported Rf and mitochondrial ATP, consistent with the presence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2) (3, 4). Newly synthesized FAD can be either efficiently incorporated into newly imported apo-flavoproteins (5, 6) or can be exported into the outer mitochondrial compartments, where it is reconverted to Rf by FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) in a recycling pathway, i.e. the Rf-FAD cycle (4, 7). This novel mitochondrial pathway is assumed to play a central role in cellular Rf homeostasis and in flavoprotein biogenesis (5,8).The origin of flavin cofactors in yeast mitochondria is still controversially discussed. It has been reported that yeast mitochondria do not contain their own FAD synthetase activity an...