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In striking analogy with Saccharomyces cerevisiae , etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by Mg-ADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae ; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel. [ABSTRACT FROM AUTHOR]

The synthesis of ATP in mitochondria is dependent on a low permeability of the inner membrane. Nevertheless, mitochondria can undergo an increased permeability to solutes, named permeability transition (PT) that is mediated by a permeability transition pore (PTP). PTP opening requires matrix Ca2+ and leads to mitochondrial swelling and release of intramembrane space proteins (e.g., cytochrome c). This feature has been initially observed in mammalian mitochondria and tentatively attributed to some components present either in the outer or inner membrane. Recent works on mammalian mitochondria point to mitochondrial ATP synthase dimers as physical basis for PT, a finding that has been substantiated in yeast and Drosophila mitochondria. In plant mitochondria, swelling and release of proteins have been linked to programmed cell death, but in isolated mitochondria PT has been observed in only a few cases and in plant cell cultures only indirect evidence is available. The possibility that mitochondrial ATP synthase dimers could function as PTP also in plants is discussed here on the basis of the current evidence. Finally, a hypothetical explanation for the origin of PTP is provided in the framework of molecular exaptation. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) is a finely tuned process of multicellular organisms. In higher plants, PCD regulates many developmental processes and the response of host plants to incompatible pathogens (hypersensitive response). Four types of PCD have been described in plants, mainly associated to vacuole rupture, that is followed by the appearance of the typical PCD hallmarks (i.e. nuclear DNA fragmentation and cell shrinkage). However, in some cases vacuole collapse is preceded by an early alteration of other subcellular organelles, such as mitochondria. In particular, the central role played by mitochondria in PCD has been largely recognised in animal cells. This review deals with the involvement of mitochondria in the manifestation of plant PCD, in comparison to that described in animal PCD. The main hallmark, connecting animal and plant PCD via mitochondria, is represented by the release of cytochrome c and possibly other chemicals such as nucleases, which may be accomplished by different mechanisms, involving both swelling and non-swelling of the organelles. [ABSTRACT FROM AUTHOR]

Pea (Pisum sativum) stem mitochondria, energized by NADH, succinate or malate plus glutamate, underwent a spontaneous low-amplitude permeability transition (PT), which could be monitored by dissipation of the electrical potential (ΔΨ) or swelling. The occurrence of the latter effects was dependent on O2 availability, because O2 shortage anticipated the manifestation of both ΔΨ dissipation and swelling. Spontaneous ΔΨ collapse was also monitored in sucrose-resuspended mitochondria and again O2 deprivation caused an anticipation of the phenomenon. However, in this case ΔΨ dissipation was not accompanied by a parallel mitochondrial swelling. The latter effect was, indeed, evident only if mitochondria were resuspended in KCl (as osmoticum), or other cations with a molecular mass up to 100 Da (choline+). PT was also induced by protonophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or free fatty acids) or valinomycin (only in KCl). The FCCP-induced dissipation of ΔΨ and swelling were inhibited by ATP and stimulated (anticipated) by cyclosporin A or O2 shortage. The FCCP-induced PT was accompanied by the release of pyridine nucleotides from the matrix and of cytochrome c from the intermembrane space of KCl-resuspended mitochondria. The spontaneous and FCCP-induced low-amplitude PT of plant mitochondria are interpreted as due to the activity of a recently identified KATP+ channel whose open/closed state is dependent on polarization of the inner membrane and on the oxidoreductive state of some sulfhydryl groups. [Copyright &y& Elsevier]

The role of a recently identified K[sup +, sub ATP] channel in preventing H[sub 2]O[sub 2] formation was examined in isolated pea stem mitochondria. The succinate-dependent H[sub 2]O[sub 2] formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15 M). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H[sub 2]O[sub 2] formation was not influenced. The succinate-sustained H[sub 2]O[sub 2] generation was also unaffected by nigericin (a H[sup +]/K[sup +] exchanger), but completely prevented by valinomycin (a K[sup +] ionophore). In addition, cyclosporin A (a K[sup +, sub ATP] channel opener) inhibited this H[sub 2]O[sub 2] formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K[sup +] channel faces the intermembrane space. Finally, the succinate-dependent H[sub 2]O[sub 2] formation was partially prevented by phenylarsine oxide (a thiol oxidant). [ABSTRACT FROM AUTHOR]

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea (Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 mM. Pyrophosphate could enter from te cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F– and Ca2+ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F– or Ca2+. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate-generated ΔΨ had reached a steady state and, again, F– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H+-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H+-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F– or Ca2+. The physiological role of the mitochondrial H+-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction. [ABSTRACT FROM AUTHOR]

The H+/PPi stoichiometry of the mitochondrial H+-PPiase from pea (Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H+/substrate stoichiometries of the mitochondrial H+-ATPase, and the vacuolar H+-PPiase and H+-ATPase. Using sub-mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate-dependent H+-transport were evaluated: by a mathematical model, describing the time-course of H+-gradient (ΔpH) formation; or by determining the rate of H+-leakage following H+-pumping inhibition by EDTA at the steady-state ΔpH. When the H+-transport rates were divided by those of PPi or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H+-PPiase and H+-ATPase, and for the vacuolar H+-ATPase. The stoichiometry of the vacuolar H+-PPiase was found to be ca 1. From these results, it is suggested that the mitochondrial H+-PPiase may, in theory, function as a primary H+-pump poised towards synthesis of PPi and, therefore, acting in parallel with the main H+-ATPase. [ABSTRACT FROM AUTHOR]

Proton pumping pyrophosphatase (H+-PPiase) of pea stem mitochondria appears to be localized on the inner surface of the inner membrane. Aminohexanediphosphonate and dichloromethylenediphosphonate exert different inhibitory effects on this activity and on that of tonoplast. Antibody raised against membrane-bound mitochondrial H+-PPiase does not react with tonoplast vesicles. Thus, plant mitochondrial H+PPiase seems to have a molecular structure different from that of vacuolar H+-PPiase. [ABSTRACT FROM AUTHOR]