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The mitochondrial F-ATP synthase is responsible for coupling the transmembrane proton gradient, generated through the inner membrane by the electron transport chain, to the synthesis of ATP. This enzyme shares a basic architecture with the prokaryotic and chloroplast ones, since it is composed of a catalytic head (F1), located in the mitochondrial matrix, a membrane-bound part (FO), together with a central and a peripheral stalk. In this review we compare the structural and functional properties of F-ATP synthase in plant mitochondria with those of yeast and mammals. We also present the physiological impact of the alteration of F-ATP synthase in plants, with a special regard to its involvement in cytoplasmic male sterility. Furthermore, we show the involvement of this enzyme in plant stress responses. Finally, we discuss the role of F-ATP synthase in shaping the curvature of the mitochondrial inner membrane and in permeability transition pore formation.

Premise Despite great attention given to the relationship between plant growth and carbon balance in alpine tree species, little is known about shrubs at the treeline. We hypothesized that the pattern of main nonstructural carbohydrates (NSCs) across elevations depends on the interplay between phenotypic trait plasticity, plant-plant interaction, and elevation. Methods We studied the pattern of NSCs (i.e., glucose, fructose, sucrose, and starch) in alpine stands of Vaccinium myrtillus (above treeline) across an elevational gradient. In the same plots, we measured key growth traits (i.e., anatomical stem features) and shrub cover, evaluating putative relationships with NSCs. Results Glucose content was positively related with altitude, but negatively related with shrub cover. Sucrose decreased at high altitude and in older populations and increased with higher percentage of vascular tissue. Starch content increased at middle and high elevations and in stands with high shrub cover. Moreover, starch content was negatively related with the number of xylem rings and the percentage of phloem tissue, but positively correlated with the percentage of xylem tissue. Conclusions We found that the increase in carbon reserves across elevations was uncoupled from plant growth, supporting the growth limitation hypothesis, which postulates NSCs accumulate at high elevation as a consequence of low temperature. Moreover, the response of NSC content to the environmental stress caused by elevation was buffered by phenotypic plasticity of plant traits, suggesting that, under climate warming conditions, shrub expansion due to enhanced plant growth would be pronounced in old but sparse stands.

In grapevine, the anatomy of xylem conduits and the non-structural carbohydrates (NSCs) content of the associated living parenchyma are expected to influence water transport under water limitation. In fact, both NSC and xylem features play a role in plant recovery from drought stress. We evaluated these traits in petioles of Cabernet Sauvignon (CS) and Syrah (SY) cultivars during water stress (WS) and recovery. In CS, the stress response was associated to NSC consumption, supporting the hypothesis that starch mobilization is related to an increased supply of maltose and sucrose, putatively involved in drought stress responses at the xylem level. In contrast, in SY, the WS-induced increase in the latter soluble NSCs was maintained even 2 days after re-watering, suggesting a different pattern of utilization of NSC resources. Interestingly, the anatomical analysis revealed that conduits are constitutively wider in SY in well-watered (WW) plants, and that water stress led to the production of narrower conduits only in this cultivar.

The mitochondrial F-ATP synthase is the principal energy-conserving nanomotor of cells that harnesses the proton motive force generated by the respiratory chain to make ATP from ADP and phosphate in a process known as oxidative phosphorylation. In the energy-converting membranes, F-ATP synthase is a multisubunit complex organized into a membrane-extrinsic F1sector and a membrane-intrinsic FOdomain, linked by central and peripheral stalks. Due to its essential role in the cellular metabolism, malfunction of F-ATP synthase has been associated with a variety of pathological conditions, and the enzyme is now considered as a promising drug target for multiple disease conditions and for the regulation of energy metabolism. We discuss structural and functional features of mitochondrial F-ATP synthase as well as several conditions that partially or fully inhibit the coupling between the F1catalytic activities and the FOproton translocation, thus decreasing the cellular metabolic efficiency and transforming the enzyme into an energy-dissipating structure through molecular mechanisms that still remain to be defined.

Grapevines store non-structural carbohydrates (NSC) during late summer to sustain plant development at the onset of the following spring’s growth. Starch is the main stored carbohydrate, found in the wood-ray parenchyma of roots and canes. Although the relationship between hydraulic and plant photosynthetic performance is well-recognized, little research has been done on the long-term effects of drought in grapevines adopting different strategies to cope with water stress (i.e. isohydric and anisohydric). We performed our study by exposing two different grape cultivars (Syrah and Cabernet Sauvignon) to a short but severe drought stress, at two stages of the growing season (July and September). No marked differences in the physiological and hydraulic responses of the two varieties were found, probably due to our experimental conditions. However, anatomical and biochemical characterization of overwintering canes pointed out several interesting outcomes. We found a significant and parallel increase of starch and medullar ray number in both cultivars exposed to early water stress. We hypothesize that stressed vines limited their carbon allocation to growth, while shifting it to starch accumulation, with a most evident effect in the period of intense photosynthetic activity. We also speculate that a different aptitude to osmotic adjustment may underlay variation in starch increase and the specific involvement of bark NSC in the two cultivars.

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.

Toxic metal contamination is one of the major environmental concerns of the recent decade, due to the large application of metals in industrial, healthcare and commercial products, even in the form of nanostructures and nanomaterials. Nevertheless, the effects of silver (Ag+) on plants have not yet thoroughly elucidated. Therefore, suspension cell cultures of grapevine were used as a model for investigating silver toxicity. To do this, oxidative stress and programmed cell death (PCD), evaluated as reactive oxygen species production, caspase-3-like activity and ubiquitin-proteasome system, were investigated. As a result, the highest concentration (10 μM) of Ag+ caused a rapid (within 24 h) induction of PCD (approx. 80%), accompanied by generation of reactive oxygen species and activation of caspase-3-like activity. In the presence of specific inhibitor of this enzyme, a partial recovery of cell viability and a strong inhibition of caspase-3-like activity was observed. In addition, silver-induced PCD was accompanied either by increase of poly-ubiquitin conjugated proteins and degradation of subunit PBA1 of the proteasome 20S core, similarly to what found for metal-induced neurotoxicity in animals. The present study shows that silver could induce PCD in grapevine suspension cell cultures, mediated by caspase-3-like activity and oxidative stress. These effects were associated to accumulation of poly-ubiquitin conjugated proteins, suggesting the impairment of ubiquitin-proteasome complex, confirmed by the decrease of the PBA1 subunit. These findings indicate that animal and plant cells could share a common pathway in response to toxic metal, which involves PCD and disassembling of proteasome complex.

Enhanced shrub growth and expansion are widespread responses to climate warming in many arctic and alpine ecosystems. Warmer temperatures and shrub expansion could cause major changes in plant community structure, affecting both species composition and diversity. To improve our understanding of the ongoing changes in plant communities in alpine tundra, we studied interrelations among climate, shrub growth, shrub cover and plant diversity, using an elevation gradient as a proxy for climate conditions. Specifically, we analyzed growth of bilberry (Vaccinium myrtillus L.) and its associated plant communities along an elevation gradient of ca. 600 vertical meters in the eastern European Alps. We assessed the ramet age, ring width and shoot length of V. myrtillus, and the shrub cover and plant diversity of the community. At higher elevation, ramets of V. myrtillus were younger, with shorter shoots and narrower growth rings. Shoot length was positively related to shrub cover, but shrub cover did not show a direct relationship with elevation. A greater shrub cover had a negative effect on species richness, also affecting species composition (beta-diversity), but these variables were not influenced by elevation. Our findings suggest that changes in plant diversity are driven directly by shrub cover and only indirectly by climate, here represented by changes in elevation.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here we establish live MgATP2−assessment in plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensorin vitrounder conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2−changesin planta. A MgATP2−map of the Arabidopsis seedling highlights different MgATP2−concentrations between tissues and in individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.One-sentence SummarySensing of MgATP2−by fluorimetry and microscopy allows dissection of ATP fluxes of isolated organelles, and dynamics of cytosolic MgATP2−in vivo.Funding AgenciesThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Emmy-Noether programme (SCHW1719/1-1; M.S. and GR4251/1-1; C.G.), the Research Training Group GRK 2064 (M.S.; A.J.M.), the Priority Program SPP1710 (A.J.M.) and a grant (SCHW1719/5-1; M.S.) as part of the package PAK918. The Seed Fund grant CoSens from the Bioeconomy Science Center, NRW (A.J.M.; M.S.) is gratefully acknowledged. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13). A.Co. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca through the FIRB 2010 programme (RBFR10S1LJ_001) and Piano di Sviluppo di Ateneo 2015 (Università degli Studi di Milano). M.Z. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca (Italy) through the PRIN 2010 programme (PRIN2010CSJX4F). S.W. and T.N. received travel support by the Deutscher Akademischer Austauschdienst (DAAD). V.D.C. was supported by the European Social Fund, Operational Programme 2007/2013, and an Erasmus+ Traineeship grant. M.D.F was supported by The Human Frontier Science Program (RPG0053/2012), and the Leverhulme Foundation (RPG-2015-437). I.M.M. was supported by a grant from the Danish Council for Independent Research - Natural Sciences. V.C.P. was supported by the Innovation and Technology Fund (Funding Support to Partner State Key Laboratories in Hong Kong) of the HKSAR.AbbreviationsAAC – ADP/ATP carrier; AK – adenylate kinase; cAT – carboxyatractyloside; CCCP – carbonyl cyanide m-chlorophenyl hydrazone; CFP – cyan fluorescent protein; CLSM – confocal laser scanning microscopy; ETC – electron transport chain; FRET – Förster Resonance Energy Transfer; LSFM – light sheet fluorescence microscopy.

This paper aims at analysing the synthesis of flavonoids, their import and export in plant cell compartments, as well as their involvement in the response to stress, with particular reference to grapevine (Vitis vinifera L.). A multidrug and toxic compound extrusion (MATE) as well as ABC transporters have been demonstrated in the tonoplast of grape berry, where they perform a flavonoid transport. The involvement of a glutathione S-transferase (GST) gene has also been inferred. Recently, a putative flavonoid carrier, similar to mammalian bilitranslocase (BTL), has been identified in both grape berry skin and pulp. In skin the pattern of BTL expression increases from véraison to harvest, while in the pulp its expression reaches the maximum at the early ripening stage. Moreover, the presence of BTL in vascular bundles suggests its participation in long distance transport of flavonoids. In addition, the presence of a vesicular trafficking in plants responsible for flavonoid transport is discussed. Finally, the involvement of flavonoids in the response to stress is described.

In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤1500Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins.In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role.Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.

Apple trees (Malus domestica Borkh.) may be affected by apple proliferation (AP), caused by 'Candidatus Phytoplasma mali'. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. In this article it is shown that NAD(P)H peroxidases of leaf plasma membrane-enriched fractions exhibited a higher activity in samples from both AP-diseased and recovered plants. In addition, an increase in endogenous SA was characteristic of the symptomatic plants, since its content increased in samples obtained from diseased apple trees. In agreement, phenylalanine ammonia lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway, was increased too. Jasmonic acid (JA) increased only during recovery, in a phase subsequent to the pathological state, and in concomitance to a decline of salicylic acid (SA). Oxylipin pathway, responsible for JA synthesis, was not induced during the development of AP-disease, but it appeared to be stimulated when the recovery occurred. Accordingly, lipoxygenase (LOX) activity, detected in plasma membrane-enriched fractions, showed an increase in apple leaves obtained from recovered plants. This enhancement was paralleled by an increase of hydroperoxide lyase (HPL) activity, detected in leaf microsomes, albeit the latter enzyme was activated in either the disease or recovery conditions. Hence, a reciprocal antagonism between SA- and JA-pathways could be suggested as an effective mechanism by which apple plants react to phytoplasma invasions, thereby providing a suitable defense response leading to the establishment of the recovery phenomenon.

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 Ca(2+) 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.

To investigate the role of natural humic substances on plant cell phosphate level and metabolism, tobacco BY-2 suspension cell cultures were grown in the presence of humic samples of different chemical composition: soil humic acid and its three size-fractions (I–III) separated by High Pressure Size Exclusion Chromatography. The humic samples were characterized by CPMAS-NMR spectroscopy and on-line pyrolysis–gas chromatography–mass spectrometry. Suspension cell cultures, after 7 days of incubation, were facing Pi starvation. The fraction III, the most hydrophilic and smallest in molecular size among humic samples, induced a partial relief from Pi starvation, increasing total cell phosphate amount, ATP and glucose-6-phosphate levels, as well as the activity of secreted acid phosphatases. Furthermore, fraction III induced a decrease of KCN-insensitive respiration, evaluated in both suspension cells and isolated mitochondria. The low amount of acidic groups in fraction III excluded that its observed effect in relieving cells from Pi deficiency may be attributed to a partial replacement of the chelating ability of secreted acids (mainly citric and malic acids) in releasing Pi from metal–phosphate complexes. The molecular characteristics of fraction III are conducive to a flexible conformational structure due to hydrophilic domains, which are still contoured by hydrophobic moieties such as alkyl and aromatic compounds. Such flexible molecular associations may induce an efficient release of Pi from organic sources (e.g. nucleic acids), released in the media by damaged or dead cells, exerting a sort of positive effect on either the production or activity of extracellular Pi hydrolytic enzymes. This work shows that only by combining advanced molecular characterization of natural humic molecules with their effect on plant cells, it is possible to formulate sound hypotheses for structure–activity relationships.

Iron sequestration inside the plant cell may be accomplished by ferritins, a class of proteins able to buffer and store this metal in a safe and soluble form. This protein has been shown in plastids and only recently in plant mitochondria. In this paper, we examined the localization of mitochondrial ferritin in different plant organs and its possible involvement in iron uptake and/or release. Ferritin was found only in mitochondria from young organs, such as roots, etiolated stems and, although to a lesser extent, developing leaves. Its presence was not influenced by iron in the hydroponic medium of culture, as well as by the induction of senescence through ethylene treatment. Lipoperoxidation, evaluated as formation of thiobarbituric acid reactive substances (TBARS), was determined in mitochondria isolated from etiolated stems and leaves of plants incubated with FeSO 4 /citrate and ascorbate. The phenomenon was lower in young stem mitochondria, where ferritin was detected, than in those from leaves, where the protein was undetectable. Therefore, these results indicate that the mitochondrial ferritin was able to sequestrate iron within its shell. A significant increase of TBARS formation was also found in etiolated stem mitochondria incubated, in the absence of supplied iron, with 5 mM ascorbate, which was paralleled by a decrease of iron in mitochondrial ferritin, detected by Inductively Coupled Plasma Mass Spectrum (ICP-MS) measurements after immunoprecipitation. These results indicate that plant mitochondrial ferritin plays a key role in iron buffering through the uptake and release of iron that could be modulated by ascorbate.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.

A soluble protein with a molecular mass of 55 kDa has been purified from etiolated pea stem mitochondria. The protein exhibits a Mg2+-requiring PPiase activity, with an optimum at pH 9.0, which is not stimulated by monovalent cations, but inhibited by F−, Ca2+, aminomethylenediphosphate and imidodiphosphate. The protein does not cross-react with polyclonal antibodies raised against vacuolar, mitochondrial or soluble PPiases, respectively. Conversely, it cross-reacts with an antibody for the α/β-subunit of the ATP synthase from beef heart mitochondria. The purified protein has been analyzed by MALDI-TOF mass spectrometry and the results, covering the 30% of assigned sequence, indicate that it corresponds to the β-subunit of the ATP synthase of pea mitochondria. It is suggested that this enzymatic protein may perform a dual function as soluble PPiase or as subunit of the more complex ATP synthase.

In this paper, lipase activity was characterized in coffee (Coffea arabica L.) seeds to determine its involvement in lipid degradation during germination. The lipase activity, evaluated by a colorimetric method, was already present before imbibition of seeds and was further induced during the germination process. The activity showed a biphasic behaviour, which was similar in seeds either with or without endocarp (parchment), even though the phenomenon showed a delay in the former. The enzymatic activity was inhibited by tetrahydrolipstatin (THL), a selective and irreversible inhibitor of lipases, and by a polyclonal antibody raised against purified alkaline lipase from castor bean. The immunochemical analysis evidenced a protein of ca. 60 kDa, cross-reacting with an anti-lipase antibody, in coffee samples obtained from seeds of both types. Gas chromatographic analyses of free fatty acid (FFA) content confirmed the differences shown in the lipolytic activity of the samples with or without parchment, since FFA levels increased more rapidly in samples without parchment. Finally, the analyses of the antioxidant capacity showed that the presence of parchment was crucial for lowering the oxidation of the lipophylic fraction, being the seeds with parchment less prone to oxidation processes.

The influence of light on fresh-cut vegetables during storage is controversial, since both positive and negative effects on shelf-life and quality of such products have been observed. In this work, the effect of low-intensity light treatments on lamb's lettuce, a fresh-cut leafy and ready-to-eat vegetable, was investigated during storage at low temperature (6 °C), in comparison with conventional storage (in the dark at 4 °C). Although continuous light treatment (1 cycle of 8 h per day) was deleterious, cycles of light treatments (8 cycles of 1 h per day; 16 cycles of 0.5 h per day) showed positive effects, assessed by evaluating the content of chlorophylls, carotenoids, ATP, glucose and ascorbate. These analyses were performed at the beginning and after 6 days of storage, in comparison with samples stored in the dark at 4 °C. Under low-intensity light treatments, even if performed at a higher temperature (6 °C), the content of such bioactive compounds increased or was at least similar to that found in samples stored in the dark at the same temperature. We suggest that continuous low-intensity light treatments during cold storage of lamb's lettuce are able to promote photosynthesis but, at the same time, induce photo-damage. On the contrary, under intermittent low-intensity light cycles, photosynthesis is only partially activated, while the metabolism of the green tissues is still able to provide carbon moieties for the synthesis of bioactive molecules involved in delaying senescence. Therefore, low-intensity light cycles at 6 °C could contribute to maintain quality of lamb's lettuce, with respect to samples stored in the dark at both 6 and 4 °C. Finally, setting the temperature at 6 °C allows reduction of refrigerator energy consumption during storage.

The role of free Mg2+ and its chelated forms as a fine mechanism to control succinate dehydrogenase (SDH) and the phosphorylative activities, in particular that of H+-PPiase, of pea stem mitochondria was examined. Free pyrophosphate (PPi) was found to be a competitive inhibitor of SDH in sub-mitochondrial particles (SMP), but this inhibition was relieved when PPi was chelated by Mg2+. Neither Mg2+ alone in SMP, nor Mg2+ depletion in intact mitochondria affected SDH activity. Chelation of Mg2+ by ATP, ADP, EDTA or citrate restored PPi-induced inhibition of SDH activity. Being (Mg)PPi or (Mg)2PPi the substrate of the H+-PPiase, the subtraction of Mg2+ from this complex by stronger chelators, such as ATP or ADP, liberates free PPi causing an inhibition of the H+-PPiase activity. In particular, the formation of the (Mg)ADP chelate could favor the H+-ATPase, being its substrate. Therefore, the activities of SDH, H+-PPiase and H+-ATPase appear to be interconnected and regulated by the equilibrium between free Mg2+ and its chelates with PPi, ADP and ATP.

The H + /PP i stoichiometry of the mitochondrial H + -PP i ase 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 + -PP i ase 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 PP i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H + -PP i ase and H + -ATPase, and for the vacuolar H + -ATPase. The stoichiometry of the vacuolar H + -PP i ase was found to be ca 1. From these results, it is suggested that the mitochondrial H + -PP i ase may, in theory, function as a primary H + -pump poised towards synthesis of PP i and, therefore, acting in parallel with the main H + -ATPase.

Proton pumping pyrophosphatase (H-PP,ase) 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 HPPjase does not react with tonoplast vesicles. Thus, plant mitochondrial H-PPjase seems to have a molecular structure different from that of vacuolar H-PPiase.

Highly purified soya bean plasma membranes exhibited peroxidase activity. Divalent copper strongly inhibited NADH oxidation (but not H 2 O 2 oxidation) catalysed by this enzyme, whereas Cd 2+ , Ni 2+ or Zn 2+ had little or no effect. This inhibition did not depend on a reaction by sulphydryl groups, or on a replacement of ions in the enzyme by Cu 2+ . The effect of Cu 2+ may be explained by its scavenging capability towards O 2 − which is produced during NADH-dependent peroxidase activity.

The effect of metabolic inhibitors (KCN and 2-deoxy- d -glucose) on the vacuolar proton gradient, monitored by acridine orange, was assayed in Acer pseudoplatanus cells. Potassium cyanide plus 2-deoxy- d -glucose slightly lowered this gradient, while cellular ATP level was strongly decreased and inorganic pyrophosphate (PP i ) content was halved. Two phosphatase inhibitors (imidodiphosphate and KF) restored the PP i level in KCN-treated cells, but decreased the vacuolar proton gradient by inhibiting H+-PP i ase. These results, hence, suggest that tonoplast H + -PP i ase is especially responsible for the maintenance of vacuolar ΔpH and that this enzyme is the major scavenger of cytoplasmic PP i in cells treated with metabolic inhibitors.

The catalytic subunit of a membrane-bound pyrophosphatase was purified by electroendosmotic preparative electrophoresis from etiolated pea stem mitochondria. The enzyme was identified as a single peak relatively pure, because only a very limited number of polypeptides were detectable by SDS/PAGE of the active fractions. The pyrophosphatase was associated to a band with a molecular mass of 35 kDa, showing a specific activity of 0.7 mumol Pi . mg-1 protein . min-1 (37 degrees C, pH 8.0) and an apparent Km value of 200 microM. The hydrolytic activity required Mg2+, was inhibited by imidodiphosphate (HNO6P2Na4), Ca2+, F- and was stimulated by phospholipids. Cardiolipin, phophatidylcholine and phosphatidylethanolamine had the maximal activating effect. The isolated protein is very similar to the catalytic subunit of pyrophosphatases isolated from rat liver (beta-subunit) and Saccharomyces cerevisiae mitochondria.

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.

Flavonoids are a class of secondary metabolites present in large amounts in grapevine (Vitis vinifera L.), which are involved in several aspects of its physiology (e.g. protection against biotic and abiotic stress). Even if the biosynthetic pathways of flavonoid sub-classes have been largely characterised, the mechanisms of their transport and accumulation to the final target sites are still not completely understood. Unanticipated insights have been obtained by probing plant tissues with pure antibodies targeting bilitranslocase (BTL, TCDB # 2.A.65.1.1), a mammalian transporter involved in the absorption and tissue distribution of dietary flavonoids. The occurrence of a BTL homologue has also been found in grape berries, in both tegumental layers of skin and pulp vascular bundles. In the skin, the expression of this protein starts from véraison (starting of the change in colour and softening of berries) and increases up to a maximum at the harvest stage, matching the same temporal pattern of flavonoid accumulation.

Flavonoids are a class of secondary metabolites present in large amounts in grapevine (Vitis vinifera L.), which are involved in several aspects of its physiology (e.g. protection against biotic and abiotic stress). Even if the biosynthetic pathways of flavonoid sub-classes have been largely characterised, the mechanisms of their transport and accumulation to the final target sites are still not completely understood. Unanticipated insights have been obtained by probing plant tissues with pure antibodies targeting bilitranslocase (BTL, TCDB # 2.A.65.1.1), a mammalian transporter involved in the absorption and tissue distribution of dietary flavonoids. The occurrence of a BTL homologue has also been found in grape berries, in both tegumental layers of skin and pulp vascular bundles. In the skin, the expression of this protein starts from veraison (starting of the change in colour and softening of berries) and increases up to a maximum at the harvest stage, matching the same temporal pattern of flavonoid accumulation.

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O−2, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H2O2 that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H2O2 (catalase) or preventing H2O2 formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O−2, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H2O2 that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).

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 n 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.

To investigate the possible role of basic residues in H+ translocation through vacuolar-type H+-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a “decoupled” phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.

In this paper, the relationship between adenylate kinase and apyrase activities in controlling the adenylate pool of pea stem mitochondria was examined. Non-purified mitochondria possess an adenylate kinase, localised on the outer surface of the inner membrane, and an apyrase on the surface of the outer membrane. These organelles, energised by succinate, exhibit a cyanide-insensitive ATP synthesis, which is inhibited by P 1 ,P 5 -di(adenosine-5′)pentaphosphate (Ap5A) and carboxyatractylate (CAtr). This synthesis can also be followed either as AMP-induced dissipation of the electrical potential, that is reversed by CAtr and prevented by Ap5A, or as state 4 respiration which is partially inhibited by Ap5A and CAtr. However, when Percoll-purified mitochondria are used, state 4 respiration is insensitive to both inhibitors. Commercial apyrase exogenously supplied to purified mitochondria (which do not have apyrase activity) restores Ap5A- and CAtr-sensitive state 4 oxygen consumption. These results can be interpreted as follows. Apyrase, probably of cytoskeleton or endoplasmic reticulum origin and still adhering to non-purified mitochondria, produces AMP by hydrolysing ADP. AMP, acting as co-substrate with endogenous ATP, is utilised by adenylate kinase (reverse reaction) to generate ADP which, once entered the mitochondrial matrix, is available for ATP synthase.

Summary In the presence of hydrogen peroxide, soybean root plasma membranes oxidize caffeic acid, ferulic acid and coniferyl alcohol, exhibiting a pattern very similar to that described for horseradish peroxidase (EC 1.11.1.7). Ascorbic acid, rysteine, and dithioerythritol, added during the course of the reaction, caused inhibition that, in particular for ascorbic acid and cysteine, had a transient effect, being followed by a recovery of activity, as observed for cell wall-bound guaiacol peroxidase. This transient effect is paralleled by ascorbate oxidation: the oxidation of phenolics could restart only when ascorbate was completely oxidized. A similar delay of phenolic oxidation was also observed in the presence of rysteine or dithioerythritol. The reduction of hydrogen peroxide to water by horseradish peroxidase or soybean root plasma membrane, in the presence of phenolics, was enhanced by the addition of ascorbic acid, rysteine or dithioerythritol. At pH 7.0, horseradish peroxidase was reduced to Compound III by thiols but not by ascorbic acid, while at pH 5.5 ascorbate, rysteine or dithioerythritol did not exert any effect. These results confirm that plasma membranes possess peroxidase(s) similar to the well-known horseradish peroxidase, probably localized on the apoplastic side of the membrane, which is regulated by ascorbic acid availability. This enzyme could have a dual role in the generation/breakdown of H 2 O 2 depending on the physiological circumstances.

Pea stem submitochondrial particles have an inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) which is able to couple PP i hydrolysis with the generation of an inside positive proton gradient. The activity is Mg 2+ -requiring, is stimulated by monovalent cations, inhibited by imidodiphosphate and, partially, by dicyclohexylcarbodiimide, diethylstilbestrol and oligomycin. This mitochondrial H + -PP i ase is distinguishable from the tonoplast H + -PP i ase and appears to be an additional coupling factor, which works in parallel with the well-known H + -ATPase.

The effect of free fatty acids (FFA) and lysophosphatidylcholine-oleoyl (lyso-PC) on proton gradients of pea stem microsomes and submitochondrial particles was studied. Linolenic (18:3), linoleic (18:2), oleic (18:1), palmitic (16:0) and stearic (18:0) acids collapsed the proton gradient generated by addition of ATP or PP to microsomes. When an artificial ΔpH was generated by NaOH, FFA did not induce any effect, but the subsequent addition of valinomycin dissipated the proton gradient. FFA were also able to discharge the ΔpH built up by the oligomycin-sensitive H + -ATPase of submitochondrial particles and the electrical potential generated by NADH oxidation in intact mitochondria. Free fatty acids stimulated NADH-dependent oxygen consumption by mitochondria and this effect was not abolished by ADP or carboxyatractyloside (CAtr). The effect of FFA increased with an increasing unsaturation of the acyl chain, while the length of the chain did not influence the activity. Lysophosphatidylcholine dissipated the proton gradient generated by H + -PPase of microsomes and H + -ATPase of submitochondrial particles, while the H + -ATPase of microsomes was slightly affected. In addition, lyso-PC stimulated NADH-dependent oxygen uptake by mitochondria. Also in this case, neither ADP nor CAtr inhibited this stimulated O 2 consumption. These results show that FFA uncoupled oxidative phosphorylation of pea mitochondria and collapsed only proton electrochemical gradients in pea microsomes and submitochondrial particles. Therefore, in this regard FFA are similar to artificial protonophores, acting as proton carriers. The mechanism of action of lyso-PC appears to be more complex and different possible explanations are proposed.