Search

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.

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.

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.

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.

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.

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.

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).

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.