Properties of the Permeability Transition of Pea Stem Mitochondria.

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.

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

BACKGROUND: Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. RESULTS: Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. CONCLUSIONS: Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

The Permeability Transition in Plant Mitochondria: The Missing Link.

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.

Lipase activity and antioxidant capacity in coffee (Coffea arabica L.) seeds during germination.

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.

Plant flavonoids--biosynthesis, transport and involvement in stress responses.

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.

Involvement of plasma membrane peroxidases and oxylipin pathway in the recovery from phytoplasma disease in apple (Malus domestica).

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 mitochondrial permeability transition pore (PTP) - an example of multiple molecular exaptation?

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.

Immunohistochemical localisation of a putative flavonoid transporter in grape berries.

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.

Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.).

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.

Lipoxygenase distribution in coffee (Coffea arabica L.) berries.

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.