Search for compounds able to modulate fof1 atp synthase in switching from life enzyme to cell death executor

Mitochondria are the main site of energy power in eukaryotic cells. The enzyme FOF1 synthase is responsible for ATP production driven by the transmembrane proton gradient. The maintenance of a very low permeability of the inner mitochondrial membrane is crucial for this mechanism, since sudden opening of the permeability transition pore (PTP) leads to matrix swelling and outer membrane rupture, with release of proapoptotic factors. Recently, it has been suggested that dimers of ATP synthase in mammals could represent the main component of the mitochondrial PTP, a feature modulated by calcium and involving the matrix protein Cyclophilin D (CyPD). This study would help to develop new tools for the identification of plant secondary metabolites, in particular flavonoids, able to modulate PTP and therefore acting on the programmed cell death mediated by mitochondria. Therefore, this project would represent the first screening for plant molecules able to interfere with programmed cell death, as a preliminary study for the development of drugs active in PTPrelated pathologies

Caspase-3-like activity and proteasome degradation in grapevine suspension cell cultures undergoing silver-induced programmed cell death

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 \u3bcM) 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

Structural and functional properties of plant mitochondrial F-ATP synthase

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

Biochemical and immunochemical similarities among mammalian bilitranslocase and a plant flavonoid translocator

Flavonoids are a large class of plant secondary metabolites, belonging to polyphenol family, which possess pharmacological and nutritional properties. Their synthesis takes place only in plants, while mammals can acquire them only with diet. It has been demonstrated that flavonoid uptake occurs in rat also by the activity of bilitranslocase, a carrier that is involved in anion transport in liver cell, vascular endothelium and gastric mucosa. A sequence of bilitranslocase interacting with flavonoid moieties is already known and characterized. Antibody raised against such protein epitope were shown to exhibit cross-reactivity against plant membrane proteins in tissues involved in flavonoid transport and accumulation, such as teguments of carnation petals and skin of grape berries. Further immunolocalization studies allowed to demonstrate the presence of cross-reacting protein not only at the level of tegumental tissues, but also associated to sieve elements and seed teguments in grape berries

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

CHARACTERIZATION OF LIPASE ACTIVITY IN GREEN COFFEE BEANS DURING STORAGE AND GERMINATION

Lipase, green coffee, germination, storage, lipid Coffee seeds possess an intermediate storage pattern, showing a non-quiescent behaviour characterized by various metabolic reactions occurring during storage. In particular, lipase is the main enzyme involved in the mobilization of triacylglycerols, providing energy and a source of carbon skeleton during early stages of germination. During storage, the triacylglycerols might be involved in the generation of undesirable compounds (known as \u201coff-flavours\u201d), lowering both the viability of coffee seeds and the cup quality. In this work, the soluble protein fraction from coffee seeds and plantlets was extracted by acetone and then was utilised to assay lipase activity. Green coffee beans, harvested in Colombia, were stored at room temperature for 2-3 months, in order to verify the influence of prolonged storage on lipase activity. For germination experiments, the seeds were imbibed for 7 days at 30 \ub0C and transferred in perlite at 28 \ub0C and 90% R.H. for further 3 weeks. Lipase activity was detected by a colorimetric method based on specific degradation of a chromogenic substrate, at pH 8.2. Green coffee seeds exhibited an appreciable lipase activity that was slightly increased during storage. Such an activity was inhibited by tetrahydrolipstatin (THL) in a concentration-dependent manner, while it was slightly stimulated by both EGTA and EDTA. During the germination, after 10, 14, 17 and 21 days, lipase activity showed an initial increase that was followed by a gradual decrease. The effect of the presence or absence of the parchment (seed coat), during the first stages of germination, has also been investigated

Phospholipase activities in green coffee beans (Coffea arabica L.) harvested in different countries

Triacylglycerols (TAGs) are accumulated in specialised organelles called \u201coil bodies\u201d, which are enclosed in a phospholipid monolayer embedded with some unique proteins. Upon germination, such membranes are modified to allow the availability of TAGs as an energy source during early stages of seedling growth in oilseeds. This process occurs by the sequential and/or collective action of many hydrolytic enzymes, such as phospholipases, lipoxygenases and lipases that are associated to oil body membranes. In contrast, during seed storage, oilseed lipids may undergo lipolytic degradation processes leading to a wide range of metabolites potentially harmful for seed viability. In particular, green coffee endosperm consist of approx. 99% of the mature seed mass and contains many polyunsaturated fatty acids whose degradation leads to volatile compound formation through the oxylipin pathway. In spite of this, the enzymes involved in TAGs degradation (particularly lipases) are poorly studied. Therefore, the aim of this work was to evaluate the involvement of phospholipase activity in oil body membrane degradation during storage of green coffee (Coffea arabica L.) and to determine the correlations between storage lipid mobilization and maintenance of seed viability in beans harvested in different countries (Ethiopia, India, Kenya and Tanzania). Green coffee beans were frozen and powdered in liquid nitrogen and oil bodies were extracted with cold acetone. Phospholipase A1 and A2 activities were assayed in crude extracts by a fluorimetric method, using different probes. Such activities were just partially stimulated by free Ca2+, in contrast with what reported by others. Furthermore, PLA2 activity was assayed in a wide range of pH, evidencing two peaks of pH optimum. These results suggests that green coffee bean presents at least two isoforms of PLA2. Phospholipase profiles (PLA2 and total) were correlated with the provenience of the beans, showing a higher activity in those harvested in Ethiopia, while the lower was associated to beans from India

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 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

Analysis of Non-Structural Carbohydrates and Xylem Anatomy of Leaf Petioles Offers New Insights in the Drought Response of Two Grapevine Cultivars

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

Investigation of non-structural carbohydrates and xylem anatomy in petiole of grapevine varieties during water limitation and after re-irrigation

Water shortage (WS) during growing of Vitis vinifera L. can limit shoot growth and affect yield and fruit quality, as well as allocation of carbon reserves into perennial organs for the upcoming years. Varietal anatomical differences, such as specific mean xylem vessel diameter in petiole, are expected to influence water transport in canes facing water limitation. Several authors have also evidenced that non-structural carbohydrates (NSC) of adjacent living parenchyma are involved in the repair mechanism of embolized vessels. In this work, we evaluated NSC level and xylem anatomy in petiole of Cabernet Sauvignon and Syrah varieties, subjected to WS and subsequent water refilling in the summer of 2017. The anatomical analysis highlighted that Syrah had high frequency of classes of large vessels, and that the xylem differentiation of vascular bundles was also affected by WS. Moreover, petiole NSC content was significantly influenced by WS and recovery, supporting the hypothesis that starch mobilization was associated to an elevated concentration in soluble NSC. This effect was determinant for Cabernet Sauvignon, whose stress response seemed to be based mainly on NSC metabolism. Finally, Syrah, differently to Cabernet Sauvignon, sustained the WS-induced increase in soluble NSC of petiole also 18 h after re-watering