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.

Fruits and vegetables are rich in flavonoids, and ample epidemiological data show that diets rich in fruits and vegetables confer protection against cardiovascular, neurodegenerative and inflammatory diseases, and cancer. However, flavonoid bioavailability is reportedly very low in mammals and the molecular mechanisms of their action are still poorly known. This review focuses on membrane transport of flavonoids, a critical determinant of their bioavailability. Cellular influx and efflux transporters are reviewed for their involvement in the absorption of flavonoids from the gastro-intestinal tract and their subsequent tissue distribution. A focus on the mammalian bilirubin transporter bilitranslocase (TCDB 2.A.65.1.1) provides further insight into flavonoid bioavailability and its relationship with plasma bilirubin (an endogenous antioxidant). The general function of bilitranslocase as a flavonoid membrane transporter is further demonstrated by the occurrence of a plant homologue in organs (petals, berries) where flavonoid biosynthesis is most active. Bilitranslocase appears associated with sub-cellular membrane compartments and operates as a flavonoid membrane transporter.

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca 2؉ and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.Mitochondria are the focus of intense research as major integrators and regulators of cell death pathways (7,8,10,15). Release of death-promoting factors by mitochondria has been reported as a regulatory event in apoptotic death mediated by different signals (20,35,37,39). Mitochondrial dysfunction may also cause necrotic death, and the role played by mitochondria has been well documented in several experimental systems (1,21,23,40). The central role of mitochondria as integrators of the death effector mechanisms is suggested by the fact that several signals derived either from external stimuli or from the cytosol or nucleus converge on mitochondria to trigger cell death (8).The human antimicrobial peptide hystatin 5 was recently added to the number of toxic agents that act through mitochondria. This molecule is cytotoxic to Candida albicans in a manner dependent on functionally active mitochondria, as inhibition of respiration protects the cells from its toxicity (16). Likewise, de-energized human cell lines are protected from the cytotoxic effects of other antimicrobial peptides such as the human defensins (25, 26) and the bovine BMAP-28, a cationic peptide of the cathelicidin family (34,36,45). The latter agent causes membrane permeabilization and death of activated human lymphocytes and tumor cells (34). The cytotoxic activity has been related to the structural features of the peptide, which consists of a cationic N-terminal sequence predicted to assume an amphipathic ␣-helical conformation (residues 1 to 18) and a C-terminal hydrophobic tail (residues 19 to 27). The C-terminal sequence seems to be crucial for the cytotoxic activity, as a great reduction of this effect is observed with the synthetic analogue BMAP-28(1-18) comprising the 18 N-terminal residues (36). A second requirement for cytotoxicity is an active metabolism of the target cells, as BMAP-28 is ineffective in nonrespiring cells (34).In view of these observations, we have investigated whether mitochondria are targets of the peptide itself and/or mediators of i...

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.

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

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high‐affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono‐ and di‐glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein‐based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (KM = 5.3 µm), which is competitively inhibited by cyanidine 3‐glucoside (Ki = 51.6 µm) and mainly noncompetitively by cyanidin (Ki = 88.3 µm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin‐binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high‐affinity binding site for cyanidine 3‐glucoside (Kd = 1.7 µm) on the carnation carrier only, and a high‐affinity bilirubin‐binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabelled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.

During maturation, Vitis vinifera berries accumulate a large amount of several anthocyanins in the epidermal tissue, whereas their precursors and intermediates are ubiquitously synthesized within the fruit. Up to date, several mechanisms of flavonoid transport at subcellular level have been hypothesized, but it is not possible to identify a general model applicable in every plant tissue and organ. Recently, a putative anthocyanin carrier, homologue to mammalian bilitranslocase (BTL) (TC 2.A.65.1.1), was found in Dianthus caryophyllus petal microsomes. In the present paper, an immunohistochemical and immunochemical analysis, using an antibody raised against a BTL epitope, evidences the expression and function of such a transporter in V. vinifera berries (cv. Merlot). Specific localisations of the putative carrier within berry tissues together with expression changes during different developmental stages are shown. Water stress induces an increase in protein expression in both skin and pulp samples. A bromosulfalein (BSP) uptake activity, inhibitable by the BTL antibody, is detected in berry mesocarp microsomes, with K (m) = 2.39 microM BSP and V (max) = 0.29 micromol BSP min(-1) mg(-1) protein. This BSP uptake is also competitively inhibited by quercetin (K (i) = 4 microM). A putative role for this carrier is discussed in relation to the membrane transport of secondary metabolites.

The generation of H 2 O 2 by isolated pea stem mitochondria, oxidizing either malate plus glutamate or succinate, was examined. The level of H 2 O 2 was almost one order of magnitude higher when mitochondria were energized by succinate. The succinate-dependent H 2 O 2 formation was abolished by malonate, but unaffected by rotenone. The lack of effect of the latter suggests that pea mitochondria were working with a proton motive force below the threshold value required for reverse electron transfer. The activation by pyruvate of the alternative oxidase was reflected in an inhibition of H 2 O 2 formation. This effect was stronger when pea mitochondria oxidized malate plus glutamate. Succinate-dependent H 2 O 2 formation was ca. four times lower in Arum sp. mitochondria (known to have a high alternative oxidase) than in pea mitochondria. An uncoupler (FCCP) completely prevented succinate-dependent H 2 O 2 generation, while it only partially (40^50%) inhibited that linked to malate plus glutamate. ADP plus inorganic phosphate (transition from state 4 to state 3) also inhibited the succinate-dependent H 2 O 2 formation. Conversely, that dependent on malate plus glutamate oxidation was unaffected by low and stimulated by high concentrations of ADP. These results show that the main bulk of H 2 O 2 is formed during substrate oxidation at the level of complex II and that this generation may be prevented by either dissipation of the electrochemical proton gradient (uncoupling and transition state 4-state 3), or preventing its formation (alternative oxidase). Conversely, H 2 O 2 production, dependent on oxidation of complex I substrate, is mainly lowered by the activation of the alternative oxidase.z 1999 Federation of European Biochemical Societies.