Marco Zancani

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.

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.

Guaiacol peroxidase associated to soybean root plasma membranes oxidizes ascorbate

Summary Soybean ( Glycine max L. (Merr.)) root plasma membranes exhibited a guaiacol peroxidase activity that was resolved into two bands after SDS-PAGE of solubilized membranes. The activity was more or less tightly bound to membranes, because treatment with NaCl or EGTA resulted in a 29 or 59 % decrease, respectively, whereas CaCl 2 induced a 42 % increase. The membrane-bound peroxidase was ca. 40 % lowered by treatment of roots with tunicamycin, thus indicating the glycoprotein nature of the protein. Isolated plasma membranes exhibited a negligible level of ascorbate oxidation, which, however, was strongly stimulated by phenolic acids (caffeic and ferulic acids) at 50 μmol/L. It is suggested that plasma membrane-associated peroxidase oxidizing ascorbate in the presence of phenols may contribute in detoxifying hydrogen peroxide at the interface cell wall/plasmalemma.

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.

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles.

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fentons reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.

Phenol-dependent H2O2 Breakdown by Soybean Root Plasma Membrane-bound Peroxidase is Regulated by Ascorbate and Thiols

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.

Copper-inhibited NADH-Dependent peroxidase activity of purified soya bean plasma membranes

Abstract 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.

Free fatty acids dissipate proton electrochemical gradients in pea stem microsomes and submitochondrial particles

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.

Pyrophosphate and H+-pyrophosphatase maintain the vacuolar proton gradient in metabolic inhibitor-treated Acer pseudoplatanus cells

Abstract 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.

Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells.

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.