Short- and long-term effects of dehydroascorbate in Lupinus albus and Allium cepa roots.

Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.

A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds.

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3, 4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular "secretory" peroxidase activity (-73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed.

Inhibition of galactonolactone dehydrogenase activity by lycorine.

Galactonolactone dehydrogenase, a mitochondrial enzyme catalyzing the last step in ascorbate biosynthesis, is strongly inhibited by lycorine. A concentration of 10 microM of the alkaloid fully inhibits the activity of the enzyme. The high sensitivity of this enzyme to lycorine supports the hypothesis that the lycorine specifically inhibits ascorbate biosynthesis and that all the other metabolic responses to lycorine treatment depend on this primary inhibition of ascorbate biosynthesis.

The multiplicity of enzymatic DNA reduction: a new purification procedure for a DHA reducing protein from potato tubers.

The multiplicity of DHA reducing proteins has been observed by means of a native-PAGE technique in several plant species, thus demonstrating, in accordance with recent literature, that several differently evolved proteins are likely to perform DHA reduction. Moreover, a research strategy coupling the use of native-PAGE with chromatographic separation procedure, tentatively performed in Solanum tuberosum, proved to be a useful tool for the separation and partial identification of the proteins involved in DHA reduction.

An electrophoretic procedure to detect three key enzymes of the ascorbate system.

A method for detection of ascorbic acid oxidase, ascorbic acid peroxidase and dehydroascorbic acid reductase is reported. This method allows the qualitative determination of the presence of these enzymes, also in conditions where the commonly used spectrophotometric assays are unreliable.

Ascorbate system in Dasypyrum villosum from different environments.

Ascorbic acid content and redox-enzymes activities of AA system are determined in four population of Dasypyrum villosum adapted to live in different environments. In D. villosum from the driest and warmest environment (Pachino), AFR reductase and AA peroxidase have activities lower than in the other populations. The results point out the role of AA peroxidase as the main "scavenger" of the H2O2 produced by cell metabolism.

Reduction of cytochrome C by ascorbic free radical.

The rate of "in vivo" reduction of cytochrome c by ascorbic acid (AA) increases from 69 nmoles of cytochrome c for minute, to 202 nanomoles when ascorbate oxidase is added. Since the AA oxidation by AA oxidase is a system to generate ascorbic free radical (AFR), data suggest that AFR is a better reducing compound than ascorbate in cytochrome c reduction. Since the addition of oxidized glutathione and human immunoglobulins (-S-S- bridge containing compounds) in the medium produces a remarkable decrease in cytochrome c reduction, it is suggested that AFR could also reduce -S-S- groups.