Silvio Dipierro

Ascorbate free radical reductase, a key enzyme of the ascorbic acid system

Following the finding that lycorine (an alkaloid from Amarillidaceae) is a powerful inhibitor of ascorbic acid (AA) biosynthesis, it was possible to establish that large amounts of AA are utilized in plant cell metabolism [ 1,2], similarly to what happens in animals [3,4]. Cell metabolism utilizes ascorbic acid essentially as an electron donor, so that AA undergoes continuous oxidation and reduction. Oxidation products are ascorbic free radical (AFR) and dehydroascorbic acid (DHA). They can be reconverted to AA by two enzymes: AFR-reductase (NADH:AFRoxidoreductase = NADH:monodehydroascorbate oxidoreductase, EC 1.6.5.4) [5-81 and DHA-reductase (GSH:dehydroascorbate oxidoreductase, EC 1.8.5.1) [9]. Here we report data concerning the relative contribution of these two enzymes in maintaining the ascorbic acid system in the reduced state.

Purification and properties of ascorbate free-radical reductase from potato tubers

Ascrobate free-radical reductase (EC 1.6.5.4) from potato tubers was purified to apparent homogencity by a method which included ammonium-sulfate precipitation, gel filtration and chromatography on diethylaminoethyl cellulose and hydroxylapatite. Gel filtration and gel electrophoresis showed that the purified enzyme was monomeric with a molecular weight of about 42 000. Enzyme activity was heat lable and severely inhibited by thiol reagents. The Km values for enzyme substrates were estimated.

Dehydroascorbate reductase from potato tubers

Abstract Dehydroascorbate reductase from the cytosol of potato tubers was purified to a nearly homogeneous state. The enzyme had a M r of ca 23 000, a pH optimum of ica 8, a K m for dehydroascorbate of 0.39 mM and a K m for GSH of 4.35 mM. GSH could not be replaced by other electron donors such as cysteine or dithiothreitol. The enzyme was rapidly inhibited by compounds which react with thiol groups. Inhibition could be either prevented, or partially reversed by GSH, thus suggesting that thiol groups are involved in the reaction.

A Specific Ascorbate Free Radical Reductase Isozyme Participates in the Regeneration of Ascorbate for Scavenging Toxic Oxygen Species in Potato Tuber Mitochondria

Ascorbate free radical (AFR) reductase from isolated potato tuber (Solanum tuberosum L.) mitochondria was studied. The enzyme was purified to homogeneity and its physico-chemical and kinetic properties were compared to those of the cytosolic enzyme. The molecular mass of the mitochondrial enzyme was about 54 kD, whereas that of the cytosolic enzyme was about 42 kD. The Km values of mitochondrial AFR reductase for NADH, NADPH, and AFR were higher than those of the cytosolic enzyme. Moreover, the mitochondrial enzyme proved to be less sensitive to inhibition by sulfhydryl reagents. It was concluded that the ascorbate involved in the scavenging of toxic oxygen species in potato tuber mitochondria is regenerated via the ascorbate-glutathione pathway, in which a specific AFR reductase isozyme participates.

The Role of the Ascorbic Acid System in Delaying the Senescence of Oat (Avena sativa L.) Leaf Segments

Summary The changes in the ascorbic acid system during the onset of senescence in oat leaf segments were studied. The ascorbic acid level was found to decrease while chlorophyll was being lost. The incubation of the segments in the presence of L-galactono-1,4-lactone resulted in both an increased level of ascorbic acid and a lower rate of chlorophyll loss. The ascorbate peroxidase activity was also higher, appearing to be related to the ascorbic acid content. The treatment with L-galactono-1,4-lactone also decreased lipid peroxidation, measured as malondialdehyde level, which suggested a direct role for ascorbic acid in delaying senescence in oat leaf segments.

Interaction of ascorbate free radical reductase with sulphhydryl reagents

Abstract The involvement of SH-group(s) in ascorbate free radical (AFR) reductase activity was investigated. The incubation of the enzyme with either N -ethylmaleimide or mersalyl, or both, resulted in different levels of inhibition. Only inhibition by mersalyl could be partially reversed by thiol-containing compounds, such as cysteine. However, the ability of cysteine to reverse the inhibition was progressively diminished by increasing times of incubation of the enzyme with the inhibitor. Pyridine nucleotides elicited a protecting effect on AFR-reductase when added prior to the inhibitors. This finding was consistent with the presence of a SH-group proximal to the flavin microenvironment of the enzyme.

Soluble ascorbate peroxidase from potato tubers

Abstract Ascorbate peroxidase (EC 1.11.1.11) from potato tubers was purified to a nearly homogeneous state. The enzyme occurred only in the soluble fraction and showed to be labile in the absence of ascorbate. It had a r of ∼30 kDa, a pH optimum of ∼7, a K m for hydrogen peroxide of 30 μM and a K m for ascorbate of 55 μM. Ascorbatee was specifically required as the electron donor. The enzyme was inhibited by compounds which react with thiol groups, thus suggesting that thiol group(s) are involved in the reaction.

Effects of selenite and selenate on the antioxidant systems in Senecio scandens L

Abstract Selenate and selenite are the most prevalent bioavailable selenium (Se) forms and most easily taken up by plants. Some studies indicate that they are differently absorbed and accumulated in plants and that selenium is toxic if accumulated at high concentrations. Toxicity is due to substitution of sulphur by selenium in cysteine and methionine aminoacids with alteration of the tertiary structure and catalytic activity of proteins and with inhibition of enzymes involved in chlorophyll biosynthesis. Moreover, the interaction between Se and thiol groups induces loss of efficiency of plant defence systems and increases the reactive oxygen species (ROS) production thus enhancing the oxidative stress. To further elucidate the role of Se in higher plants, in this study the antioxidative response to the phytotoxicity of selenite and selenate in Senecio scandens L. was evaluated. The data indicate that while selenite induces oxidative stress enhancing ROS production, lipid peroxidation and the oxidised forms of ascorbate and glutathione, selenate does not significantly affect the analysed pathways. This article outlines that the synergistic action of different antioxidant components is necessary to overcome the phytotoxicity of selenium in Senecio.

Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells

The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.