Rosalia Liso

Relationship between ascorbic acid and cell division

Proliferating cells require large amounts of ascorbic acid to reach cell division. The decrease in ascorbic acid caused by adding lycorine, an inhibitor of ascorbic acid biosynthesis, induces profound inhibition of cell division: the cell cycle is arrested in G1 and G2 phase, more than 90% of the cells being accumulated in G1 after some time. The effect of lycorine on mitotic index (MI) has been reversed by increasing experimentally the concentration of ascorbic acid in tissues. Ascorbic acid control on cell division is found to be specific, since isoascorbic acid is wholly ineffective. It is suggested that the principal role of ascorbic acid in the cell cycle may be related to its action in controlling the synthesis of hydroxyproline-containing proteins, which can be essential requirements for development of G1 and G2.

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

Abstract. 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.

Correlation between changes in cell ascorbate and growth of Lupinus albus seedlings

Summary The growth of Lupinus albus seedlings is stimulated when ascorbate (Asc) content is experimentally raised; on the contrary, when Asc content is lowered by lycorine — an alkaloid inhibiting Asc biosynthesis — the growth is inhibited. The stimulation of growth by Asc is due to the promotion of cell division and cell expansion. Redox enzymes of the Asc system respond in different manners to the Asc changes. Asc peroxidase is the enzyme that is most affected by Asc changes in the cell: its activity increases or decreases in parallel with the Asc content. Dehydroascorbic acid (DHA) reduction capability is affected in the opposite way: when Asc content rises, the DHA reduction ability of the cells drops, while it rises when Asc decreases. Ascorbate free radical (AFR) reductase activity does not vary with changing Asc content. A possible regulatory function of Asc redox enzymes is discussed.

Lycorine as an inhibitor of ascorbic acid biosynthesis

LYCORINE, an alkaloid extracted from Amarillidaceae1,2, is a powerful inhibitor of growth in higher plants, algae and yeasts. At the very low concentration of 10−6 M, lycorine inhibits cell division in higher plants, red algae and yeasts, and the cell cycle is arrested in the interphase stage3. No inhibition of cell division was observed in Escherichia coli (O. A., unpublished).

Ascorbic Acid Oxidase: An Enzyme in Search of a Role

Ascorbic acid oxidase (AAO) has been fully characterized at molecular level, yet its functional role is unclear. The properties of the enzyme and the main hypotheses on its function are discussed. Recent data and reappraisal of previous observations suggest that AAO could be part of a dynamic mechanism operating whenever plant cells have to control oxygen availability.

Lycorine : A powerful inhibitor of L-galactono-γ-lactone dehydrogenase activity

Summary In vivo conversion of L-galactono-γ-lactone to ascorbate is carried out in all of the plants that we have analysed. The enzyme catalysing this reaction, i.e. L-galactono-γ-lactone dehydrogenase, which appears to be localised in the mitochondrial membrane, is strongly inhibited by lycorine: 5 μM lycorine almost completely inhibits the activity of the enzyme. The alkaloid does not affect the activity of the ascorbate free radical reductase, dehydroascorbate reductase or ascorbate peroxidase. The results presented here confirm that lycorine is a specific inhibitor of ascorbate biosynthesis in plants, and, consequently, that it can be a useful tool to bener understand the ascorbate metabolism in ascorbate synthesising organisms.

Ascorbic acid utilization by prolyl hydroxylase in vivo

Abstract The treatment with 3,4-dehydro- d , l -proline, an analogous compound of proline which specifically inhibits prolyl hydroxylase, reduces the quantity of the hydroxyproline residues present in the polypeptide chains and at the same time leads to an increase in the ascorbic acid content in different plant tissues analysed (pea embryos, carrot and potato slices). The increase of ascorbic acid induced by dehydroproline treatment can be interpreted as a consequence ofthe reduced activity of the prolyl hydroxylase and suggests that in vivo the ascorbic acid acts as electron donor in the process of proline hydroxylation.

In vivo inhibition of galactono-γ-lactone conversion to ascorbate by lycorine

Summary Maize embryos are endowed with the ascorbate biosynthetic system and the last enzyme of the pathway, galactono oxidase (or dehydrogenase), is very active; L-galactono-γ-lactone addition gives rise to a 3-fold increase in cellular ascorbate content. Lycorine, an alkaloid extracted from members of the Amaryllidaceae, strongly inhibits the in vivo conversion of L-galactono-γ-lactone to ascorbic acid. Data reported here seem to suggest that lycorine forms a relatively stable association with galactono oxidase; incubation with 50 μM lycorine shows a marked inhibitory effect that persists when the alkaloid is removed from the incubation medium. The inhibitory effect of lycorine is significantly higher in onion roots and pea embryos in comparison with maize embryos. This different sensitivity to the alkaloid can be explained by the inability of onion and pea to overcome the decrease in ascorbate biosynthesis by means of dehydroascorbate reductase, which has a significantly lower activity in these two species than that in maize embryos. Galactono oxidase also efficiently utilizes L-gulono-γ-lactone, the physiological substrate of the animal enzyme. Considering that lycorine induces scurvy-like symptoms in ascorbic acid-synthesizing animals, it is reasonable to suppose that lycorine inhibits ascorbate biosynthesis in both plants and animals by acting on the last step in the biosynthetic pathway leading from sugar to ascorbate.

Correlation between Ascorbate Peroxidase Activity and some Anomalies of Seedlings from Aged Caryopses of Dasypyrum villosum (L.) Borb.

Summary Dasypymm villosum (L.) Borb. produces two types of caryopses in the same ear: some are «clear». and slightly larger, others «dark» and smaller. In this paper we demonstrate that the germination capacity of «clear» caryopses drops strongly during aging and some anomalies appear in the seedlings. On the contrary, the «dark» caryopses maintain an elevated germination capacity for a long time and normal seedlings are formed. Analysis of the ascorbic acid system shows that caryopsis aging does not affect either AA biosynthesis or AFR reductase activity; a drop in DHA reductase activity, similar in both seedlings from «clear» and «dark» caryopses, is observed. AA peroxidase activity, which is high and similar in both seedlings, strongly decreases in the «clear» ones during caryopsis aging, while remaining unchanged in the seedlings from the aged «dark» caryopses. A possible correlation between AA peroxidase decrease and the anomalies occurring in the seedlings from aged «clear» caryopses is indicated.

Further experiments on structure-activity relationships among the lycorine alkaloids

Abstract Synthetic lycorine analogues, five Amaryllidaceae alkaloids and narciclasine, all structurally related to lycorine, were tested for their ability to inhibit ascorbic acid biosynthesis in vivo . The highest potency observed was displayed by narciclasine followed by compounds having an aromatic C-ring. Derivatives modified at C-1 and/or C-2 were inactive, while the compound with a double bond between these positions is a weak inhibitor. Also lutessine and its deacetyl derivative having an α-methoxyl group bonded to C-4 of the D-ring appeared completely inactive. These results confirm that the presence of an appropriately substituted C-ring is a necessary requirement for optimal ‘response-triggering’ contact between the lycorine derivatives and the specific receptor. Functional groups jutting out from the α-side of the molecule do not allow a good fit with the binding sites.