Costantino Paciolla

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.

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.

Dehydroascorbate-reducing proteins in maize are induced by the ascorbate biosynthesis inhibitor lycorine

Abstract Dehydroascorbate (DHA) reductase (glutathaone: dehydroascorbate oxidoreductase, EC 1.8.5.1) has been generally considered a specific enzyme of the ascorbate-glutathione cycle. However, at least four distinct proteins can catalyze in vitro both glutathione-dependent DHA reduction and other reactions mainly related to thiol-disulphide exchange. These data have raised questions both on the existence of specific DHA reductase and the actual physiological role of DHA-reducing proteins (DRP). We have observed characteristic electrophoretic patterns of DRP in dark-germinating embryos of different plant species. Marked differences were observed not only in the number, but also in the migration rate of DRP under non-denaturing conditions. In order to evaluate the actual contribution of DRP activity to ascorbate (ASC) regeneration under conditions limiting ASC biosynthesis, Z. mays germinating embryos excised from endosperm were either incubated in distilled water or treated with the alkaloid lycorine, an inhibitor of ASC biosynthesis. In parallel with the decrease in ASC content, a strong enhancement in DRP activity occurred. The increase in DRP activity was prevented by cycloheximide, and thus seems to be due to de novo protein synthesis. The possible involvement of DRP in avoiding DHA accumulation under adverse environmental conditions is discussed.

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.

Effect of rare earth elements on growth and antioxidant metabo- lism in Lemna minor L.

Abstract Lemna minor is frequently used in bioremediation processes to remove nutrients and contaminants from waste water. In this work the response of L. minor to treatments with lanthanum nitrate and with a mix of several light rare earth elements (REE) nitrates was investigated. Preliminary results indicate that L. minor shows an overall good tolerance to the presence of REE in the media. Toxic effects were observed after prolonged exposition to high concentration of REE. An increase in ascorbate and glutathione content as well as in ascorbate peroxidase, dehy-droascorbate reductase and ascorbate free radical (AFR) reductase activity was observed in treated plants.

The Ascorbate System in Two Bryophytes: Brachythecium velutinum and Marchantia polymorpha

The ascorbate system, one of the major antioxidant systems, has been studied in two bryophytes; a moss, Brachythecium velutinum (Hedw.) B., S. & G., and a liverwort, Marchantia polymorpha L. The moss and liverwort gametophytes contain ascorbate both in the reduced and oxidized form; utilize ascorbate in removing hydrogen peroxide by means of ascorbate peroxidase and reconvert to ascorbate its oxidation products by means of dehydroascorbate reductase and monodehydroascorbate reductase. Ascorbate oxidase activity was measured in the cytosolic fraction suggesting a localization of the enzyme different from more evolved organisms. The ascorbate content was maintained in the moss after drought stress while it declines in the liverwort, which seems more sensitive to water stress. Since ascorbate recycling is more efficient in the moss than in the liverwort, this seems to suggest a correlation between efficiency of ascorbate recycling and water stress tolerance.

Defense responses to mycotoxin-producing fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in kernels of susceptible and resistant maize genotypes

Developing kernels of resistant and susceptible maize genotypes were inoculated with Fusarium proliferatum, F. subglutinans, and Aspergillus flavus. Selected defense systems were investigated using real-time reverse transcription-polymerase chain reaction to monitor the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes protective from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) at 72 h postinoculation. The study was also extended to the analysis of the ascorbate-glutathione cycle and catalase, superoxide dismutase, and cytosolic and wall peroxidases enzymes. Furthermore, the hydrogen peroxide and malondialdehyde contents were studied to evaluate the oxidation level. Higher gene expression and enzymatic activities were observed in uninoculated kernels of resistant line, conferring a major readiness to the pathogen attack. Moreover expression values of PR genes remained higher in the resistant line after inoculation, demonstrating a potentiated response to the pathogen invasions. In contrast, reactive oxygen species-scavenging genes were strongly induced in the susceptible line only after pathogen inoculation, although their enzymatic activity was higher in the resistant line. Our data provide an important basis for further investigation of defense gene functions in developing kernels in order to improve resistance to fungal pathogens. Maize genotypes with overexpressed resistance traits could be profitably utilized in breeding programs focused on resistance to pathogens and grain safety.

Light qualities and dose influence ascorbate pool size in detached oat leaves

In this work, we studied the mechanism of light influence on AsA pool size in Avena sativa L. under the effects of low intensity light at different wavelengths. Exposure to low intensity light of oat leaf segments incubated in water or in l-galactono-1,4-lactone (GL), resulted in an increase in AsA content compared with the dark control. This increase was due to modulation of l-galactono-1,4-lactone dehydrogenase (GLDH; EC 1.3.2.3) light-dependent activity and was dependent on the size of the endogenous GL pool. Both blue and red light were effective in increasing AsA, and this increase depended on both exposure time and light intensity. Protein biosynthesis, photosynthesis and calcium were involved in controlling the level of light-dependent AsA. We suggest that multiple checkpoints correlated to the presence of light underlie the ascorbate pool size. The presence of a light-activated switch for the maintenance of an adequate AsA level seems to be necessary for the various tasks of scavenging reactive oxygen species, in response to the dark-light cycle which plants experience under natural conditions.

Ascorbate metabolism in mature pollen grains of Dasypyrum villosum (L.) Borb. during imbibition

Summary A mature pollen grain of Dasypyrum villosum , roundish in shape with a single germinal pore, is tricellular and rich in starch granules. Pollen grains are endowed with the ascorbate biosynthetic pathway and actively synthesize ascorbic acid. In D. villosum pollen the two main oxido-reduction enzymes of the ascorbate system, AFR reductase and AA peroxidase, are present with a high activity; DHA reductase activity is very low and there is no AA oxidase. Ascorbate peroxidase activity is shown to be 10 times higher than that of catalase, indicating that ascorbate peroxidase, rather than catalase, is the key enzyme to remove H 2 O 2 produced in the cell metabolism. AA peroxidase and AFR reductase are released in the germinating medium; their possible role is discussed.

Constitutive expression of pathogenesis-related proteins and antioxydant enzyme activities triggers maize resistance towards Fusarium verticillioides

Fusarium verticillioides is a fungal pathogen of maize that causes ear rot and contaminates the grains with fumonisin mycotoxins. Breeding for resistance to Fusarium emerged as the most economic and environmentally safe strategy; therefore the discovery of resistant sources and effective molecular markers are a priority. Ears of resistant (CO441 and CO433) and susceptible (CO354 and CO389) maize lines were inoculated with F. verticillioides and the expression of pathogenesis-related (PR) genes (PR1, PR5, PRm3, PRm6) and genes that protect from oxidative stress (peroxidase, catalase, superoxide dismutase and ascorbate peroxidase) were evaluated in the kernels at 72h post inoculation. In addition, the oxidation level and the enzymatic activity of ascorbate-glutathione cycle, catalase, superoxide dismutase and cytosolic and wall peroxidases were investigated. The uninoculated kernels of the resistant lines showed higher gene expression and enzymatic activities, highlighting the key role of constitutive resistance in limiting pathogen attack. In contrast, the susceptible lines activated defensive genes only after pathogen inoculation, resulting in increased levels of H2O2 and lipid peroxidation, as well as lower enzymatic activities. The constitutive defenses observed in this study from seed could be profitably exploited to develop markers to speed up conventional breeding programs in the selection of resistant genotypes.