Laura De Gara

Ascorbate and glutathione: guardians of the cell cycle, partners in crime?

Abstract Besides the implication of ascorbate and glutathione in the defence against oxidative stress, these two compounds are involved in plant growth and cell cycle control. Ascorbate metabolism is closely linked to the development of embryos and seedlings. Furthermore, ascorbate stimulates cell cycle activity in competent cells, while the oxidised form, dehydroascorbate, blocks normal cell cycle progression. Several possible mechanisms have been proposed to explain the effect of these compounds. The links between glutathione and the cell cycle are less clear. It has long been assumed that both compounds are closely linked by way of the Halliwell–Asada cycle. Any hypothesis concerning the pathways by which ascorbate or glutathione influence cell division, should take this connection into account. However, other mechanisms have been proposed for ascorbate-mediated cell cycle control, e.g. via the thioredoxin pathway.

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.

Selection and use of autochthonous mixed starter for lactic acid fermentation of carrots, French beans or marrows.

Strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella soli/Weissella koreensis, Enterococcus faecalis, Pediococcus pentosaceus and Lactobacillus fermentum were identified from raw carrots, French beans and marrows by partial 16S rRNA gene sequence. L. plantarum M1, Leuc. mesenteroides C1 and P. pentosaceus F4 were selected based on the rates of growth and acidification in vegetable juice media, and used as the autochthonous mixed starter for the fermentation of carrots, French beans or marrows. An allochthonous starter, consisting of the same species, was also used for fermentation. A two-step fermentation process (1 day at 25 degrees C and 7 days at 15 degrees C) in brine (1% w/v) followed by storage at room temperature in olive oil until 40 days was set up. Unstarted vegetables subjected to the same treatments were used as the controls. Cell numbers of lactic acid bacteria in the started vegetables were ca. 10,000 (autochthonous starter) and 1000 (allochthonous starter) times higher than unstarted samples throughout the process. When fermented with the autochthonous starter, carrots, French beans or marrows were characterized by the rapid decrease of pH (<4.5), marked consumption of fermentable carbohydrates, and inhibition of Enterobacteriaceae and yeasts. Fermentation with the allochthonous starter did not acidify and inhibit bacteria and yeasts so rapidly. After 40 days, carrots, French beans and marrows fermented with the autochthonous starter had significantly (P<0.05) higher total concentration of vitamin C (ascorbate+dehydroascorbate) with respect to those fermented with the allochthonous starter and, especially unstarted vegetables. The same was found for the indexes of color. Firmness of both started vegetables was higher than unstarted vegetables. Sensory analysis differentiated started vegetables. Carrots and French beans fermented with the autochthonous starter were, especially, appreciated for fragrance. Appearance was the sensory attribute that mainly distinguished marrows fermented with the autochthonous starter.

Effect of autochthonous lactic acid bacteria starters on health-promoting and sensory properties of tomato juices.

Strains of Lactobacillus plantarum, Weissella cibaria/confusa, Lactobacillus brevis, Pediococcus pentosaceous, Lactobacillus sp. and Enterococcus faecium/faecalis were identified from raw tomatoes by Biolog System, partial 16S rRNA gene sequence and subjected to typing by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis. Ten autochthonous strains were singly used to ferment tomato juice (TJ) via a protocol which included fermentation at 25 degrees C for 17 h and further storage at 4 degrees C for 40 days. Unstarted TJ and TJ fermented with an allochthonous strain of L. plantarum were used as the controls. All autochthonous strains grew well in TJ reaching cell densities ca. 10,000 and 10 times higher than unstarted TJ and TJ fermented with the allochthonous strain. Viscosity of TJs fermented with autochthonous strains was generally the highest, especially when started with W. cibaria/confusa which synthesized exo-polysaccharides. Overall, unstarted TJ and TJ fermented with the allochthonous strain showed marked decreases of ascorbic acid (ASC), glutathione (GSH) and total antioxidant activity (TTA) during storage. On the contrary, several TJs fermented with autochthonous strains, especially with L. plantarum POM1 and POM 35, maintained elevated values of ASC, GSH and TAA. The variation of color indexes mirrored the above behavior. TJs fermented with the above two autochthonous strains were compared to controls based on volatile components through Purge and Trap or Solid Phase Microextraction Gas Chromatography-Mass Spectrometry (PT or SPME-GC/MS) analysis. As shown by Principal Component Analysis a large number of volatiles belonging to various chemical classes markedly differentiated TJs fermented with autochthonous strains with respect to controls.

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.

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.

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells.

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS‐PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase‐3‐like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS‐PCD upstream of the caspase‐like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase‐3‐like protease activation.

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.

The structure of the cytochrome oxidase subunit II gene and its use as a new character in the construction of the phylogenetic tree of Angiospermae

Abstract With the aim of correlating the structure of the intron present in the mitochondrial gene coding for subunit II of the cytochrome oxidase complex ( coxII gene) with some aspects of Angiosperm evolution, we analysed the organization of the coxII gene from 30 plants belonging to 18 different orders. In all the monocotyledons tested, the gene contains an intron with a composite structure. Among dicot plants, 16 contain a split gene, 5 a continuous gene. The presence or the absence of intervening sequences in the genes of dicot plants does not seem to be a casual event, on the contrary it seems to be a feature of specific phylogenetic lines. Our results suggest that this approach, extended to a wider number of plant species, could make a contribution to the construction of the phylogenetic tree of Angiosperms.