L. De Gara

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells.

SummaryThe effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.

Redox regulation in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary.The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O2− and H2O2) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O2− production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl3 assays, which monitor H2O2 production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl3) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H2O2 is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H2O2 in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H2O2 was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H2O2 produced by this in vitro culture system is destined for use during lignin biosynthesis.

Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

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.

Galactone-γ-lactone-dependent ascorbate biosynthesis alters wheat kernel maturation

Kernel development and maturation involve several well-characterised events, such as changes in ascorbate (ASC) metabolism, protein synthesis and storage, programmed cell death (PCD) of starchy endosperm and tissue dehydration. Despite many studies focusing on these events, whether and how they are metabolically related to each other, remains to be elucidated. In the present investigation, the changes in ASC-related metabolism, PCD occurrence, kernel filling and dehydration have been analysed during kernel maturation, over a 3-year period in plants grown under normal conditions and in plants displaying modified ASC synthesis. The obtained results suggest that ASC plays a pivotal role in the network of events characterising kernel maturation. During this process, a decrease in ASC content occurs. When ASC biosynthesis is improved in the kernel, by feeding the plants with its immediate precursor, L-galactone-γ-lactone (GL), the decrease in ASC, observed during kernel maturation, is delayed. As a consequence, ascorbate peroxidase (APX) activity is also enhanced. Moreover, a delay in the ASC decrease permits a delay in PCD occurring in kernel storage tissues and in kernel dehydration. Interestingly, the data emerging from the present investigation suggest that the delay in the decrease in ASC content and APX activity also improves kernel filling. The relevance of the ascorbate-dependent redox regulation for kernel productivity is discussed.

Ascorbic acid requirement for increased peroxidase activity during potato tuber slice aging

The increase in peroxidase activity during aging is a well known process depending on de novo enzyme synthesis. The enhancement of peroxidase is strongly inhibited by lycorine, an ascorbic acid biosynthesis inhibitor. The inhibitory effect of lycorine can be abolished by experimentally increasing the ascorbic acid concentration in the tissues. Conversely, glucose‐6‐phosphate dehydrogenase enhancement ‐ which also occurs during the aging of potato slices ‐ depends on protein synthesis but does not require ascorbic acid. It is suggested that the role of ascorbic acid in the development of peroxidase activity may be related to controlling the synthesis of hydroxyproline‐containing proteins. A possible relationship between peroxidase increase and hydroxyproline‐containing proteins biosynthesis is discussed.

Variation in fructooligosaccharide contents during plant development and in different cultivars of durum wheat

Abstract Fructooligosaccharides (FOS) have received a great deal of attention for their properties as prebiotic components. Several plants store FOS in their tissues as carbohydrate source or as osmoregulators. In comparison with the other natural FOS sources, cereals, in particular durum wheat, store a significant amount of these oligosaccharides during their growth. To gain a deeper insight on the relationships between FOS and plant/kernel development, FOS levels were analysed in the grains and stems of a widely cultivated durum wheat cultivar (Simeto), during the whole period of grain filling. In addition, a broad analysis of FOS contents was carried out by comparing their levels in the stems and grains of 45 cultivars of durum wheat harvested at two development stages. Results show a large variation among cultivars in their capability to metabolise FOS.

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H2O2. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.