Enrique Olmos

Salt-induced oxidative stress in chloroplasts of pea plants

Abstract The possible involvement of activated oxygen species in the mechanism of damage by NaCl strees was studied in chloroplasts from leaves of two cultivars of pea ( Pisum sativum L.) with differential sensitivity to NaCl. Intact organelles were purified by centrifugation in density-gradients of Percoll. In chloroplasts from tolerant plants, NaCl stress produced a significant increase of CuZn-SOD II and ascorbate peroxidase activities as well as in ascorbate content, while in those from sensitive plants NaCl produced increases in the H 2 O 2 content and lipid peroxidation and no changes were observed in the enzymatic activities. Chlorophyll content significantly decreased in chloroplasts from sensitive plants and chloroplast integrity was lower in sensitive than in tolerant plants. Electron microscopy showed that the thylakoidal structure of chloroplast was notably disorganized in the NaCl-treated leaves. In purified chloroplasts, an increase in the number and size of plastoglobuli was produced by NaCl in chloroplasts from tolerant plants and to a lesser extent, in chloroplasts from sensitive plants. The relative starch content only decreased in chloroplasts from tolerant plants by NaCl-treatment. Results obtained suggest that in the cellular toxicity of NaCl in pea plants, superoxide- and H 2 O 2 -mediated oxidative damage in chloroplasts may play an important role.

Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus

In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13–15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.

Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies

The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.

Induction of Several Antioxidant Enzymes in the Selection of a Salt-Tolerant Cell Line of Pisum sativum

Summary Using the in vitro culture technique, we selected a cell line of Pisum sativum cv. Challis adapted to 85.5 mM NaCl. The possible relationships between the activity of enzymes related to oxygen metabolism and the salt adaptation of pea calli were analysed. The induction of two new Cu,Zn-SOD isozymes in salt-resistant calli was observed. Peroxidase activity was greatly increased in selected calli and catalase did not show a significant variation. The activity changes observed are discussed in terms of their possible relevance to pea calli adaptation to salt-stress.

A different role for hydrogen peroxide and the antioxidative system under short and long salt stress in Brassica oleracea roots

Salinity affects normal growth and development of plants depending on their capacity to overcome the induced stress. The present study was focused on the response and regulation of the antioxidant defence system in Brassica oleracea roots under short and long salt treatments. The function and the implications of hydrogen peroxide as a stressor or as a signalling molecule were also studied. Two different zones were analysed—the elongation and differentiation zone and the fully differentiated root zone—in order to broaden the knowledge of the different effects of salt stress in root. In general, an accumulation of hydrogen peroxide was observed in both zones at the highest (80 mM NaCl) concentration. A higher accumulation of hydrogen peroxide was observed in the stele of salt-treated roots. At the subcellular level, mitochondria accumulated hydrogen peroxide in salt-treated roots. The results confirm a drastic decrease in the antioxidant enzymes catalase, ascorbate peroxidase, and peroxidases under short salt treatments. However, catalase and peroxidase activities were recovered under long salt stress treatments. The two antioxidant molecules analysed, ascorbate and glutathione, showed a different trend during salt treatments. Ascorbate was progressively accumulated and its redox state maintained, but glutathione was highly accumulated at 24 h of salt treatment, but then its concentration and redox state progressively decreased. Concomitantly, the antioxidant enzymes involved in ascorbate and glutathione regeneration were modified under salt stress treatments. In conclusion, the increase in ascorbate levels and the maintenance of the redox state seem to be critical for root growth and development under salt stress.

Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants

Abstract The anatomy of normal and hyperhydric leaves of Dianthus caryophyllus plantlets regenerated from leaves was compared using scanning electron microscopy and transmission electron microscopy. The hyperhydric leaves had large vacuolated mesophyll cells, showing a hypertrophy of the cells and presented larger intercellular spaces. This leaf type lacked cuticular wax and chloroplasts presented abundant plastoglobuli. The guard cells were different in morphology and X-ray microanalysis demonstrated high levels of K + . The alteration of guard cells could be a mechanical impediment to stomatal function.

Cd-induced oxidative burst in tobacco BY2 cells: time course, subcellular location and antioxidant response.

The relation between Cd and oxidative stress in BY2 cell cultures of tobacco was studied. In response to 5 mM Cd, a rapid generation of H2O2 has been detected in tobacco cell cultures by the oxidative quenching of the fluorescent reporter dye pyranine. This oxidative burst reached the maximum production of H2O2 after 10 min of treatment with Cd. This response could be considered as short term hypersensitive response previous to the oxidative stress caused by the metal at the cell plasma membrane. The observed antioxidant enzymatic response to the oxidative burst was preceded by an increased peroxidation of lipids with a significant increase in the activities of superoxide dismutase and ascorbate peroxidase. The results presented in this study point out to the plasma membrane as the primary target for the short term production of activated oxygen species in response to Cd in BY2 tobacco cells followed by a coordinated activation of the antioxidant enzymatic system.

Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

Changes in antioxidant enzymes and organic solutes associated with adaptation of citrus cells to salt stress

Embryogenic callus cultures of lemon (Citrus limon L. Burm f. cv Verna), were selected for resistance to salt stress (170 mM NaCl). Inorganic analysis showed that selected callus accumulated more Na+ and Cl- ions than the non-selected one. Moreover, the salt-tolerant C. limon callus exhibited an increase in the activity of antioxidant enzymes involved in oxygen metabolism, with the induction of a new superoxide dismutase isozyme and an increase of the peroxidase activity while the catalase activity was unchanged. Proline and total sugar, mainly sucrose, concentrations increases significantly in salt-tolerant cells as compared to control cells. On the other hand, the selected cell line also showed an increase in choline and glycine betaine, but to lesser extent.