A Different Pattern of Production and Scavenging of Reactive Oxygen Species in Halophytic Eutrema salsugineum (Thellungiella salsuginea) Plants in Comparison to Arabidopsis thaliana and Its Relation to Salt Stress Signaling.

Isolated thylakoids from halophytic Eutrema salsugineum (Thellungiella salsuginea) produces more H2O2 in comparison to glycophytic Arabidopsis thaliana. The first objective of this study was to verify whether this feature is relevant also to the intact chloroplasts and leaves. Enhanced H2O2 levels in chloroplasts and leaves of E. salsugineum were positively verified with several methods (electron microscopy, staining with Amplex Red and with diaminobenzidine). This effect was associated with a decreased ratio of [Formula: see text]/H2O2 in E. salsugineum in comparison to A. thaliana as detected by electron paramagnetic resonance method. As a next step, we tested how this specific ROS signature of halophytic species affects the antioxidant status and down-stream components of ROS signaling. Comparison of enzymatic antioxidants revealed a decreased activity of ascorbate peroxidase (APX), enhanced activity of glutathione peroxidase, and the presence of thylakoid-bound forms of iron superoxide dismutase (FeSOD) and APX in E. salsugineum. These cues were, however, independent from application of salt stress. The typical H2O2-dependent cellular responses, namely the levels of glucosinolates and stress-related hormones were determined. The total glucosinolate content in E. salsugineum water-treated leaves was higher than in A. thaliana and increased after salinity treatment. Treatment with salinity up-regulated all of tested stress hormones, their precursors and catabolites [abscisic acid (ABA), dihydrophaseic acid, phaseic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, jasmonic acid, cis-(+)-12-oxo-phytodienoic acid and jasmonoyl-L-isoleucine] in A. thaliana, whereas in E. salsugineum only a stimulation in ethylene synthesis and ABA catabolism was noted. Obtained results suggest that constitutively enhanced H2O2 generation in chloroplasts of E. salsugineum might be a crucial component of stress-prepardeness of this halophytic species. It shapes a very efficient antioxidant protection (in which glucosinolates might play a specific role) and a fine tuning of hormonal signaling to suppress the cell death program directed by jasmonate pathway.

Changes in production of reactive oxygen species in illuminated thylakoids isolated during development and senescence of barley.

This paper presents a detailed analysis of thylakoids isolated from secondary barley leaves harvested 18, 22, 25, 29, 32, 35 and 39 days after sowing (DAS). Goal of the analysis was to investigate the production of different reactive oxygen species (ROS) during development and senescence of barley. Generation of superoxide anion (O2-•) and hydrogen peroxide (H2O2) increases during development of barley reaching the highest value right after the onset of senescence (between 25 and 29 DAS), thereafter the levels of both ROS start to decrease until 35 DAS when production of H2O2 increases again. In comparison with O2-• and H2O2, generation of singlet oxygen ((1)O2) showed continuous production of low amounts thought the duration of experiment. Oxidative damage to the thylakoid membrane was assessed by measuring lipid peroxidation. Results showed gradual increase in lipid peroxidation with progress of plant development with highest increase occurring at the late stages of senescence. A possible factor contributing to the elevation in the production of ROS could be an increase in membrane fluidity observed in our previous study. Fluidization of the membrane, allows for better penetration of oxygen inside the membrane, which can lead to an increase in the production of ROS. Indeed, the production of ROS started to increase together with observed fluidization of the membrane from 22 to 29 DAS. Thereafter, production of ROS started to decline till 35th DAS. On the last day of the measurement, chl is at 25% of its initial value, lipid peroxidation reaches the highest value and H2O2 increases again.

Senescence, Stress, and Reactive Oxygen Species.

Generation of reactive oxygen species (ROS) is one of the earliest responses of plant cells to various biotic and abiotic stresses. ROS are capable of inducing cellular damage by oxidation of proteins, inactivation of enzymes, alterations in the gene expression, and decomposition of biomembranes. On the other hand, they also have a signaling role and changes in production of ROS can act as signals that change the transcription of genes that favor the acclimation of plants to abiotic stresses. Among the ROS, it is believed that H2O2 causes the largest changes in the levels of gene expression in plants. A wide range of plant responses has been found to be triggered by H2O2 such as acclimation to drought, photooxidative stress, and induction of senescence. Our knowledge on signaling roles of singlet oxygen (¹O2) has been limited by its short lifetime, but recent experiments with a flu mutant demonstrated that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. In this review we summarize the latest progress on the signaling roles of ROS during senescence and abiotic stresses and we give a short overview of the methods that can be used for their assessment.

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence.

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25°C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.

Comparison of acute effects of red wine, beer and vodka against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans.

OBJECTIVE: We determined and compared acute effects of different alcoholic beverages on oxygen-induced increase in oxidative stress plasma marker and arterial stiffness in healthy humans. METHODS: Ten males randomly consumed one of four tested beverages: red wine (RW), vodka, beer (0.32 g ethanol/kg body wt) and water as control. Every beverage was consumed once, a week apart, in a cross-over design. The volunteers breathed 100% normobaric O(2) between 60th and 90th min of 3h study protocol. Plasma lipid peroxides (LOOH) and uric acid (UA) concentration, blood alcohol concentration (BAC) and arterial stiffness (indicated by augmentation index, AIx) were measured before and 30, 60, 90, 120 and 180 min after beverage consumption. RESULTS: Intake of all alcoholic beverages caused a similar increase of BAC. The oxygen-induced elevation in AIx was similarly reduced in all three groups relative to the control (3.4 ± 1.3%, 5.4 ± 2.2% and 0.2 ± 1.6% vs. 13.7 ± 2.6% for red wine, vodka, beer and control, respectively, 60 min after intake). Exposure to oxygen resulted in increased plasma LOOH in all groups. However, in RW group this increase was lowest (1.1 ± 0.5) in comparison to the vodka (2.1 ± 0.5), beer (1.6±0.3) and control (2.5 ± 0.4µM/L H(2)O(2)). 60 min after intake of RW and beer plasma UA significantly increased (34 ± 4 and 15 ± 3) in contrast to vodka and control (-6 ± 2 and -8 ± 2µmol/L). CONCLUSION: All three alcoholic beverages provided similar protection against oxygen-induced increase in arterial stiffness, probably due to central vasodilatatory effect of alcohol itself, but only RW provided protection against oxygen-induced oxidative stress.

Thermally treated wine retains vasodilatory activity in rat and guinea pig aorta.

In contrast to the intact wine, cardiovascular effects of the thermally treated wine have not been studied, despite widespread habits of cooking with wine and consumption of mulled wine. Vasodilatory effects of the red wine heated at 75 and 125°C were examined in the isolated rat and guinea pig aorta and compared with the intact and wine dealcoholized without thermal stress. Samples were analyzed for their phenolic content, antioxidant capacity, resveratrol and ethanol contents. Heating-induced degradation of individual phenolic fraction was observed only in the samples treated at 125°C, although total phenolic concentration and related antioxidant activity increased in the thermally treated samples due to the reduction in their volume. All wine samples regardless of treatment caused similar maximal relaxation in both species, but the response was stronger in aortas from guinea pigs. At the lowest concentrations up to 1‰, dealcoholized wine produced vasodilation greater than that produced by intact wine and wines treated at 75 and 125°C, which showed similar vasodilating activity at all concentrations. Our results indicate that wine thermally treated under heating conditions applicable to the preparation of a mulled wine and cooking with wine largely retains vasodilatory activity in vitro despite significant heat-induced changes in its composition.