Reactive Oxygen Species Attack Research Articles

Methylene Blue Doped Films of Wool Keratin with Antimicrobial Photodynamic Activity.

In this work, keratin films doped with different amounts of methylene blue (MB) were developed in order to prepare new biodegradable and biocompatible materials for tissue engineering and wound healing, able to exert antimicrobial photodynamic activity upon irradiation with visible light. Preliminary results indicated that the swelling ratio, as well as the MB release, increases by increasing the pH. Moreover, the generation of reactive oxygen species (ROS) and singlet oxygen can be easily triggered and controlled by a fine-tuning of the irradiation time and MB concentration in the films. As concerns the photodynamic effects on keratin, the ROS attack does not induce any significant photodegradation on the protein, even if a slight photo-oxidation of sulfonated amino acids occurs. Finally, the film with the highest MB concentration (400 μg per gram of keratin) displays a significant photobactericidal activity against Staphylococcus aureus with a bacterial reduction that increases by increasing the irradiation time. In particular, the irradiation of KFMB400 film incubated with S. aureus at a concentration of 10(8) cfu mL(-1) determined the 99.9% killing rate and the killing effect increased proportionally with irradiation time.

The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products

In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H–>CH3O–>CH3OC(O)CH2–>HOC(O)CH2NHCH2–≈ CH3NHCH2–, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2′-endo sugar ring conformation, as opposed to the preferred C3′-endo conformation of the parent R5S2Us.

5-Carboxamido-5-formamido-2-iminohydantoin, in Addition to 8-oxo-7,8-Dihydroguanine, Is the Major Product of the Iron-Fenton or X-ray Radiation-Induced Oxidation of Guanine under Aerobic Reducing Conditions in Nucleoside and DNA Contexts.

Exogenously and endogenously produced reactive oxygen species attack the base and sugar moieties of DNA showing a preference for reaction at 2′-deoxyguanosine (dG) sites. In the present work, dG was oxidized by HO• via the Fe(II)-Fenton reaction or by X-ray radiolysis of water. The oxidized lesions observed include the 2′-deoxynucleosides of 8-oxo-7,8-dihydroguanine (dOG), spiroiminodihydantoin (dSp), 5-guanidinohydantoin (dGh), oxazolone (dZ), 5-carboxamido-5-formamido-2-iminohydantoin (d2Ih), 5′,8-cyclo-2′-deoxyguanosine (cyclo-dG), and the free base guanine (Gua). Reactions conducted with ascorbate or N-acetylcysteine as a reductant under aerobic conditions identified d2Ih as the major lesion formed. Studies were conducted to identify the role of O2 and the reductant in product formation. From these studies, mechanisms are proposed to support d2Ih as a major oxidation product detected under aerobic conditions in the presence of the reductant. These nucleoside observations were then validated in oxidations of oligodeoxynucleotide and λ-DNA contexts that demonstrated high yields of d2Ih in tandem with dOG, dSp, and dGh. These results identify dG oxidation to d2Ih to occur in high yields leading to a hypothesis that d2Ih could be found from in cells stressed with HO•. Further, the distorted ring structure of d2Ih likely causes this lesion to be highly mutagenic.

Magnetite nanoparticles induced adaptive mechanisms counteract cell death in human pulmonary fibroblasts

Magnetite nanoparticles (MNP) have attracted great interest for biomedical applications due to their unique chemical and physical properties, but the MNP impact on human health is not fully known. Consequently, our study proposes to highlight the biochemical mechanisms that underline the toxic effects of MNP on a human lung fibroblast cell line (MRC-5). The cytotoxicity generated by MNP in MRC-5 cells was dose and time-dependent. MNP-treated MRC-5 cells accumulated large amount of iron and reactive oxygen species (ROS) and exhibited elevated antioxidant scavenger enzymes. Reduced glutathione (GSH) depletion and enhanced lipid peroxidation (LPO) processes were also observed. The cellular capacity to counteract the oxidative damage was sustained by high levels of heat shock protein 60 (Hsp60), a protein that confers resistance against ROS attack and inhibition of cell death. While significant augmentations in nitric oxide (NO) and prostaglandine E2 (PGE2) levels were detected after 72h of MNP-exposure only, caspase-1 was activated earlier starting with 24h post-treatment. Taken together, our results suggest that MRC-5 cells have the capacity to develop cell protection mechanisms against MNP. Detailed knowledge of the mechanisms induced by MNP in cell culture could be essential for their prospective use in various in vivo biochemical applications.

Cysteines under ROS attack in plants: a proteomics view.

Plants generate reactive oxygen species (ROS) as part of their metabolism and in response to various external stress factors, potentially causing significant damage to biomolecules and cell structures. During the course of evolution, plants have adapted to ROS toxicity, and use ROS as signalling messengers that activate defence responses. Cysteine (Cys) residues in proteins are one of the most sensitive targets for ROS-mediated post-translational modifications, and they have become key residues for ROS signalling studies. The reactivity of Cys residues towards ROS, and their ability to react to different oxidation states, allow them to appear at the crossroads of highly dynamic oxidative events. As such, a redox-active cysteine can be present as S-glutathionylated (-SSG), disulfide bonded (S-S), sulfenylated (-SOH), sulfinylated (-SO2H), and sulfonylated (-SO3H). The sulfenic acid (-SOH) form has been considered as part of ROS-sensing pathways, as it leads to further modifications which affect protein structure and function. Redox proteomic studies are required to understand how and why cysteines undergo oxidative post-translational modifications and to identify the ROS-sensor proteins. Here, we update current knowledge of cysteine reactivity with ROS. Further, we give an overview of proteomic techniques that have been applied to identify different redox-modified cysteines in plants. There is a particular focus on the identification of sulfenylated proteins, which have the potential to be involved in plant signal transduction.

Wisdom from the Human Eye: A Synthetic Melanin Radical Scavenger for Improved Cycle Life of Li–O2 Battery

Li–O2 batteries are attractive systems because they can deliver much higher energy densities than those of conventional lithium-ion batteries by engaging light gas-phase oxygen as a cathode active material. However, the inevitable generation of residual superoxide radicals gives rise to irreversible side reactions and consequently causes severe capacity degradation over cycling. To address this chronic issue, herein, we have taken a lesson from the human eye. Analogous to Li–O2 batteries, the human eye is liable to attack by reactive oxygen species (ROS), from its lifetime exposure to sunlight. However, it protects itself from the ROS attack by using melanin as a radical scavenger. To mimic such a defense mechanism against radical attack, we included polydopamine (pD), which is one of the most common synthetic melanins, in the ether-based electrolyte. As an outcome of the superoxide radical scavenging by the pD additive, the irreversible side reaction products were alleviated significantly, resulting in superior cycling performance. The present investigation provides a message that simple treatments inspired by the human body or nature could be effective solutions to the problems in various energy devices.

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.

Mitochondrial function and mitochondrial DNA maintenance with advancing age.

We review the impact of mitochondrial DNA (mtDNA) maintenance and mitochondrial function on the aging process. Mitochondrial function and mtDNA integrity are closely related. In order to create a protective barrier against reactive oxygen and nitrogen species (RONS) attacks and ensure mtDNA integrity, multiple cellular mtDNA copies are packaged together with various proteins in nucleoids. Regulation of antioxidant and RONS balance, DNA base excision repair, and selective degradation of damaged mtDNA copies preserves normal mtDNA quantities. Oxidative damage to mtDNA molecules does not substantially contribute to increased mtDNA mutation frequency; rather, mtDNA replication errors of DNA PolG are the main source of mtDNA mutations. Mitochondrial turnover is the major contributor to maintenance of mtDNA and functionally active mitochondria. Mitochondrial turnover involves mitochondrial biogenesis, mitochondrial dynamics, and selective autophagic removal of dysfunctional mitochondria (i.e., mitophagy). All of these processes exhibit decreased activity during aging and fall under greater nuclear genome control, possibly coincident with the emergence of nuclear genome instability. We suggest that the age-dependent accumulation of mutated mtDNA copies and dysfunctional mitochondria is associated primarily with decreased cellular autophagic and mitophagic activity.

Current Methods Used in the Protein Carbonyl Assay

Some oxidative stress effects, such as lipid peroxidation or the DNA damage, have been extensively assessed and reliable biomarkers have been associated to characterize the degree of the damage, while the attack of reactive oxygen species on proteins and the formation of protein carbonyls were investigated only in the recent years. Taking into account that protein carbonyls may play an important role in the early diagnosis of pathologies associated with reactive oxygen species overproduction, a robust and reliable method to quantify the protein carbonyls in complex biological samples is also required. This minireview investigates up-to-date methods used for the separation, identification and quantitative assay of protein carbonyls, a special attention being paid to the advantages and disadvantages of each technique.

Alterations in rat pulmonary phosphatidylcholines after chronic exposure to ambient fine particulate matter.

This study elucidated the underlying pathophysiological changes that occur after chronic ambient fine particulate matter (PM2.5) exposure via a lipidomic approach. Five male Sprague-Dawley rats were continually whole-body exposed to ambient air containing PM2.5 at 16.7 ± 10.1 μg m(-3) from the outside of the building for 8 months, whereas a control group (n = 5) inhaled filtered air. Phosphorylcholine-containing lipids were extracted from lung tissue and profiled using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The phosphatidylcholine (PC) signal features of the two groups were compared using partial least squares discriminant analysis (PLS-DA) and Wilcoxon rank sum tests. The PC profile of the exposure group differed from that of the control group; the R(2)Y and Q(2) were 0.953 and 0.677, respectively, in the PLS-DA model. In the exposure group, a significant 0.66- to 0.80-fold reduction in lyso-PC levels, which may have resulted from repeated inflammation, was observed. Decreased surfactant PCs by 16% at most may indicate injuries to alveolar type II cells. Cell function and cell signalling are likely to be altered because the decrease in unsaturated PCs may reduce membrane fluidity. Accompanied by the decline in plasmenylcholines, decreased unsaturated PCs may indicate the attack of reactive oxygen species generated by PM2.5 exposure. The physiological findings conformed to the histopathological changes in the exposed animals. PC profiling using UPLC-MS/MS-based lipidomics is sensitive for reflecting pathophysiological perturbations in the lung after long-term and low concentration PM2.5 exposure.

Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is today considered the most common form of chronic liver disease, affecting a high proportion of the population worldwide. NAFLD encompasses a large spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis and cirrhosis. Obesity, hyperglycemia, type 2 diabetes and hypertriglyceridemia are the most important risk factors. The pathogenesis of NAFLD and its progression to fibrosis and chronic liver disease is still unknown. Accumulating evidence indicates that mitochondrial dysfunction plays a key role in the physiopathology of NAFLD, although the mechanisms underlying this dysfunction are still unclear. Oxidative stress is considered an important factor in producing lethal hepatocyte injury associated with NAFLD. Mitochondrial respiratory chain is the main subcellular source of reactive oxygen species (ROS), which may damage mitochondrial proteins, lipids and mitochondrial DNA. Cardiolipin, a phospholipid located at the level of the inner mitochondrial membrane, plays an important role in several reactions and processes involved in mitochondrial bioenergetics as well as in mitochondrial dependent steps of apoptosis. This phospholipid is particularly susceptible to ROS attack. Cardiolipin peroxidation has been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including NAFLD. In this review, we focus on the potential roles played by oxidative stress and cardiolipin alterations in mitochondrial dysfunction associated with NAFLD.

Changes in the Physico-Chemical Properties of Human Kidney Cell Membranes during the Cancer Transformation

Kidney tissue is particularly susceptible to reactive oxygen species attack which leads to development of cancer. During oxidative stress, membrane lipids and proteins are major targets of re-active oxygen species (ROS). This work is focused on changes of phospholipids, proteins content and electric charge that occur in cell membranes of kidney cancer of pT3 stage, grade G3 and with metastasis. Qualitative and quantitative phospholipid composition and the presence of integral membrane proteins were determined by high-performance liquid chromatography. Electrophoresis was used to determine the surface charge density of the human kidney cell membrane. It was shown that the process of cancer transformation was accompanied by an increase phospholipid levels and altered the level of integral proteins as determined by decrease phenylalanine, tyrosine, cysteine and arginine. Moreover, the process of cancer transformation significantly enhanced changes in the surface charge density of the human kidney cell membrane. Cell membrane structure and function are modified by neoplasm lesion.

Suppressive effects of Hydroxytyrosol on oxidative stress in erythrocytes

We investigated the oxidative modifications of lipids, proteins and antioxidants enzymes activities, in erythrocytes treated with H2O2 in the presence or absence of hydroxytyrosol (HT) at 37°C for 30 min. Conjugated dienes level was measured as biomarker of lipid peroxidation. Protein carbonyl level was measured as biomarker of protein oxidation. The antioxidants enzymes activities were evaluated by superoxide dismutase and catalase activities determinations. Conjugated dienes levels decreased in erythrocytes after hydroxytyrosol treatment as compared to erythrocytes treated with 0.2 mM H2O2. Protein carbonyl levels increased in erythrocytes after hydroxytyrosol treatment as compared to erythrocytes treated with 0.2 mM H2O2. In addition, this study showed a prospective protective action of HT of human erythrocytes from the attack of reactive oxygen species. In fact, a considerable increase of superoxide dismutase activity by 40% was showed after incubation of erythrocytes with HT at dose 5 µg/ml. Although, there were no significant differences in catalase activity in erythrocytes after hydroxytyrosol treatment as compared to erythrocytes treated with 0.2 mM H2O2. This study demonstrates that hydroxytyrosol prevents protein oxidation and lipid peroxidation caused by H2O2 treatment using erythrocytes cell model. Key words: Hydroxytyrosol, thiol, erythrocytes, conjugated dienes, malondialdehyde, superoxide dismutase, catalase, peripheral blood mononuclear (PBL), peripheral blood mononuclear.

Age‐induced hair greying – the multiple effects of oxidative stress

An obvious sign of ageing is hair greying, or the loss of pigment production and deposition within the hair shafts. Numerous mechanisms, acting at different levels and follicular locations, contribute to hair greying, ranging from melanocyte stem cells defects to follicular melanocyte death. One key issue that is in common to these processes is oxidative damage. At the hair follicle stem cells niche, oxidative stress, accelerated by B-cell lymphoma 2 gene (BCL-2) depletion, leads to selective apoptosis and diminution of melanocyte stem cells, reducing the repopulation of newly formed anagen follicles. Melanotic bulbar melanocytes express high levels of BCL-2 to enable survival from melanogenesis- and ultraviolet A (UVA)-induced reactive oxygen species (ROS) attacks. With ageing, the bulbar melanocyte expression of anti-oxidant proteins such as BCL-2, and possibly TRP-2, is reduced, and the dedicated enzymatic anti-oxidant defence system throughout the follicle weakens, resulting in enhanced oxidative stress. A marked reduction in catalase expression and activity results in millimolar accumulation of hydrogen peroxide, contributing to bulbar melanocyte malfunction and death. Interestingly, amelanotic melanocytes at the outer root sheath (ORS) are somewhat less affected by these processes and survive for longer time even within the white, ageing hair follicles. Better understanding of the overtime susceptibility of melanocytes to oxidative stress at the different follicular locations might yield clues to possible therapies for the prevention and reversal of hair greying.

Reactive oxygen species at phospholipid bilayers: Distribution, mobility and permeation

Reactive oxygen species (ROS) are involved in biochemical processes such as redox signaling, aging, carcinogenesis and neurodegeneration. Although biomembranes are targets for reactive oxygen species attack, little is known about the role of their specific interactions. Here, molecular dynamics simulations were employed to determine the distribution, mobility and residence times of various reactive oxygen species at the membrane–water interface. Simulations showed that molecular oxygen (O2) accumulated at the membrane interior. The applicability of this result to singlet oxygen (1O2) was discussed. Conversely, superoxide (O2−) radicals and hydrogen peroxide (H2O2) remained at the aqueous phase. Both hydroxyl (HO) and hydroperoxyl (HO2) radicals were able to penetrate deep into the lipid headgroups region. Due to membrane fluidity and disorder, these radicals had access to potential peroxidation sites along the lipid hydrocarbon chains, without having to overcome the permeation free energy barrier. Strikingly, HO2 radicals were an order of magnitude more concentrated in the headgroups region than in water, implying a large shift in the acid–base equilibrium between HO2 and O2−. In comparison with O2, both HO and HO2 radicals had lower lateral mobility at the membrane. Simulations revealed that there were intermittent interruptions in the H-bond network around the HO radicals at the headgroups region. This effect is expected to be unfavorable for the H-transfer mechanism involved in HO diffusion. The implications for lipid peroxidation and for the effectiveness of membrane antioxidants were evaluated.

Mass spectrometry and redox proteomics: applications in disease.

Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases.

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa

Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1-3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer-assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.

Interplay of Phosphorylated Apoptosis Repressor with CARD, Casein Kinase-2 and Reactive Oxygen Species in Regulating Endothelin-1-Induced Cardiomyocyte Hypertrophy.

The role of the Apoptosis repressor with caspase recruitment domain (ARC) in apoptosis and in certain hypertrophic responses has been previously investigated, but its regulation of Endothelin-1 induced cardiac hypertrophy remains unknown. The present study discusses the inhibitory role of ARC against endothelin-induced hypertrophy. In present study Endothelin treated cardiomyocytes were used as a hypertrophic model, that were subsequently treated with adenovirus ARC and its mutant at different multiplicity of infections. Casein-kinase-2 inhibitors were used to produce dephosphorylated ARC and to study its effect on hypertrophy. Hypertrophy was assessed by cell surface area measurement, Atrial-natriuretic-Factor mRNA analysis and total protein assay. Reactive oxygen species analysis was carried out using the dichlorofluorescin-diacetate (DCFH-DA) assay. Over expression of ARC significantly inhibits Endothelin-induced cardiomyocyte hypertrophy. The nonphosphorylated mutant ARC (T149 A) remained unable to control endothelin-induced hypertrophy, suggesting a vital role for ARC phosphorylation in regulation of its activity. Sensitization study has been carried out to check the role of endogenous ARC using casein-kinase inhibitors. Finally, the significant role of ARC in regulating reactive oxygen species -mediated control of endothelin induced hypertrophy has also been assessed. Conclusion : Conclusively, present study showed the vital and potential therapeutic interventional role of ARC in preventing endothelin-1-induced cardiomyocyte hypertrophy. The regulation of hypertrophic pathway by ARC relies on blunting the reactive oxygen species attack. This study further suggests a mediatory role of casein-kinase-2 in Endothelin-induced hypertrophy, mainly through its phosphorylation of ARC.

UVB irradiation as a tool to assess ROS-induced damage in human spermatozoa

One of the consequences of oxygen metabolism is the production of reactive oxygen species (ROS) which in a situation of imbalance with antioxidants can damage several biomolecules, compromise cell function and even lead to cellular death. The particularities of the sperm cell make it particularly vulnerable to ROS attack compromising its functionality, mirrored in terms of fertility outcome and making the study of the origin of sperm ROS, as well as the alterations they cause very important. In the present work, we used UVB irradiation, an easy experimental approach known as a potent inducer of ROS formation, to better understand the origin of ROS damage without any confounding effects that usually exist in disease models in which ROS are reported to play a role. To address these issues we evaluated sperm mitochondrial ROS production using the Mitosox Red Probe, mitochondrial membrane potential using the JC-1 probe, lipid peroxidation through BODIPY probe and vitality using PI. We observed that UVB irradiation leads to an increase in sperm mitochondrial ROS production and lipid peroxidation that occur previously to an observable mitochondrial dysfunction. We concluded that sperm UVB irradiation appears to be a good and easily manipulated in vitro model system to study mitochondria-induced oxidative stress in spermatozoa and its consequences, which may be relevant in terms of dissecting the action pathways of many other pathologies, drugs and contaminants, including endocrine disruptors.

Transgenic rice overexpressing the Brassica juncea gamma-glutamylcysteine synthetase gene enhances tolerance to abiotic stress and improves grain yield under paddy field conditions

Glutathione (GSH), a low-molecular-weight tripeptide molecule that plays an important role in cell function and metabolism as an antioxidant, is synthesized by γ-glutamylcysteine synthetase and glutathione synthetase. To investigate the functional role of GSH in the adaptation of plants to abiotic stresses, we developed Brassica juncea L. ECS (BrECS)-expressing transgenic rice plants (BrECS1 and BrECS2) under the regulation of a stress-inducible Rab21 promoter. BrECS1 and BrECS2 transgenic rice plants with BrECS overexpression tolerated high salinity by maintaining a cellular glutathione (GSH)/glutathione disulfide redox buffer, which prevented unnecessary membrane oxidation. BrECS1 and BrECS2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence than wild-type (WT) rice plants in the presence of methyl viologen (MV) and salt, resulting in enhanced tolerance to abiotic stresses. During germination, BrECS overexpression increased growth and development, resulting in an increased germination rate in the presence of salt conditions, but not under salt-free normal conditions. Furthermore, BrECS1 and BrECS2 rice plants displayed a moderate increase in biomass and rice grain yield under general paddy field conditions when compared to WT rice plants under general paddy field conditions. Therefore, our results suggest that BrECS-overexpression was critical for cellular defense from reactive oxygen species attacks produced by salt and MV, promotion of germination, and metabolic processes involved in natural environmental stress tolerance, thereby enhancing growth development and rice grain yield.