Soybean MAPK, GMK1 Is dually regulated by phosphatidic acid and hydrogen peroxide and translocated to nucleus during salt stress

Mitogen-activated protein kinase (MAPK) is activated by various biotic and abiotic stresses. Salt stress induces two well-characterized MAPK activating signaling molecules, phosphatidic acid (PA) via phospholipase D and phospholipase C, and reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. In our previous study, the activity of soybean MAPK, GMK1 was strongly induced within 5 min of 300 mM NaCl treatment and this early activity was regulated by PA. In this study, we focused on the regulation of GMK1 at the later stage of the salt stress, because its activity was strongly persistent for up to 30 min. H₂O₂ activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of the treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H₂O₂ 10 min after the treatment. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor, but was increased in protoplast overexpressing TESD-GMKK1. However, these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Taken together, we propose that soybean GMK1 is dually regulated by PA and H₂O₂ at a time dependant manner and translocated to the nucleus by the salt stress signal.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H₂O₂)-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), a chloromethyl derivative of H₂DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H₂O₂-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H₂O₂-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.     

Ascorbate plays a key role in alleviating low temperature-induced oxidative stress in Arabidopsis

Low temperature has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). In order to study the role of ascorbate under chilling stress, the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc2-1 to low temperature (2°C) was investigated. After chilling stress, vtc2-1 mutants exhibited oxidative damage. An increase in the H₂O₂ generation and the production of thiobarbituric acid reactive substances (TBARS), and a decrease in chlorophyll content, the maximal photochemical efficiency of PSII (F_v/F_m) and oxidizable P₇₀₀ were also noted. The ratio of ascorbate/dehydroascorbate and reduced glutathione/oxidzed glutathione in the vtc2-1 mutants were reduced, compared with the wild type (WT) plants. The activities of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and soluble antioxidants were lower in the vtc2-1 mutants than those in WT plants. These results suggested that the ascorbate-deficient mutant vtc2-1 was more sensitive to chilling treatment than WT plants. The low temperature-induced oxidative stress was the major cause of the decrease of PSII and PSI function in the vtc2-1 mutants. Ascorbate plays a critical role of defense without which the rest of the ROS defense network is unable to react effectively.     

A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H₂O₂) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H₂O₂ stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.     

Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity

An early light-inducible protein gene (CaELIP) was isolated from a cDNA library of hot pepper (Capsicum annuum) that showed heavy metal stress-inducible expressions. This gene contains an open reading frame (ORF) encoding a protein of 160 amino acids, and the protein has significant homology with reported early light-inducible proteins from other plant species. Topology analysis for CaELIP suggested three transmembrane domains. Genomic DNA blot analysis showed that CaELIP is a single copy gene in hot pepper. The treatment of seedling roots of hot pepper with Cu induced ROS generation in the root, and the level of ROS generation was paralleled to the concentration of Cu that again was matched to the increase in the CaELIP expression level. Results suggested that expression of CaELIP can be induced by the ROS generated by the excessive Cu in the plant. Exogenous SA treatment significantly alleviated Cu-induced expression of CaELIP, while exogenous JA treatment aggravated expression of CaELIP under Cu stress. CaELIP showed a transient expression when exposing the plant to light for 1 h. CaELIP also showed an endogenous circadian rhythmicity with high expression level in the morning and decreased expression level thereafter. The expression of CaELIP was also induced by high or low temperature, high salinity, drought, and stress hormone ABA. Taken together, the results suggest that CaELIP would function in responding to environmental signals and possibly regulating the response to the abiotic stresses that can be related to the abiotic stress tolerance in plants.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Silencing of tomato mitochondrial uncoupling protein disrupts redox poise and antioxidant enzymes activities balance under oxidative stress

Plant mitochondrial uncoupling proteins (pUCPs) play important roles in generation of metabolic thermogenesis, response to stress situation, and regulation of energy metabolism. Although the signaling pathways for the pUCPs-regulated plant energy metabolism and thermogenesis are well studied, the role of pUCPs in the regulation of plant stress tolerance has not been fully substantiated. Here we showed that mitochondrial uncoupling protein was required for effective antioxidant enzymes activities, chlorophyll fluorescence and redox poise in tomato under oxidative stress using virusinduced gene silencing approach. Silencing of LeUCP gene reduced maximal quantum yield of PSII (Fv/Fm) and photochemical quenching coefficient (qP), as well as mitigated activation of antioxidant enzymes and related genes expression. The content of reduced ascorbate and reduced glutathione, redox ratio of ascorbate and L-galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) activity were all decreased in the leaves of LeUCP gene-silenced plant. However, malondialdehyde content was increased under methylviologen (MV) stress. ROS accumulation was increased significantly following MV and heat stress treatments. Meanwhile, LeUCP gene silencing aggravated accumulation of H₂O₂ and O ₂ ^·? in leaves. Taken together, these results strongly suggest that LeUCP gene plays critical role in maintaining the redox homeostasis and balance in antioxidant enzyme system under oxidative stress.     

Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H₂O₂, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.