Pseudomonas aeruginosa H3-T6SS Combats H2O2 Stress by Diminishing the Amount of Intracellular Unincorporated Iron in a Dps-Dependent Manner and Inhibiting the Synthesis of PQS.

The type VI secretion system (T6SS), a protein translocation nanomachine, is widely distributed in Gram-negative bacteria and delivers effectors directly into target cells or the extracellular environment to help the bacteria gain a competitive fitness advantage and promote bacterial survival in harmful environments. In this study, we demonstrated that the synthesis of the Pseudomonas quinolone signal (PQS) in Pseudomonas aeruginosa PAO1 was inhibited by the H3-T6SS gene cluster under iron-rich conditions, and that this inhibition was relieved under iron starvation conditions. Conversely, PQS differentially regulated the expression of the H3-T6SS structural genes and the effector protein gene tseF. The expression of tseF was inhibited by PQS, while the expressions of the H3-T6SS structural genes were positively regulated by PQS. Further studies showed that the H3-T6SS was involved in the resistance of P. aeruginosa to oxidative stress caused by hydrogen peroxide (H2O2). Interestingly, H3-T6SS expression was neither induced by H2O2 stress nor regulated by OxyR (a global anti-oxidative transcriptional regulator) but was positively regulated by RpoS (a major transcription regulator of the stress response). In addition, we found that the clpV3 (a structural gene of H3-T6SS) mutation resulted in upregulation of two proteins related to PQS synthesis and many proteins related to oxidative stress resistance, while the expression of some iron storage proteins, especially Dps, were significantly downregulated. Furthermore, the clpV3 mutation led to an increase in the intracellular free Fe2+ content of P. aeruginosa. Further studies showed that both the PQS deficient mutation and overexpression of dps effectively restored the H2O2 sensitive phenotype of the H3-T6SS mutant. Finally, we proposed the following model of H3-T6SS-mediated resistance to H2O2 stress in P. aeruginosa. H3-T6SS not only reduces the intracellular free Fe2+ level by upregulating the expression of ferritin Dps, but also inhibits the synthesis of PQS to mediate the resistance of P. aeruginosa to H2O2 stress. This study highlights the important role of H3-T6SS in the ability of P. aeruginosa to combat H2O2 stress and provides a perspective for understanding the stress response mechanism of bacteria.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress.

BACKGROUND: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2-induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Antioxidant Potential of Diosmin and Diosmetin against Oxidative Stress in Endothelial Cells.

Phlebotropic flavonoids, including diosmin and its aglycone diosmetin, are natural polyphenols widely used in the prevention and treatment of chronic venous insufficiency (CVI). As oxidative stress plays an important role in the development of pathophysiology of the cardiovascular system, the study aimed to investigate the protective effects of diosmin and diosmetin on hydrogen peroxide (H2O2)-induced oxidative stress in endothelial cells. The cells were pretreated with different concentrations of the flavonoid prior to the H2O2 exposure. The cell viability, the level of intracellular reactive oxygen species (ROS), the activity of cellular antioxidant enzymes-including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase GPx-and the malondialdehyde (MDA) level were assessed. It was found that the H2O2-induced oxidative stress was ameliorated by diosmin/diosmetin in a concentration-dependent manner. The flavonoids restored the activity of cellular antioxidant enzymes and lowered the MDA level upregulated by the H2O2 exposure. These results indicate that diosmin and diosmetin may prevent oxidative stress in endothelial cells; therefore, they may protect against the development and progression of oxidative-stress-related disorders.

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase.

Cysteine desulfurases, which supply sulfur for iron-sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe-S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon. The purified SufS protein existed as a pyridoxal 5' phosphate (PLP)-containing dimer with a relatively low desulfurase activity. Interestingly, in the presence of the AsaE protein, the catalytic efficiency of this reaction increased 10-fold. In particular, for sulfur mobilization, the AsaE protein partnered only SufS and not other cysteine desulfurases from Anabaena. The SufS protein was found to physically interact with the AsaE protein, demonstrating that AsaE was indeed the missing partner of Anabaena SufS. The conserved cysteine of the SufS or the AsaE protein was essential for activity but not for their physical association. Curiously, overexpression of the SufS protein in Anabaena caused reduced formation of reactive oxygen species on exposure to hydrogen peroxide (H2O2), resulting in superior oxidative stress tolerance to the oxidizing agent when compared with the wild-type strain. Overall, the results highlight the functional interaction between the two proteins that mediate sulfur mobilization, in the cyanobacterial SUF pathway, and further reveal that overexpression of SufS can protect cyanobacteria from oxidative stress.

Composition Analysis by UPLC-PDA-ESI (-)-HRMS and Antioxidant Activity Using Saccharomyces cerevisiae Model of Herbal Teas and Green Teas from Hainan.

Different teas from everywhere are very useful and have been extensively studied. We studied the antioxidant activity of herbal teas and green teas from Hainan, Mallotus oblongifolius Muell. Arg. (MO), Ilex kudingcha C.J. Tseng (KD), Camellia sinensis var. assamica (J. W. Mast.) Kitam. Hainan Dayezhong (DY), and Camellia sinensis (L.) O. Ktze. (produced from Hainan Baisha (BS)). The total phenol content and total flavonoid content from water extracts, resin extracts and fractions of herbal teas and green teas were compared. Later, eight fractions of herbal teas and green teas were subjected to UPLC-PDA-ESI-(-)-HRMS. We determined 1-diphenyl -2-picryl-hydrazyl radical and hydroxyl free radical scavenging activity by electron paramagnetic resonance spectroscopy. We subjected Saccharomyces cerevisiae to hydrogen peroxide, stress and evaluated antioxidant activity of herbal teas and green teas in cellulo. The experiment identified more than 14 potential antioxidant compounds from herbal teas and green teas. The herbal teas and green teas had a clearance rate higher than ferulic acid at the same concentrations. MO best reduced intracellular oxidation levels and increased catalase, glutathione reductase activities, glutathione reduced and glutathione oxidized content. KD had the highest cell survival rate and reduced cell lipid peroxidation. DY best improved superoxide dismutase activity and BS was the most active in the halo test. Therefore, we concluded that MO had stronger antioxidant activity than other herbal teas and green teas from Hainan, especially, which reduce S. cerevisiae oxidative stress under H2O2 stress.

Hydrogen Peroxide Response in Leaves of Poplar (Populus simonii × Populus nigra) Revealed from Physiological and Proteomic Analyses.

Hydrogen peroxide (H2O2) is one of the most abundant reactive oxygen species (ROS), which plays dual roles as a toxic byproduct of cell metabolism and a regulatory signal molecule in plant development and stress response. Populus simonii × Populus nigra is an important cultivated forest species with resistance to cold, drought, insect and disease, and also a key model plant for forest genetic engineering. In this study, H2O2 response in P. simonii × P. nigra leaves was investigated using physiological and proteomics approaches. The seedlings of 50-day-old P. simonii × P. nigra under H2O2 stress exhibited stressful phenotypes, such as increase of in vivo H2O2 content, decrease of photosynthetic rate, elevated osmolytes, antioxidant accumulation, as well as increased activities of several ROS scavenging enzymes. Besides, 81 H2O2-responsive proteins were identified in the poplar leaves. The diverse abundant patterns of these proteins highlight the H2O2-responsive pathways in leaves, including 14-3-3 protein and nucleoside diphosphate kinase (NDPK)-mediated signaling, modulation of thylakoid membrane structure, enhancement of various ROS scavenging pathways, decrease of photosynthesis, dynamics of proteins conformation, and changes in carbohydrate and other metabolisms. This study provides valuable information for understanding H2O2-responsive mechanisms in leaves of P. simonii × P. nigra.

Lack of a functional methionine sulfoxide reductase (MsrB) increases oxacillin and H2O2 stress resistance and enhances pigmentation in Staphylococcus aureus.

Staphylococcus aureus produces 3 MsrA enzymes (MsrA1, MsrA2, and MsrA3) and 1 MsrB enzyme. The genes encoding MsrA1 and MsrB are the first and second genes of a 4-gene operon in S. aureus. In a previous study, MsrA1-deficient S. aureus cells showed increased sensitivity to oxidative stress conditions in spite of a higher production of MsrB. In this study, an msrB mutant of S. aureus was created by site-directed mutagenesis that left the first gene of this locus, msrA1, intact. Studies with this mutant suggest that a deletion of MsrB increases resistance of S. aureus to H2O2 and oxacillin and that the mutant cells produce a higher level of carotenoids relative to wild-type S. aureus cells.

iTRAQ-based quantitative proteomic analysis reveals new metabolic pathways of wheat seedling growth under hydrogen peroxide stress.

As an abundant ROS, hydrogen peroxide (H2 O2 ) plays pivotal roles in plant growth and development. In this work, we conducted for the first time an iTRAQ-based quantitative proteomic analysis of wheat seedling growth under different exogenous H2 O2 treatments. The growth of seedlings and roots was significantly restrained by increased H2 O2 concentration stress. Malondialdehyde, soluble sugar, and proline contents as well as peroxidase activity increased with increasing H2 O2 levels. A total of 3,425 proteins were identified by iTRAQ, of which 157 showed differential expression and 44 were newly identified H2 O2 -responsive proteins. H2 O2 -responsive proteins were mainly involved in stress/defense/detoxification, signal transduction, and carbohydrate metabolism. It is clear that up-regulated expression of signal transduction and stress/defence/detoxification-related proteins under H2 O2 stress, such as plasma membrane intrinsic protein 1, fasciclin-like arabinogalactan protein, and superoxide dismutase, could contribute to H2 O2 tolerance of wheat seedlings. Increased gluconeogenesis (phosphoenol-pyruvate carboxykinase) and decreased pyruvate kinase proteins are potentially related to the higher H2 O2 tolerance of wheat seedlings. A metabolic pathway of wheat seedling growth under H2 O2 stress is presented.

Heterologous Expression of a Ferritin Homologue Gene PpFer1 from Prunus persica Enhances Plant Tolerance to Iron Toxicity and H2O2 Stress in Arabidopsis thaliana.

In plants, ferritin proteins play an important role in iron (Fe) storage which contributes to plant growth and development. However, the biological functions of ferritins in fruit trees are essentially unknown. In this study, three Ferritin genes were isolated from 'Zhentong No. 3' peach, which were named PpFer1-PpFer3. The expression levels of these genes were different in distinct tissues/organs. Notably, PpFer1 was the most abundantly expressed Ferritin family gene in all tested tissues of 'Zhentong No. 3' peach; its expression levels were significantly enhanced throughout the entire peach seedling under Fe toxicity and H2O2 stress, particularly in the leaves. In addition, over-expression of PpFer1 was effective in rescuing the retarded growth of Arabidopsis fer1-2 knockout mutant, embodied in enhanced fresh weight, primary root length, lateral root numbers, total root length, total leaf chlorophyll, stomatal conductance (Gs), net photosynthetic rate (Pn), transpiration rate, and tissue Fe concentration. This study provides insights into understanding the molecular mechanisms of Fe storage and sequestration in perennial fruit trees.

Glutathione reductase, a biomarker of pollutant and stress in Pacific abalone.

Abalone are frequently exposed to several environmental factors including heavy metal toxicity, thermal stress, H2O2-stress, starvation, viral and bacterial infection that can induce oxidative stress. Glutathione reductase is a vital enzyme in the antioxidant defense system that catalyzes the reduction of oxidized glutathione to reduced glutathione. The present study aimed to identify and localize glutathione reductase in Pacific abalone (Hdh-GR) and assess its potential role in stress physiology, heavy metal toxicity, immune response, gonadal development, and metamorphosis. The mRNA expression of Hdh-GR was upregulated in response to thermal stress, starvation, H2O2-stress, and cadmium-exposed toxicity. The induced mRNA expression was also quantified in immune-challenged abalone. Moreover, the Hdh-GR expression was significantly higher during metamorphosis. The Hdh-GR mRNA expression showed an inverse relationship with ROS production in heat stressed Pacific abalone. These results suggest that Hdh-GR has central role in the stress physiology, immune response, gonadal development, and metamorphosis of Pacific abalone.

Time-series responses in photosynthetic activity, 2-cysteine peroxiredoxin gene expression, and proteomics of Chattonella marina var. antiqua under different oxidative stress.

Chattonella spp. are known to produce large amounts of reactive oxygen species (ROS); however, little is known about the mechanisms involved in mitigating the intracellular accumulation of ROS. In this study, a time-series of biological responses in C. marina var. antiqua under different oxidative stress conditions, induced by adding H2O2 at the initial concentrations of 100 and 500 µM, was investigated. Although the added exogenous H2O2 was rapidly consumed at 3 h post-exposure (hpe), intracellular ROS levels were enhanced in the 500 µM H2O2 group but decreased in the 100 µM H2O2 group. Accompanied by increased intracellular ROS levels, the photosynthetic activity of C. marina var. antiqua was considerably inhibited in the 500 µM H2O2 group, but not in the 100 µM H2O2 group. The Fv/Fm ratio and PIABS were negatively correlated with the intracellular ROS level, while the ABS/RC, TR0/RC, and DI0/RC were positively correlated with the intracellular ROS level. Expression of the gene encoding 2-cysteine peroxiredoxin (2-Cys Prx) was up-regulated in 100 µM H2O2 group at 6 hpe, but was down-regulated in 100 µM H2O2 group at 3 and 6 hpe. A negative relationship between the 2-Cys Prx transcript levels and intracellular ROS levels was detected. Results of the 2-DE proteomic analysis confirmed that the 500 µM H2O2 treatment down-regulated the expression of 2-Cys Prx and induced more damage to photosynthetic abilities of C. marina var. antiqua.

Role of ClpX and ClpP in Streptococcus suis serotype 2 stress tolerance and virulence.

Streptococcus suis has received increasing attention for its involvement in severe infections in pigs and humans; however, their pathogenesis remains unclear. ClpX and ClpP, two subunits of the ATP-dependent caseinolytic protease Clp, play key roles in bacterial adaptation to various environmental stresses. In this study, a virulent S. suis serotype 2 strain, ZY05719, was employed to construct clpX and clpP deletion mutants (ΔclpX and ΔclpP, respectively) and their complementation strains. Both ΔclpX and ΔclpP displayed significantly reduced adaptability compared with the wild-type strain, evident through several altered phenotypes: formation of long cell chains, tendency to aggregate in culture, and reduced growth under acidic pH and H2O2-induced oxidative stress. ClpP and ClpX were required for the optimal growth during heat and cold stress, respectively. An in vitro experiment on RAW264.7 macrophage cells showed significantly increased sensitivity of ΔclpX and ΔclpP to phagocytosis compared with the wild-type strain. Mouse infection assays verified the deletion of clpX and clpP led to not only fewer clinical symptoms and lower mortality but also to a marked attenuation in bacterial colonization. These virulence-related phenotypes were restored by genetic complementation. Furthermore, the deletion of clpX or clpP caused a significant decrease in the expression of sodA, tpx, and apuA compared with the wild-type strain, suggesting that these genes may be regulated by ClpX and ClpP as downstream response factors to facilitate the bacterial tolerance against various environmental stresses. Taken together, these results suggest that ClpX and ClpP play important roles in stress tolerance for achieving the full virulence of S. suis serotype 2 during infection.

Stimulation of Vibrio vulnificus Pyruvate Kinase in the Presence of Glucose to Cope With H2O2 Stress Generated by Its Competitors.

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates a variety of cellular processes in addition to catalyzing the coupled transport and phosphorylation of carbohydrates. We recently reported that, in the presence of glucose, HPr of the PTS is dephosphorylated and interacts with pyruvate kinase A (PykA) catalyzing the conversion of PEP to pyruvate in Vibrio vulnificus. Here, we show that this interaction enables V. vulnificus to survive H2O2 stress by increasing pyruvate production. A pykA deletion mutant was more susceptible to H2O2 stress than wild-type V. vulnificus without any decrease in the expression level of catalase, and this sensitivity was rescued by the addition of pyruvate. The H2O2 sensitivity difference between wild-type and pykA mutant strains becomes more apparent in the presence of glucose. Fungi isolated from the natural habitat of V. vulnificus retarded the growth of the pykA mutant more severely than the wild-type strain in the presence of glucose by glucose oxidase-dependent generation of H2O2. These data suggest that V. vulnificus has evolved to resist the killing action of its fungal competitors by increasing pyruvate production in the presence of glucose.

Snf1 cooperates with the CWI MAPK pathway to mediate the degradation of Med13 following oxidative stress.

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCFGrr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second SCFGrr1 responsive degron in Med13. Deletion of Snf1 resulted in nuclear retention of cyclin C and failure to induce mitochondrial fragmentation. This degron was able to confer oxidative-stress-induced destruction when fused to a heterologous protein in a Snf1 dependent manner. Although snf1∆ mutants failed to destroy Med13, deleting the degron did not prevent destruction. These results indicate that the control of Med13 degradation following H2O2 stress is complex, being controlled simultaneously by CWI and MAPK pathways.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

The Copper Homeostasis Transcription Factor CopR Is Involved in H2O2 Stress in Lactobacillus plantarum CAUH2.

Transcriptional factors (TFs) play important roles in the responses to oxidative, acid, and other environmental stresses in Gram-positive bacteria, but the regulatory mechanism of TFs involved in oxidative stress remains unknown in lactic acid bacteria. In the present work, homologous overexpression strains with 43 TFs were constructed in the Lactobacillus plantarum CAUH2 parent strain. The strain overexpressing CopR displayed the highest sensitivity and a 110-fold decrease in survival rate under H2O2 challenge. The importance of CopR in the response to H2O2 stress was further confirmed by a 10.8-fold increase in the survival of a copR insertion mutant. In silico analysis of the genes flanking copR revealed putative CopR-binding "cop box" sequences in the promoter region of the adjacent gene copB encoding a Cu2+-exporting ATPase. Electrophoretic mobility shift assay (EMSA) analysis demonstrated the specific binding of CopR with copB in vitro, suggesting copB is a target gene of CopR in L. plantarum. The role of CopB involved in oxidative stress was verified by the significantly decreased survival in the copB mutant. Furthermore, a growth defect in copper-containing medium demonstrated that CopB functions as an export ATPase for copper ions. Furthermore, EMSAs revealed that CopR functions as a regulator that negatively regulates copB gene and Cu2+ serves as inducer of CopR to activate the expression of CopB in response to H2O2 stress in L. plantarum CAUH2. Our findings indicated that CopR plays an important role in enhancing oxidative resistance by regulating copB to modulate copper homeostasis.

Analysis of Copper-Binding Proteins in Rice Radicles Exposed to Excess Copper and Hydrogen Peroxide Stress.

Copper (Cu) is an essential micronutrient for plants, but excess Cu can inactivate and disturb the protein function due to unavoidable binding to proteins at the cellular level. As a redox-active metal, Cu toxicity is mediated by the formation of reactive oxygen species (ROS). Cu-binding structural motifs may alleviate Cu-induced damage by decreasing free Cu(2+) activity in cytoplasm or scavenging ROS. The identification of Cu-binding proteins involved in the response of plants to Cu or ROS toxicity may increase our understanding the mechanisms of metal toxicity and tolerance in plants. This study investigated change of Cu-binding proteins in radicles of germinating rice seeds under excess Cu and oxidative stress using immobilized Cu(2+) affinity chromatography, two-dimensional electrophoresis, and mass spectra analysis. Quantitative image analysis revealed that 26 protein spots showed more than a 1.5-fold difference in abundances under Cu or H2O2 treatment compared to the control. The identified Cu-binding proteins were involved in anti-oxidative defense, stress response and detoxification, protein synthesis, protein modification, and metabolism regulation. The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to low concentration Cu (20 µM Cu) and H2O2 stress, and 5 out of 24 were increased under low and high concentration Cu (100 µM Cu) but unaffected under H2O2 stress, which hint Cu ions can regulate Cu-binding proteins accumulation by H2O2 or no H2O2 pathway to cope with excess Cu in cell. The change pattern of these Cu-binding proteins and their function analysis warrant to further study the roles of Cu ions in these Cu-binding proteins of plant cells.

High-Throughput Sequencing Reveals H2O2 Stress-Associated MicroRNAs and a Potential Regulatory Network in Brachypodium distachyon Seedlings.

Oxidative stress in plants can be triggered by many environmental stress factors, such as drought and salinity. Brachypodium distachyon is a model organism for the study of biofuel plants and crops, such as wheat. Although recent studies have found many oxidative stress response-related proteins, the mechanism of microRNA (miRNA)-mediated oxidative stress response is still unclear. Using next generation high-throughput sequencing technology, the small RNAs were sequenced from the model plant B. distachyon 21 (Bd21) under H2O2 stress and normal growth conditions. In total, 144 known B. distachyon miRNAs and 221 potential new miRNAs were identified. Further analysis of potential new miRNAs suggested that 36 could be clustered into known miRNA families, while the remaining 185 were identified as B. distachyon-specific new miRNAs. Differential analysis of miRNAs from the normal and H2O2 stress libraries identified 31 known and 30 new H2O2 stress responsive miRNAs. The expression patterns of seven representative miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which produced results consistent with those of the deep sequencing method. Moreover, we also performed RT-qPCR analysis to verify the expression levels of 13 target genes and the cleavage site of 5 target genes by known or novel miRNAs were validated experimentally by 5' RACE. Additionally, a miRNA-mediated gene regulatory network for H2O2 stress response was constructed. Our study identifies a set of H2O2-responsive miRNAs and their target genes and reveals the mechanism of oxidative stress response and defense at the post-transcriptional regulatory level.

Integrative proteome analysis of Brachypodium distachyon roots and leaves reveals a synergetic responsive network under H2O2 stress.

The plant oxidative stress response is vital for defense against various abiotic and biotic stresses. In this study, ultrastructural changes and the proteomic response to H2O2 stress in roots and leaves of the model plant Brachypodium distachyon were studied. Transmission electron microscopy (TEM) showed that the ultrastructural damage in roots was more serious than in leaves. Particularly, the ultrastructures of organelles and the nucleus in root tip cells were damaged, leading to the inhibition of normal biological activities of roots, which then spread throughout the plant. Based on two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS, 84 and 53 differentially accumulated protein (DAP) spots representing 75 and 45 unique proteins responsive to H2O2 stress in roots and leaves, respectively, were identified. These protein species were mainly involved in signal transduction, energy metabolism, redox homeostasis/stress defense, protein folding/degradation, and cell wall/cell structure. Interestingly, two 14-3-3 proteins (GF14-B and GF14-D) were identified as DAPs in both roots and leaves. Protein-protein interaction (PPI) analysis revealed a synergetic H2O2-responsive network.

Danhong injection protects cardiomyocytes against hypoxia/reoxygenation- and H2O2-induced injury by inhibiting mitochondrial permeability transition pore opening.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI), a Chinese medical product extracted from Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese), has been widely used for the treatment of ischemic heart disease, and clinical and experimental studies have demonstrated the protective effects against myocardial ischemia/reperfusion injury. Nevertheless, the underlying cellular mechanisms responsible for this protective effect are poorly understood. AIM OF THE STUDY: The present study aimed to examine the mechanism of DHI in regulating hypoxia/reoxygenation- and H2O2-induced cardiomyocytes injury. MATERIALS AND METHODS: Neonatal rat cardiomyocytes were subjected to hypoxia (9h)-reoxygenation (2h) or H2O2 (100 µM) in the presence or absence of DHI (2.5, 5, 10 µg/mL). Intracellular reactive oxygen species (ROS), cytosolic and mitochondrial Ca(2+) concentrations, mitochondrial membrane potential (ΔΨm) and mitochondrial permeability transition pore (mPTP) opening were monitored using CMH2DCFDA, Fluo-4 and rhod-2, JC-1 and calcein, respectively. Cell survival was evaluated using the 2-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and apoptosis was detected by Annexin V/propidium iodide (PI) staining. RESULTS: DHI improved cell survival following H/R and H2O2 injury and reduced H/R-induced cytochrome c release and apoptosis when compared with non-DHI treated cells. In addition, DHI attenuated H/R-induced ROS generation, H2O2-induced cytosolic and mitochondrial Ca(2+) overload, and cellular ROS generation when compared with H/R- and H2O2-only groups. Moreover, DHI significantly inhibited both mPTP opening and ΔΨm depolarization. CONCLUSION: These data demonstrate that the protective mechanism of DHI against H/R- and H2O2-induced injury is mediated by the inhibition of mPTP opening via mitigating Ca(2+) overload and ROS generation.