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1.
Pol J Microbiol ; 68(4): 527-539, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880896

RESUMO

Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.


Assuntos
Microalgas/isolamento & purificação , Clorófitas/classificação , Clorófitas/genética , Diatomáceas/classificação , Diatomáceas/genética , Diatomáceas/isolamento & purificação , Água Doce/análise , Ilhas , Microalgas/classificação , Microalgas/genética , Filogenia , Análise de Sequência de DNA
2.
Front Plant Sci ; 9: 1848, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619416

RESUMO

Cyanobacterial 2-Cys peroxiredoxin (thioredoxin peroxidase, TPX) comprises a family of thiol antioxidant enzymes critically involved in cell survival under oxidative stress. In our previous study, a putative TPX was identified using a proteomics analysis of rice (Oryza sativa L. japonica, OsTPX) seedlings exposed to oxidative stress. This OsTPX gene is structurally similar to the Synechococcus elongatus TPX gene in the highly conserved redox-active disulfide bridge (Cys114, Cys236) and other highly conserved regions. In the present study, the OsTPX gene was cloned into rice plants and S. elongatus PCC 7942 strain to study hydrogen peroxide (H2O2) stress responses. The OsTPX gene expression was confirmed using semi-quantitative RT-PCR and western blot analysis. The OsTPX gene expression increased growth under oxidative stress by decreasing reactive oxygen species and malondialdehyde level. Additionally, the OsTPX gene expression in S. elongatus PCC 7942 (OT) strain exhibited a reduced loss of chlorophyll and enhanced photosynthesis efficiency under H2O2 stress, thereby increasing biomass yields twofold compared with that of the control wild type (WT) strain. Furthermore, redox balance, ion homeostasis, molecular chaperone, and photosynthetic systems showed upregulation of some genes in the OT strain than in the WT strain by RNA-Seq analysis. Thus, OsTPX gene expression enhances oxidative stress tolerance by increasing cell defense regulatory networks through the cellular redox homeostasis in the rice plants and S. elongatus PCC 7942.

3.
Biotechnol Lett ; 39(10): 1499-1507, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28667417

RESUMO

OBJECTIVES: To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H2O2, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR). RESULTS: Under H2O2 stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H2O2 level decreased. In addition, under H2O2 stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains. CONCLUSION: By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H2O2 stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.


Assuntos
Mostardeira/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Synechococcus/crescimento & desenvolvimento , Ácido Ascórbico/metabolismo , Biomassa , Clorofila/metabolismo , Clonagem Molecular , Ácido Desidroascórbico , Peróxido de Hidrogênio/metabolismo , Mostardeira/genética , Estresse Oxidativo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Synechococcus/genética
4.
J Plant Physiol ; 215: 39-47, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28527337

RESUMO

Reactive oxygen species, which increase under various environmental stresses, have deleterious effects on plants. An important antioxidant, glutathione, is used to detoxify reactive oxygen species in plant cells and is mainly produced by two enzymes: gamma-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the glutathione synthetase gene (OsGS) in rice, we generated four independent transgenic rice plants (TG1-TG4) that overexpressed OsGS under the control of the constitutively expressed OsCc1 promoter. When grown under natural paddy field conditions, the TG rice plants exhibited greater growth development, higher chlorophyll content, and higher GSH/GSSH ratios than control wild-type (WT) rice plants. Subsequently, the TG rice plants enhanced redox homeostasis by preventing hydroperoxide-mediated membrane damage, which improved their adaptation to environmental stresses. As a result, TG rice plants improved rice grain yield and total biomass following increases in panicle number and number of spikelets per panicle, despite differences in climate during the cultivation periods of 2014 and 2015. Overall, our results indicate that OsGS overexpression improved redox homeostasis by enhancing the glutathione pool, which resulted in greater tolerance to environmental stresses in the paddy fields.


Assuntos
Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia
6.
Sci Rep ; 6: 33903, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27652777

RESUMO

Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63-80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.

7.
PLoS One ; 11(7): e0158841, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27392090

RESUMO

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascorbate recycling. To examine whether heterologous expression of MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.


Assuntos
Expressão Gênica , NADH NADPH Oxirredutases , Organismos Geneticamente Modificados , Oryza/genética , Proteínas de Plantas , Saccharomyces cerevisiae , Estresse Fisiológico , Etanol/metabolismo , NADH NADPH Oxirredutases/biossíntese , NADH NADPH Oxirredutases/genética , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Oryza/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
8.
Biochem Biophys Res Commun ; 477(3): 395-400, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27329814

RESUMO

The cyanobacterial aldehyde deformylating oxygenase (cADO) is a key enzyme that catalyzes the unusual deformylation of aliphatic aldehydes for alkane biosynthesis and can be applied to the production of biofuel in vitro and in vivo. In this study, we determined crystal structures of two ADOs from Limnothrix sp. KNUA012 (LiADO) and Oscillatoria sp. KNUA011 (OsADO). The structures of LiADO and OsADO resembled those of typical cADOs, consisting of eight α-helices found in ferritin-like di-iron proteins. However, structural comparisons revealed that while the LiADO active site was vacant of iron and substrates, the OsADO active site was fully occupied, containing both a coordinated metal ion and substrate. Previous reports indicated that helix 5 is capable of adopting two distinct conformations depending upon the existence of bound iron. We observed that helix 5 of OsADO with an iron bound in the active site presented as a long helix, whereas helix 5 of LiADO, which lacked iron in the active site, presented two conformations (one long and two short helices), indicating that an equilibrium exists between the two states in solution. Furthermore, acquisition of a structure having a fully occupied active site is unique in the absence of higher iron concentrations as compared with other cADO structures, wherein low affinity for iron complicates the acquisition of crystal structures with bound iron. An in-depth analysis of the ADO apo-enzyme, the enzyme with substrate bound, and the enzyme with both iron and substrate bound provided novel insight into substrate-binding modes in the absence of a coordinated metal ion and suggested a separate two-step binding mechanism for substrate and iron co-factors. Moreover, our results provided a comprehensive structural basis for conformational changes induced by binding of the substrate and co-factor.


Assuntos
Aldeídos/metabolismo , Cianobactérias/enzimologia , Oxigenases/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Oxigenases/genética , Oxigenases/metabolismo , Homologia de Sequência de Aminoácidos
9.
Sci Rep ; 6: 19498, 2016 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-26775680

RESUMO

Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate (dehydroascorbate, DHA). As a result, DHAR regenerates a pool of reduced ascorbate and detoxifies reactive oxygen species (ROS). In previous experiments involving transgenic rice, we observed that overexpression of DHAR enhanced grain yield and biomass. Since the structure of DHAR is not available, the enzymatic mechanism is not well-understood and remains poorly characterized. To elucidate the molecular basis of DHAR catalysis, we determined the crystal structures of DHAR from Oryza sativa L. japonica (OsDHAR) in the native, ascorbate-bound, and GSH-bound forms and refined their resolutions to 1.9, 1.7, and 1.7 Å, respectively. These complex structures provide the first information regarding the location of the ascorbate and GSH binding sites and their interacting residues. The location of the ascorbate-binding site overlaps with the GSH-binding site, suggesting a ping-pong kinetic mechanism for electron transfer at the common Cys20 active site. Our structural information and mutagenesis data provide useful insights into the reaction mechanism of OsDHAR against ROS-induced oxidative stress in rice.


Assuntos
Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Oryza/metabolismo , Oxirredução , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Expressão Gênica , Glutationa/química , Glutationa/genética , Redes e Vias Metabólicas , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico
10.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 9): 1244-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25195901

RESUMO

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate-glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Šresolution and contained one molecule in the asymmetric unit (the Matthews coefficient VM is 1.98 Å(3) Da(-1), corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model.


Assuntos
Cristalografia por Raios X/métodos , NADH NADPH Oxirredutases/química , Oryza/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/isolamento & purificação
11.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 6): 781-5, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24915093

RESUMO

Dehydroascorbate reductase from Oryza sativa L. japonica (OsDHAR), a key enzyme in the regeneration of vitamin C, maintains reduced pools of ascorbic acid to detoxify reactive oxygen species. In previous studies, the overexpression of OsDHAR in transgenic rice increased grain yield and biomass as well as the amount of ascorbate, suggesting that ascorbate levels are directly associated with crop production in rice. Hence, it has been speculated that the increased level of antioxidants generated by OsDHAR protects rice from oxidative damage and increases the yield of rice grains. However, the crystal structure and detailed mechanisms of this important enzyme need to be further elucidated. In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 M potassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Šon beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space group P21 with unit-cell parameters a=47.03, b=48.38, c=51.83 Å, ß=107.41°.


Assuntos
Oryza/enzimologia , Oxirredutases/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
12.
Mol Cells ; 35(3): 210-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23512334

RESUMO

Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and alcohol dehydrogenase), antioxidant enzymes (thioredoxin 3, thioredoxin reductase, and porin), and molecular chaperones and its cofactors (Hsp104, Hsp82, Hsp60, Hsp42, Hsp30, Hsp26, Cpr1, Sti1, and Zpr1) are upregulated during fermentation, in comparison to S. cerevisiae S288C (S288C). Expression of glyceraldehyde-3-phosphate dehydrogenase increased significantly in KNU5377 cells. In addition, cellular hydroperoxide and protein oxidation, particularly lipid peroxidation of triosephosphate isomerase, was lower in KNU5377 than in S288C. Thus, KNU5377 activates various cell rescue proteins through transcription activators, improving tolerance and increasing alcohol yield by rapidly responding to fermentation stress through redox homeostasis and proteostasis.


Assuntos
Etanol/metabolismo , Fermentação , Resposta ao Choque Térmico , Saccharomyces cerevisiae/metabolismo , Adaptação Fisiológica , Biocombustíveis , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Viabilidade Microbiana , Oxirredução , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulação para Cima
13.
Planta ; 237(6): 1613-25, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23519921

RESUMO

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintains redox pools of ascorbate (AsA) by recycling oxidized AsA to reduced AsA. To investigate whether DHAR affects rice yield under normal environmental conditions, cDNA-encoding DHAR (OsDHAR1) was isolated from rice and used to develop OsDHAR1-overexpressing transgenic rice plants, under the regulation of a maize ubiquitin promoter. Incorporation and expression of the transgene in transgenic rice plants was confirmed by genomic polymerase chain reaction (PCR), semi-quantitative reverse transcription PCR (RT-PCR), western blot, and enzyme activity. The expression levels were at least twofold higher in transgenic (TG) rice plants than in control wild-type (WT) rice plants. In addition, OsDHAR1-overexpression in seven-independent homologous transgenic plants, as compared to WT plants, increased photosynthetic capacity and antioxidant enzyme activities under paddy field conditions, which led to an improved AsA pool and redox homeostasis. Furthermore, OsDHAR1 overexpression significantly improved grain yield and biomass due to the increase of culm and root weights and to enhance panicle and spikelet numbers in the same seven independent TG rice plants during the farming season (2010 and 2011) in South Korea. The OsDHAR protein contained the redox-active site (Cys20), as well as the conserved GSH-binding region, GSH-binding motif, glutathione-S-transferase (GST) N-terminal domain, C-terminal domain interface, and GST C-terminal domain. Therefore, our results indicate that OsDHAR1 overexpression, capable of functioning in AsA recycling, and protein folding increases environmental adaptation to paddy field conditions by the improving AsA pool and redox homeostasis, which enhances rice grain yield and biomass.


Assuntos
Biomassa , Citosol/enzimologia , Oryza/enzimologia , Oryza/genética , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Agricultura , Sequência de Aminoácidos , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homeostase , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Fotossíntese/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Sementes/enzimologia , Alinhamento de Sequência
14.
Appl Microbiol Biotechnol ; 97(20): 8997-9009, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23377791

RESUMO

We investigated Arctic plants to determine if they have a specific mechanism enabling them to adapt to extreme environments because they are subject to such conditions throughout their life cycles. Among the cell defense systems of the Arctic mouse-ear chickweed Cerastium arcticum, we identified a stress-responsive dehydrin gene CaDHN that belongs to the SK5 subclass and contains conserved regions with one S segment at the N-terminus and five K segments from the N-terminus to the C-terminus. To investigate the molecular properties of CaDHN, the yeast Saccharomyces was transformed with CaDHN. CaDHN-expressing transgenic yeast (TG) cells recovered more rapidly from challenge with exogenous stimuli, including oxidants (hydrogen peroxide, menadione, and tert-butyl hydroperoxide), high salinity, freezing and thawing, and metal (Zn(2+)), than wild-type (WT) cells. TG cells were sensitive to copper, cobalt, and sodium dodecyl sulfate. In addition, the cell survival of TG cells was higher than that of WT cells when cells at the mid-log and stationary stages were exposed to increased ethanol concentrations. There was a significant difference in cultures that have an ethanol content >16 %. During glucose-based batch fermentation at generally used (30 °C) and low (18 °C) temperatures, TG cells produced a higher alcohol concentration through improved cell survival. Specifically, the final alcohol concentrations were 13.3 and 13.2 % in TG cells during fermentation at 30 and 18 °C, respectively, whereas they were 10.2 and 9.4 %, respectively, in WT cells under the same fermentation conditions. An in vitro assay revealed that purified CaDHN acted as a reactive oxygen species scavenger by neutralizing H2O2 and a chaperone by preventing high temperature-mediated catalase inactivation. Taken together, our results show that CaDHN expression in transgenic yeast confers tolerance to various abiotic stresses by improving redox homeostasis and enhances fermentation capacity, especially at low temperatures (18 °C).


Assuntos
Caryophyllaceae/genética , Engenharia Genética , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/fisiologia , Regiões Árticas , Etanol/metabolismo , Fermentação , Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Estresse Fisiológico
15.
J Plant Physiol ; 170(6): 610-8, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23294545

RESUMO

Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10µM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions.


Assuntos
Epistasia Genética , Regulação da Expressão Gênica de Plantas , Glutamato-Cisteína Ligase/genética , Oryza/fisiologia , Proteínas de Plantas/genética , Aclimatação , Agrobacterium/genética , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa/genética , Glutationa/metabolismo , Homeostase , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Tolerância ao Sal , Estresse Fisiológico
16.
Appl Microbiol Biotechnol ; 97(8): 3519-33, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23053072

RESUMO

Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 °C) and at the general cultivation temperature (30 °C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g(-1)) and 21 % (0.12 g g(-1)) during fermentation at 40 and 30 °C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature.


Assuntos
Oryza/enzimologia , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Clonagem Molecular , Etanol/metabolismo , Fermentação , Expressão Gênica , Glucose/metabolismo , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Oryza/genética , Peroxirredoxinas/genética , Folhas de Planta/química , Folhas de Planta/enzimologia , Proteoma/análise , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética
17.
J Microbiol Biotechnol ; 22(11): 1557-67, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23124348

RESUMO

Glutathione reductase (GR, E.C. 1.6.4.2) is an important enzyme that reduces glutathione disulfide (GSSG) to a sulfydryl form (GSH) in the presence of an NADPH-dependent system. This is a critical antioxidant mechanism. Owing to the significance of GR, this enzyme has been examined in a number of animals, plants, and microbes. We performed a study to evaluate the molecular properties of GR (OsGR) from rice (Oryza sativa). To determine whether heterologous expression of OsGR can reduce the deleterious effects of unfavorable abiotic conditions, we constructed a transgenic Saccharomyces cerevisiae strain expressing the GR gene cloned into the yeast expression vector p426GPD. OsGR expression was confirmed by a semiquantitative reverse transcriptase polymerase chain reaction (semiquantitative RT-PCR) assay, Western-blotting, and a test for enzyme activity. OsGR expression increased the ability of the yeast cells to adapt and recover from H2O2-induced oxidative stress and various stimuli including heat shock and exposure to menadione, heavy metals (iron, zinc, copper, and cadmium), sodium dodecyl sulfate (SDS), ethanol, and sulfuric acid. However, augmented OsGR expression did not affect the yeast fermentation capacity owing to reduction of OsGR by multiple factors produced during the fermentation process. These results suggest that ectopic OsGR expression conferred acquired tolerance by improving cellular homeostasis and resistance against different stresses in the genetically modified yeast strain, but did not affect fermentation ability.


Assuntos
Expressão Gênica , Glutationa Redutase/metabolismo , Oryza/enzimologia , Estresse Oxidativo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Engenharia Genética , Glutationa Redutase/genética , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética
18.
World J Microbiol Biotechnol ; 28(5): 1901-15, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22806013

RESUMO

To determine whether the exogenous expression of glutathione reductase (GR) from Brassica rapa subsp. pekinensis (BrGR) can reduce the deleterious effects of unfavorable conditions, we constructed a transgenic Saccharomyces cerevisiae strain bearing the GR gene cloned into the yeast expression vector, pVTU260. BrGR expression was confirmed by semi reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, immunoblotting analysis and an enzyme assay. Ectopic BrGR-expression improved cellular glutathione (GSH) homeostasis after higher GSH accumulation in the transgenic yeast than in the wild-type yeast under H(2)O(2)-induced oxidative stress. The BrGR-expressing yeast strain induced the activation of metabolic enzymes (Hxt, G6PDH, GAPDH and Ald), antioxidant systems (Gpx, Trx2, Trx3, Trr1, Tsa1 and porin) and molecular chaperones (Hsp104, Hsp90, Hsp70, Hsp42, Hsp26, Grp, Sti1 and Zpr1), which led to lower oxidative protein damage after a reduction in the level of cellular ROS in the BrGR-expressing yeast strain exposed to H(2)O(2) than in the wild-type yeast strain. BrGR-expression increased the ability to adapt and recover from H(2)O(2)-induced oxidative stress and various stressors, including heat shock, menadione, tert-butyl hydroperoxide, heavy metals, sodium dodecyl sulfate, ethanol and NaCl, but did not affect fermentation capacity. These results suggest that ectopic BrGR expression confers acquired tolerance by improving proteostasis and redox homeostasis through co-activation of various cell rescue proteins against ROS-induced oxidative stress in yeast cells.


Assuntos
Brassica rapa/enzimologia , Glutationa Redutase/metabolismo , Estresse Oxidativo , Saccharomyces cerevisiae/fisiologia , Brassica rapa/genética , Clonagem Molecular , Fermentação , Expressão Gênica , Vetores Genéticos , Glutationa/metabolismo , Glutationa Redutase/genética , Peróxido de Hidrogênio/toxicidade , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Organismos Geneticamente Modificados/fisiologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
19.
Mol Cells ; 33(3): 285-93, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22382682

RESUMO

Abscisic acid stress ripening (ASR1) protein is a small hydrophilic, low molecular weight, and stress-specific plant protein. The gene coding region of ASR1 protein, which is induced under high salinity in rice (Oryza sativa Ilmi), was cloned into a yeast expression vector pVTU260 and transformed into yeast cells. Heterologous expression of ASR1 protein in transgenic yeast cells improved tolerance to abiotic stresses including hydrogen peroxide (H(2)O(2)), high salinity (NaCl), heat shock, menadione, copper sulfate, sulfuric acid, lactic acid, salicylic acid, and also high concentration of ethanol. In particular, the expression of metabolic enzymes (Fba1p, Pgk1p, Eno2p, Tpi1p, and Adh1p), antioxidant enzyme (Ahp1p), molecular chaperone (Ssb1p), and pyrimidine biosynthesis-related enzyme (Ura1p) was up-regulated in the transgenic yeast cells under oxidative stress when compared with wild-type cells. All of these enzymes contribute to an alleviated redox state to H2O2-induced oxidative stress. In the in vitro assay, the purified ASR1 protein was able to scavenge ROS by converting H(2)O(2) to H(2)O. Taken together, these results suggest that the ASR1 protein could function as an effective ROS scavenger and its expression could enhance acquired tolerance of ROS-induced oxidative stress through induction of various cell rescue proteins in yeast cells.


Assuntos
Chaperonas Moleculares/biossíntese , Oryza/metabolismo , Estresse Oxidativo , Proteínas de Plantas/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Catalase/genética , Catalase/metabolismo , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Oxidantes/farmacologia , Oxirredução , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal
20.
J Microbiol Biotechnol ; 20(6): 974-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20622494

RESUMO

Cyclophilins contain the conserved activity of cis-trans peptidyl-prolyl isomerase that is implicated in protein folding and function as molecular chaperones. The yeast cyclophilin A gene (cpr1) was subcloned to the prokaryotic expression vector pKM260. It was found that the expression of Cpr1 drastically increased the cell viability of E. coli BL21 in the presence of abiotic stress conditions, such as cadmium, copper, hydrogen peroxide, heat, and SDS. Thus, this study illustrates the importance of Cpr1 as a molecular chaperone that improved cellular stress responses when E. coli cells were exposed to adverse conditions, and it also shows the possibility of increasing the stability of E. coli strains utilized for the production of recombinant proteins.


Assuntos
Ciclofilina A/genética , Escherichia coli/fisiologia , Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ciclofilina A/metabolismo , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico
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