Il-Sup Kim

Rice ASR1 protein with reactive oxygen species scavenging and chaperone-like activities enhances acquired tolerance to abiotic stresses in Saccharomyces cerevisiae.

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 (H2O2), 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 H2O2 to H2O. 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.

Homologous expression of cytosolic dehydroascorbate reductase increases grain yield and biomass under paddy field conditions in transgenic rice (Oryza sativa L. japonica)

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.

Stress response of plant H+-PPase-expressing transgenic Escherichia coli and Saccharomyces cerevisiae: a potentially useful mechanism for the development of stress-tolerant organisms

The simple proton-translocating inorganic pyrophosphatase (H+-PPase) found in plants and protists is an evolutionally conserved, essential enzyme that catalyzes the hydrolysis of pyrophosphate (PPi). Little is known about the functional contribution of H+-PPase to the cellular response to abiotic stresses, except its high salinity and drought stress. To investigate the role of H+-PPase during response to cellular stress, we isolated the cDNA of Arabidopsis thaliana H+-PPase (AVP1) and Oryza sativa H+-PPase (OVP1) and constructed transgenic Saccharomyces cerevisiae and Escherichia coli lines that express AVP1 and OVP1. In S. cerevisiae, the expression of a chimeric derivative of the AVP1 and OVP1 alleviated the phenotype associated with ipp2-deficient cells in the presence of high salinity (NaCl) and metal stressors (Cd, Mn, and Zn). In E. coli, AVP1 and OVP1 overexpression conferred enhanced tolerance to abiotic stresses, including heat shock and H2O2, as well as NaCl, Cd, Mn, Zn, Ca, and Al. Interestingly, AVP1 and OVP1 overexpression resulted in hypersensitivity to menadione and cobalt. These results demonstrate the cellular capacity of AVP1- and OVP1-expressing transgenic yeast and E. coli in response to physiological, abiotic stresses. Moreover, our results suggest new ways of engineering stress-tolerant plants that are capable of responding to climate change. Here, we provide an outline of an experimental system to examine the alternative roles of plant H+-PPase.

Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli

Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H2O2, menadione, and heavy metal (CdCl2, ZnCl2 and AlCl2)-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H2O2 stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.

Adaptive stress response to menadione-induced oxidative stress in Saccharomyces cerevisiae KNU5377

The molecular mechanisms involved in the ability of yeast cells to adapt and respond to oxidative stress are of great interest to the pharmaceutical, medical, food, and fermentation industries. In this study, we investigated the time-dependent, cellular redox homeostasis ability to adapt to menadione-induced oxidative stress, using biochemical and proteomic approaches in Saccharomyces cerevisiae KNU5377. Time-dependent cell viability was inversely proportional to endogenous amounts of ROS measured by a fluorescence assay with 2′,7′-dichlorofluorescin diacetate (DCFHDA), and was hypersensitive when cells were exposed to the compound for 60 min. Morphological changes, protein oxidation and lipid peroxidation were also observed. To overcome the unfavorable conditions due to the presence of menadione, yeast cells activated a variety of cell rescue proteins including antioxidant enzymes, molecular chaperones, energy-generating metabolic enzymes, and antioxidant molecules such as trehalose. Thus, these results show that menadione causes ROS generation and high accumulation of cellular ROS levels, which affects cell viability and cell morphology and there is a correlation between resistance to menadione and the high induction of cell rescue proteins after cells enter into this physiological state, which provides a clue about the complex and dynamic stress response in yeast cells.

Saccharomyces cerevisiae KNU5377 Stress Response during High-Temperature Ethanol Fermentation

Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to costeffective 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.

A Cyclophilin A CPR1 Overexpression Enhances Stress Acquisition in Saccharomyces cerevisiae

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidanr enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.

Structural understanding of the recycling of oxidized ascorbate by dehydroascorbate reductase (OsDHAR) from Oryza sativa L. japonica

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.

Decarbonylated cyclophilin A Cpr1 protein protects Saccharomyces cerevisiae KNU5377Y when exposed to stress induced by menadione

Cyclophilins are conserved cis–trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.

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 Brassicajuncea 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.