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.

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 H 2 O 2 -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.

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.

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.

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.

Abscisic acid-, stress-, and ripening-induced (ASR) genes are involved in responding to abiotic stresses, but their precise roles in enhancing grain yield under stress conditions remain to be determined. We cloned a rice (Oryza sativa) ASR gene, OsASR1, and characterized its function in rice plants. OsASR1 expression was induced by abscisic acid (ABA), salt, and drought treatments. Transgenic rice plants overexpressing OsASR1 displayed improved water regulation under salt and drought stresses, which was associated with osmolyte accumulation, improved modulation of stomatal closure, and reduced transpiration rates. OsASR1-overexpressing plants were hypersensitive to exogenous ABA and accumulated higher endogenous ABA levels under salt and drought stresses, indicating that OsASR1 is a positive regulator of the ABA signaling pathway. The growth of OsASR1-overexpressing plants was superior to that of wild-type (WT) plants under paddy field conditions when irrigation was withheld, likely due to improved modulation of stomatal closure via modified ABA signaling. The transgenic plants had higher grain yields than WT plants for four consecutive generations. We conclude that OsASR1 has a crucial role in ABA-mediated regulation of stomatal closure to conserve water under salt-and drought-stress conditions, and OsASR1 overexpression can enhance salinity and drought tolerance, resulting in improved crop yields.

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 H(2)O(2), menadione, and heavy metal (CdCl(2), ZnCl(2) and AlCl(2))-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 H(2)O(2) 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.

This study aims to characterize the potential of three strains of microalgal species ( Chlorella sorokiniana KNUA114 and KNUA122; C. vulgaris KNUA104) for use as feedstock, based on their fatty acid compositions. Each strain was molecularly identified using four marker genes ( ITS , SSU , rbcL , and tufA ) and phylogenetically characterized. C. sorokiniana and C. vulgaris collected from Ulleung Island, South Korea, were homologous with other known species groups. Samples' fatty acid components were measured using GC/MS analysis in growth temperatures of 10 °C, 25 °C, and 35 °C. The growth rate of C. sorokiniana strains was higher than that of C. vulgaris under high-temperature conditions, confirming the potential industrial applicability of the former as feedstock material. Additionally, saturated fatty acid contents and productivities increased as biological resources of the C. sorokiniana strains were higher than those of C. vulgaris under high light intensity and temperature conditions. These results suggest that the fatty acid components of C. sorokiniana strains may potentially be used as biological resources (e.g., feedstock).