Over-Expression of Dehydroascorbate Reductase Improves Salt Tolerance, Environmental Adaptability and Productivity in Oryza sativa.

Abiotic stress induces reactive oxygen species (ROS) generation in plants, and high ROS levels can cause partial or severe oxidative damage to cellular components that regulate the redox status. Here, we developed salt-tolerant transgenic rice plants that overexpressed the dehydroascorbate reductase gene (OsDHAR1) under the control of a stress-inducible sweet potato promoter (SWPA2). OsDHAR1-expressing transgenic plants exhibited improved environmental adaptability compared to wild-type plants, owing to enhanced ascorbate levels, redox homeostasis, photosynthetic ability, and membrane stability through cross-activation of ascorbate-glutathione cycle enzymes under paddy-field conditions, which enhanced various agronomic traits, including root development, panicle number, spikelet number per panicle, and total grain yield. dhar2-knockdown plants were susceptible to salt stress, and owing to poor seed maturation, exhibited reduced biomass (root growth) and grain yield under paddy field conditions. Microarray revealed that transgenic plants highly expressed genes associated with cell growth, plant growth, leaf senescence, root development, ROS and heavy metal detoxification systems, lipid metabolism, isoflavone and ascorbate recycling, and photosynthesis. We identified the genetic source of functional genomics-based molecular breeding in crop plants and provided new insights into the physiological processes underlying environmental adaptability, which will enable improvement of stress tolerance and crop species productivity in response to climate change.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice.

Drought and salinity are major important factors that restrain growth and productivity of rice. In plants, many really interesting new gene (RING) finger proteins have been reported to enhance drought and salt tolerance. However, their mode of action and interacting substrates are largely unknown. Here, we identified a new small RING-H2 type E3 ligase OsRF1, which is involved in the ABA and stress responses of rice. OsRF1 transcripts were highly induced by ABA, salt, or drought treatment. Upregulation of OsRF1 in transgenic rice conferred drought and salt tolerance and increased endogenous ABA levels. Consistent with this, faster transcriptional activation of key ABA biosynthetic genes, ZEP, NCED3, and ABA4, was observed in OsRF1-OE plants compared with wild type in response to drought stress. Yeast two-hybrid assay, BiFC, and co-immunoprecipitation analysis identified clade A PP2C proteins as direct interacting partners with OsRF1. In vitro ubiquitination assay indicated that OsRF1 exhibited E3 ligase activity, and that it targeted OsPP2C09 protein for ubiquitination and degradation. Cell-free degradation assay further showed that the OsPP2C09 protein is more rapidly degraded by ABA in the OsRF1-OE rice than in the wild type. The combined results suggested that OsRF1 is a positive player of stress responses by modulating protein stability of clade A PP2C proteins, negative regulators of ABA signaling.

Enhanced glutathione content improves lateral root development and grain yield in rice plants.

KEY MESSAGE: Enhanced glutathione content improves lateral root development by positively regulating the transcripts of root development genes responsive to glutathione treatment, thereby increasing the overall productivity of rice plants. Glutathione is primarily known as a cellular antioxidant molecule, but its role in lateral root development in rice plants has not been elucidated. Here, we have investigated its role in lateral root development of rice Oryza sativa L. Exogenous glutathione (GSH) promoted both the number and length of lateral roots in rice, and the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) significantly reduced these parameters, compared to untreated plants. The inhibition by BSO was reversed with exogenous GSH. Transcript profiling by RNA-seq revealed that expression of the transcription factor genes DREB and ERF and the hormone-related genes AOS, LOX, JAZ, and SAUR were significantly downregulated in the BSO-treated plants and, in contrast, upregulated in plants treated with GSH and with GSH and BSO together. We generated OsGS-overexpressing transgenic plants in which the transgene is controlled by the abiotic-stress-inducible OsRab21 promoter to study the effect of endogenously increased GSH levels. In cold stress, transgenic rice plants enhanced stress tolerance and lateral root development by maintaining redox homeostasis and improving upregulating the expression of transcription factors and hormone-related genes involved in lateral root development. We observed improved root growth of OsGS-overexpressing plants in paddy fields compared to the wild-type controls. These traits may have alleviated transplanting stress during early growth in the field and accounted for the increased productivity. These results provide information and perspectives on the role of GSH in gene expression, lateral root development, and grain yield in rice.

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942.

An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the glutathione (GSH) pools, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, transgenic Synechococcus elongatus PCC 7942 (TA) was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the cyanobacterium to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type S. elongatus PCC 7942 (WT). Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress.

ASR Enhances Environmental Stress Tolerance and Improves Grain Yield by Modulating Stomatal Closure in Rice.

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.

Expression of OsTPX Gene Improves Cellular Redox Homeostasis and Photosynthesis Efficiency in Synechococcus elongatus PCC 7942.

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.

Improved stress tolerance and productivity in transgenic rice plants constitutively expressing the Oryza sativa glutathione synthetase OsGS under paddy field conditions.

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.

Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica.

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.

Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) from Oryza sativa L. japonica.

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°.