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°.