Overexpression of Rat Neurons Nitric Oxide Synthase in Rice Enhances Drought and Salt Tolerance

Nitric oxide (NO) has been shown to play an important role in the plant response to biotic and abiotic stresses in Arabidopsis mutants with lower or higher levels of endogenous NO. The exogenous application of NO donors or scavengers has also suggested an important role for NO in plant defense against environmental stress. In this study, rice plants under drought and high salinity conditions showed increased nitric oxide synthase (NOS) activity and NO levels. Overexpression of rat neuronal NO synthase (nNOS) in rice increased both NOS activity and NO accumulation, resulting in improved tolerance of the transgenic plants to both drought and salt stresses. nNOS-overexpressing plants exhibited stronger water-holding capability, higher proline accumulation, less lipid peroxidation and reduced electrolyte leakage under drought and salt conditions than wild rice. Moreover, nNOS-overexpressing plants accumulated less H₂O₂, due to the observed up-regulation of OsCATA, OsCATB and OsPOX1. In agreement, the activities of CAT and POX were higher in transgenic rice than wild type. Additionally, the expression of six tested stress-responsive genes including OsDREB2A, OsDREB2B, OsSNAC1, OsSNAC2, OsLEA3 and OsRD29A, in nNOS-overexpressing plants was higher than that in the wild type under drought and high salinity conditions. Taken together, our results suggest that nNOS overexpression suppresses the stress-enhanced electrolyte leakage, lipid peroxidation and H₂O₂ accumulation, and promotes proline accumulation and the expression of stress-responsive genes under stress conditions, thereby promoting increased tolerance to drought and salt stresses.     

Expression of Indica rice <Emphasis Type="Italic">OsBADH1</Emphasis> gene under salinity stress in transgenic tobacco

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T₂ progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.     

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64)

To overcome the salinity‐induced loss of crop yield, a salinity‐tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T₁ and T₂ sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T₂ transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H₂O₂ production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity‐induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.     

Expression of Arabidopsis DREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced Arabidopsis AtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Overexpression of a ItICE1 gene from Isatis tinctoria enhances cold tolerance in rice

ItICE1, a ICE1-like gene, was isolated from a cDNA library from cold-treated woad (Isatis tinctoria L.) tissues. Expression analysis revealed that the ItICE1 gene was expressed constitutively and was predominant in the leaves of woad seedlings and that its mRNA accumulation was altered by salt stress and abscisic acid application, but not by dehydration and cold stresses. The transgenic rice lines overexpressing ItICE1 showed no growth retardation under normal growth conditions as well as enhanced tolerance to cold stress. Physiological assays showed that ItICE1 not only increased the accumulation of free proline and chlorophyll in transgenic rice lines under cold stress, but also reduced malondialdehyde content and electrolyte leakage. The analysis of gene expression in transgenic rice lines indicated that the maize ubiquitin promoter could respond to cold stress and upregulate ItICE1 gene expression level under its control. Under cold stress conditions, transgenic lines had a remarkably increased expression of OsDREB1A, J013078A14, 001-125-G03, 001-023-B08 and J023042N13 compared to wild-type plants (P < 0.05), implying that ItICE1 functions in the CBF/DREB1 cold-response pathway. These results demonstrate that ItICE1 plays an important regulatory role in the improvement of tolerance to cold stress in rice and is potentially useful for improving the cold tolerance of other plants.     

MsSAMS,a cold stress-responsive gene,provides resistance to environmental stress in T2-generation transgenic plants

The SAMS (S-adenosylmethionine synthetase) gene is known to play an important role in the mechanism of cold resistance, as overexpression of this gene results in phenotypic changes in T₁-generation transgenic plants. Accordingly, this study was conducted to test the expression of the MsSAMS gene in T₂-generation transgenic plants and to investigate the resistance of these plants and the function of the transgene in response to various environmental stresses. For the morphological analysis of T₂-generation transgenic plants overexpressing the MsSAMS gene, observations using scanning electron microscopy (SEM) were performed. T₂-generation transgenic plants were obtained by planting a total of 5 lines, and their characteristics were tested by comparisons with those of the control. SEM revealed that the thickest leaves were produced by the T6 transgenic line—161.24?±?8.05 µm. The number of stomata ranged from 20.00?±?2.65 to 34.00?±?1.00 in the T₂-generation transgenic plants, but the control had more stomata. Resistance to various factors, such as low temperature, drought, and oxidative stress, in the T₂-generation transgenic plants was also confirmed. Under cold-stress conditions, the T6 transgenic line presented the lowest value (22.73%) of ion leakage, and under drought-stress conditions, compared with the control, the transgenic lines presented lower ion leakage after being treated with various concentrations of mannitol. Even under oxidative-stress conditions, the T₂-generation transgenic plants presented ion leakage levels that were 32.91?±?4.24 to 48.33?±?3.54% lower than those of the control after treatment with various concentrations of methyl viologen. Regarding SAMS enzyme activity, as the duration of cold treatment increased, the activity in the transgenic plants tended to decrease and then increase. During 48 h of cold treatment, the control showed a decrease in SAM content, while the T₂-generation transgenic plants presented an increase in SAM content, from 13.58?±?1.04 to 22.75?±?1.95 mg protein/g FW. The results suggest that the MsSAMS gene may be important to the mechanisms of resistance to oxidative and drought stresses in addition to its previously known association with cold resistance. Based on these results, it was suggested that the MsSAMS gene, whose expression is induced by cold stress, can serve as a marker of various responses to environmental stresses, because resistance to cold damage and various environmental stresses are stably inherited in the T₂ generation.