Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor <Emphasis Type="Italic">TERF2</Emphasis>/<Emphasis Type="Italic">LeERF2</Emphasis> is modulated by ethylene biosynthesis

Increasing numbers of investigations indicate that ethylene response factor (ERF) proteins play important roles in plant stress responses via interacting with GCC box and/dehydration-responsive element/C-repeat to modulate expression of downstream genes, but the detailed regulatory mechanism is not well elucidated. Revealing the modulation pathway of ERF proteins in response to stresses is vital. Previously, we showed that tomato ERF protein TERF2/LeERF2 is ethylene inducible, and ethylene production is suppressed in antisense TERF2/LeERF2 tomatoes, suggesting that TERF2/LeERF2 functions as a positive regulator in ethylene biosynthesis. In this paper, we report that regulation of TERF2/LeERF2 in ethylene biosynthesis is associated with enhanced freezing tolerance of tobacco and tomato. Analysis of gene expression showed that cold slowly induces expression of TERF2/LeERF2 in tomato, implying that TERF2/LeERF2 may be involved in cold response through ethylene modulation. To test the hypothesis, we first observed that overexpressing TERF2/LeERF2 tobaccos not only enhances freezing tolerance via activating expression of cold-related genes, but also significantly reduces electrolyte leakage. In addition, with treatment of ethylene biosynthesis inhibitor or ethylene receptor antagonist, we then showed that blockage of ethylene biosynthesis or the ethylene signaling pathway decreases freezing tolerance of overexpressing TERF2/LeERF2 tobaccos. Moreover, the results from tomatoes showed that overexpressing TERF2/LeERF2 tomatoes enhances while antisense TERF2/LeERF2 transgenic lines decreases freezing tolerance, and application of ethylene precursor 1-aminocyclopropane-1-carboxylic acid restored freezing tolerance of antisense lines. Therefore our results establish that TERF2/LeERF2 enhances freezing tolerance of plants through ethylene biosynthesis and the ethylene signaling pathway.     

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice. Shumei Gao, Haiwen Zhang and Yun Tian contributed equally to this work.     

Overexpression of <Emphasis Type="Italic">SlERF1</Emphasis> tomato gene encoding an ERF-type transcription activator enhances salt tolerance

Ethylene-responsive factors (ERFs) play an important role in plant responses to stresses. For this study, we obtained sense- and antisense-SlERF1 transgenic tomato plants to analyze the function of the SlERF1 gene in tomato plants. Overexpression of SlERF1 in tomato plants enhanced salt tolerance during tomato seedling root development. In addition, tomato seedlings overexpressing SlERF1 showed the higher relative water content and lower MDA content and electrolyte leakage; they accumulated more free proline and soluble sugars, as compared with wild-type and antisense-SlERF1 transgenic tomato plants under salt stress. Moreover, SlERF1 activated the expression of stress-related genes, including LEA, P5CS, DREB3-1, and ltpg2 in tomato plants under salt stress. Thus, SlERF1 played a positive role in the salt tolerance of tomato plants.     

Overexpression of the trehalose-6-phosphate synthase gene <Emphasis Type="Italic">OsTPS1</Emphasis> enhances abiotic stress tolerance in rice

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.     

Role of the Rice BAHD Acyltransferase Gene OsAt10 in Plant Cold Stress Tolerance

The rice (Oryza sativa L.) BAHD acyltransferase gene OsAt10 affects growth and metabolism of cells and regulates cell response to environmental stress. However, influence of the OsAt10 gene on low-temperature stress tolerance has not been evaluated in plant cells. Here, cell suspension cultures of plant species Arabidopsis (Arabidopsis thaliana L.), cotton (Gossypium hirsutum L.), white pine (Pinus strobus L.), and rice (Oryza sativa L.) were used to generate transgenic cell lines via Agrobacterium tumefaciens-mediated genetic transformation to examine the effects of OsAt10 on cold stress tolerance. OsAt10 transgenic cell lines of A. thaliana, G. hirsutum, P. strobus, and O. sativa were confirmed by molecular analyses including Southern blotting ND northern blotting, following by physiological and biochemical analyses under cold stress. The experimental results demonstrated that growth rate, cell viability, lipid peroxidation, ion leakage, antioxidative enzyme activity, polyamines level, and cell morphology were changed in transgenic cells under cold stress, compared to the controls. In transgenic A. thaliana cells, overexpression of the OsAt10 gene increases expression of polyamines biosynthesis genes under cold stress. In transgenic A. thaliana plants, overexpression of the OsAt10 gene increased cold stress tolerance by regulating expression of stress marker genes, TBARS content, ion leakage level, antioxidant enzymes activity, and polyamines content, indicating that the OsAt10 gene could be economically important for improving low-temperature stress tolerance in plants.

     

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.     

Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system

The aim of this research was to generate selectable marker-free transgenic tomato plants with improved tolerance to abiotic stress. An estradiol-induced site-specific DNA excision of a selectable marker gene using the Cre/loxP DNA recombination system was employed to develop transgenic tomato constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase gene from Arabidopsis thaliana. Transgenic tomato plants containing a selectable marker were also produced as controls. The expression of AtIpk2β conferred improved resistance to drought, cold and oxidative stress in both sets of transgenic tomato plants. These results demonstrate the feasibility of using this Cre/loxP-based marker elimination strategy to generate marker-free transgenic crops with improved stress tolerance.     

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

<Emphasis Type="Italic">osa</Emphasis>-<Emphasis Type="Italic">MIR393</Emphasis>: a salinity- and alkaline stress-related microRNA gene

Salinity and alkalinity are the two main environmental factors that limit rice production. Better understanding of the mechanisms responsible for salinity and alkaline stress tolerance would allow researchers to modify rice to increase its resistance to salinity and alkaline stress. MicroRNAs (miRNAs) are ~21-nucleotide RNAs that are ubiquitous regulators of gene expression in eukaryotic organisms. Some miRNAs acts as an important endogenous regulator in plant responses to abiotic stressors. miR393 is a conservative miRNA family that occurs in a variety of different plants. The two members of the miR393 family found in rice are named osa-MIR393 and osa-MIR393b. We found that the osa-MIR393 expression level changed under salinity and alkaline stress, whereas that of osa-MIR393b did not. Target genes of osa-MIR393 were predicted, and some of these putative targets are abiotic related genes. Furthermore, we generated transgenic rice and Arabidopsis thaliana that over-expressed osa-MIR393, and the phenotype analysis showed that these transgenic plants were more sensitive to salt and alkali treatment compared to wild-type plants. These results illustrate that over-expression of osa-MIR393 can negatively regulate rice salt-alkali stress tolerance.