Enhancement of salt tolerance in transgenic rice expressing an Escherichia coli catalase gene, katE

Rice (Oryza sativa) is sensitive to salt stresses and cannot survive under low salt conditions, such as 50 mM NaCl. In an attempt to improve salt tolerance of rice, we introduced katE, a catalase gene of Escherichia coli, into japonica rice cultivar, Nipponbare. The resultant transgenic rice plants constitutively expressing katE were able to grow for more than 14 days in the presence of 250 mM NaCl, and were able to form flower and produce seeds in the presence of 100 mM NaCl. Catalase activity in the transgenic rice plants was 1.5- to 2.5-fold higher than non-transgenic rice plants. Our results clearly indicate that simple genetic modification of rice to express E. coli-derived catalase can efficiently increase its tolerance against salt stresses. The transformant presented here is one of the most salt-tolerant rice plants created by molecular breeding so far.     

Infrequent transposition of Ac in lettuce,Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R₁ plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2 promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.     

Improvement of freezing tolerance in tobacco plants expressing a cold-responsive and chloroplast-targeting protein WCOR15 of wheat

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.     

Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene

Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10⁴ fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30 mg g⁻¹ of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.     

Vesicular-arbuscular mycorrhizal infection in lettuce (Lactuca sativa) in relation to calcium supply

Summary Infection of lettuce roots (Lactuca sativa L.) by the vesicular-arbuscular mycorrhizal fungiGlomus caledonium andGlomus mosseae was dependent on the amount of calcium (supplied as CaCl₂·2H₂O or CaSO₄·2H₂O) in the nutrient solution; those plants growing at low calcium concentrations being poorly infected.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

<Emphasis Type="Italic">Arabidopsis rd29A::DREB1A</Emphasis> enhances freezing tolerance in transgenic potato

The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using plantlets. The lines were transgenic for the DREB1A gene under control of the rd29A promoter, both of which were derived from Arabidopsis thaliana. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv. Desiree). Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation, but freezing tolerance was not dependent on the number of insertions. Northern blot analysis using a DREB1A cDNA probe revealed high levels of DREB1A expression among the transgenic clones during the initial cold exposure at 4°C (after 2 h) and in the early stages of freezing (−20°C, 1–10 min). Furthermore, a linear correlation was detected between the level of expression and the phenotypic response for all lines except D138. Thus, in the case of potato, a significant increase in freezing tolerance was observed in vitro on a small scale following the introduction of rd29A::DREB1A. Additional testing will show whether this strategy can be used for tolerance breeding in potato and to increase the freezing tolerance of other agriculturally important crops. Babak Behnam and Akira Kikuchi equally contributed for this work.     

Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco

cDNA encoding betaine aldehyde dehydrogenase (BADH) from the halophyte Suaeda liaotungensis has been cloned, sequenced and expressed in tobacco (Nictiana tabacum 89). The full-length cDNA is 1506 base pairs (bp) long and encodes a 502 amino-acid polypeptide. The cDNA fragment coding for the mature enzyme was cloned into vector pCAMBIA-1301 for expression in tobacco. Southern blotting analysis showed that that the Badh gene was integrated into the genome of tobacco. Tobacco expressing BADH survived on MS medium containing 200 mM NaCl, whereas the untransformed plants turned yellow after about 20 d and died.     

High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3Aa gene from Bacillus thuringiensis ssp tenebrionis

Hybrid poplars (Populus tremula ×Populus tremuloides) have been genetically engineered viaAgrobacterium tumefaciens, to express a syntheticcry3Aa gene derived from the native Bacillusthuringiensis subsp. tenebrionis cry3Aa gene.The presence and the expression of the transgene have been verified in fourtransgenic poplar lines, using Southern, northern and western analyses. Thetransgenic poplar's toxicity towards the phytophagous beetleChrysomela tremulae (Coleoptera, Chrysomelidae) has beenassessed on six month-old greenhouse-grown selected plants in laboratoryconditions. Laboratory experiments consisted of feeding tests of fresh detachedleaves on C. tremulae at all developmental stages. Ourresults indicate that the transgenic poplar leaves, expressing a Cry3Aa proteinamount in a range of 0.05–0.0025% of total soluble protein, weredefinitely deleterious for C. tremulae, regardless of thedevelopmental stage.     

Heterologous expression of the mutated melon ethylene receptor gene Cm-ERS1/H70A produces stable sterility in transgenic lettuce (Lactuca sativa)

The mutated melon ethylene receptor gene Cm-ERS1/H70A was introduced into tobacco and induced stable sterility in transgenic lines. This gene contains a missense mutation that converts the His(70) residue to Ala in the melon ethylene receptor gene Cm-ERS1. To test the applicability of this inducible sterility system to other plants, lettuce (Lactuca sativa) was transformed with the gene using Agrobacterium, and putative transformants containing Cm-ERS1/H70A were obtained. Thirteen randomly selected putative transformants were grown in a growth room under constant conditions, and seven of the lines showed sterility or significantly reduced fertility. DNA gel blot analysis confirmed the integration of the Cm-ERS1/H70A gene into the genomes of the putative transformants, and RT-PCR and protein gel blot analysis confirmed the expression of Cm-ERS1/H70A mRNA and protein in all of the transformants. Five transformants showing sterility or reduced fertility when grown in a growth room under constant conditions were randomly selected to be grown in an open-air greenhouse under various environmental conditions. All five showed stable sterility under the various conditions. These results suggest that Cm-ERS1/H70A can induce sterility in heterologous transgenic plants.     

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana

Summary. Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana. Correspondence and reprints: Laboratório de Biotecnologia de Células Vegetais, ITQB, Apartado 127, Avenida da República (E.A.N.), 2781-901 Oeiras, Portugal.     

Development of insect-resistant transgenic indica rice with a synthetic cry1C* gene

Stemborers and leaffolders are two groups of lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, a cry1C* gene encoding Bacillus thuringiensis (Bt) δ-endotoxin was synthesized by codon optimization as the first step towards gene stacking in our resistance management strategy of transgenic rice. Agrobacterium-mediated transformation of this gene into Minghui 63 (Oryza sativa L.), an elite indica CMS restorer line, produced 120 independently transformed plants, 19 of which had a single-copy transgene. Preliminary screening of T₁ families of these 19 transformants in the field identified five lines showing a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Hybrids were produced by crossing these five lines with Zhenshan 97A, the male-sterile line for Shanyou 63, the most widely cultivated hybrid in China. These five lines and their hybrids were highly resistant to yellow stemborer (Tryporyza incertulas) as revealed by an insect bioassay. The content of Cry1C* protein varied considerably among the five lines as well as among the corresponding hybrids. T1c-19, a line showing the highest content of Cry1C* protein, and its hybrid were tested in the field for insect resistance and agronomic performance and found to be highly resistant to stemborers and leaffolders throughout the growth period, resulting in a significantly increased grain yield compared with the respective controls. These results indicate that T1c-19 can be used for production of insect-resistant hybrid rice and as a germplasm for gene stacking to produce rice plants with two toxins.