Elucidation of genomic organizations of transgenic soybean plants through de novo genome assembly with short paired-end reads.

Elucidation of the genomic organizations of transgene insertion sites is essential for the genetic studies of transgenic plants. Herein, we establish an analysis pipeline that identifies the transgene insertion sites as well as the presence of vector backbones, through de novo genome assembly with high-throughput sequencing data in two transgenic soybean lines, AtYUCCA6-#5 and 35S-UGT72E3/2-#7. Sequencing data of approximately 28× and 29× genome coverages for each line generated by high-throughput sequencing were de novo assembled. The databases generated from the de novo assembled sequences were used to search contigs that contained putative insertion sites and their flanking sequences (integration sites) of transgene fragments using transgenic vector sequences as queries. The predicted integration site sequences, which are located at three annotated genes that might regulate plant development or confer disease resistance, were then confirmed by local alignment against the soybean reference genome and PCR amplification. As results, we revealed the precise transgene-flanking sequences and sequence rearrangements at insertion sites in both the transgenic lines, as well as the aberrant insertion of a transgene fragment. Consequently, relative to experimental or enrichment technologies, our approach is straightforward and time-effective, providing an alternative method for the identification of insertion sites in transgenic plants.

Metabolomic changes in grains of well-watered and drought-stressed transgenic rice.

BACKGROUND: Drought induces a number of physiological and biochemical responses in cereals. This study was designed to examine the metabolite changes in grains of drought-tolerant transgenic rice (Oryza sativa L.) that overexpresses AtCYP78A7 encoding cytochrome P450 protein using proton nuclear magnetic resonance ((1)H-NMR) and gas chromatography/mass spectrometry. RESULTS: Principal component analysis showed that the (1)H-NMR-based profile was clearly separated by soil water status of well-watered and water-deficit. A discrimination of metabolites between transgenic and non-transgenic grains appeared under both watering regimes. Variations in the levels of amino acids and sugars led to the discrimination of metabolites among genotypes. In particular, drought significantly enhanced the levels of γ-aminobutyric acid (GABA, 244.6%), fructose (155.7%), glucose (211.0%), glycerol (57.2%), glycine (65.8%) and aminoethanol (192.4%) in the transgenic grains compared with the non-transgenic control grains. CONCLUSION: These changes in amounts of metabolites may assist in improving drought tolerance in transgenic rice by playing crucial roles in stress-responsive pathways including GABA biosynthesis, sucrose metabolism and antioxidant defenses.

Compositional differences in hybrids between protoporphyrinogen IX oxidase (PPO)-inhibiting herbicide-resistant transgenic rice and weedy rice accessions.

We characterized the metabolites in grains of transgenic protoporphyrinogen IX oxidase-inhibiting herbicide-resistant rice and weedy accessions using GC-MS and examined whether the chemical composition of their hybrids differed from that of the parents. We found that the metabolite profiles of transgenic rice and weedy rice were clearly separated. Although the metabolite profiles of F2 progeny were partially separated from their parents, zygosity did not affect the profiles. The F2 progeny had similar or intermediate levels of most major nutritional components compared with their parents. However, levels of galactopyranose, trehalose, xylofuranose, mannitol, and benzoic acid were higher in the F2 progeny. Some fatty acids and organic acids also showed prominent quantitative differences between the F2 progeny and the parents. Changes in the metabolite levels of transgenic crop-weed hybrids compared to their parents might influence not only the ecological consequences of the hybrids, but also the nutritional quality and food safety.

Performance of hybrids between abiotic stress-tolerant transgenic rice and its weedy relatives under water-stressed conditions.

Gene transfer from transgenic crops to their weedy relatives may introduce undesired ecological consequences that can increase the fitness and invasiveness of weedy populations. Here, we examined the rate of gene flow from abiotic stress-tolerant transgenic rice that over-express AtCYP78A7, a gene encoding cytochrome P450 protein, to six weedy rice accessions and compared the phenotypic performance and drought tolerance of their hybrids over generations. The rate of transgene flow from AtCYP78A7-overexpressing transgenic to weedy rice varied between 0% and 0.0396%. F1 hybrids containing AtCYP78A7 were significantly taller and heavier, but the percentage of ripened grains, grain numbers and weight per plant were significantly lower than their transgenic and weedy parents. The homozygous and hemizygous F2 progeny showed higher tolerance to drought stress than the nullizygous F2 progeny, as indicated by leaf rolling scores. Shoot growth of nullizygous F3 progeny was significantly greater than weedy rice under water-deficient conditions in a rainout shelter, however, that of homozygous F3 progeny was similar to weedy rice, indicating the cost of continuous expression of transgene. Our findings imply that gene flow from AtCYP78A7-overexpressing transgenic to weedy rice might increase drought tolerance as shown in the pot experiment, however, increased fitness under stressed conditions in the field were not observed for hybrid progeny containing transgenes.

Salinity affects metabolomic profiles of different trophic levels in a food chain.

Salinization is one of the most important abiotic stressors in an ecosystem. To examine how exposing a host plant to excess salt affects the consequent performance and metabolism of insects in a food chain, we determined the life history traits and the metabolite profiles in rice (Oryza sativa), the herbivore Sitobion avenae, and its predator Harmonia axyridis. When compared with performance under normal (non-stressed) conditions, exposing plants to 50mM NaCl significantly delayed the timing of development for S. avenae fed on rice and H. axyridis and also reduced the body mass of the latter. Our GC-MS-based analysis revealed clear differences in metabolite profiles between trophic levels or treatment conditions. Salinity apparently increased the levels of main components in rice, but decreased levels of major components in S. avenae and H. axyridis. In addition, 16 metabolites showed salinity-related contrasts in this trophic interaction for our rice-S. avenae-H. axyridis system. Salinity impeded the accumulation of metabolites, especially several sugars, amino acids, organic acids, and fatty acids in both insects, a response that was possibly associated with the negative impacts on their growth and reproduction under stress conditions.

Comparative analysis of chemical compositions between non-transgenic soybean seeds and those from plants over-expressing AtJMT, the gene for jasmonic acid carboxyl methyltransferase.

Transgenic overexpression of the Arabidopsis gene for jasmonic acid carboxyl methyltransferase (AtJMT) is involved in regulating jasmonate-related plant responses. To examine its role in the compositional profile of soybean (Glycine max), we compared the seeds from field-grown plants that over-express AtJMT with those of the non-transgenic, wild-type (WT) counterpart. Our analysis of chemical compositions included proximates, amino acids, fatty acids, isoflavones, and antinutrients. Overexpression of AtJMT in the seeds resulted in decreased amounts of tryptophan, palmitic acid, linolenic acid, and stachyose, but increased levels of gadoleic acid and genistein. In particular, seeds from the transgenic soybeans contained 120.0-130.5% more genistein and 60.5-82.1% less stachyose than the WT. A separate evaluation of ingredient values showed that all were within the reference ranges reported for commercially available soybeans, thereby demonstrating the substantial equivalence of these transgenic and non-transgenic seeds.

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.

Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions.

Phosphoinositide 3-kinase activity leads to silica-induced NF-kappaB activation through interacting with tyrosine-phosphorylated I(kappa)B-alpha and contributing to tyrosine phosphorylation of p65 NF-kappaB.

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-kappaB activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85alpha subunit of PI3-kinase interacted with tyrosine-phosphorylated I(kappa)B-alpha in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-kappaB activation. The inhibition of NF-KB activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-kappaB p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-kappaB activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-kappaB activation through interaction with tyrosine-phosphorylated I(kappa)B-alpha and contributing to tyrosine phosphorylation of p65 NF-kappaB.

Time course for inhibition of lipopolysaccharide-induced lung injury by genistein: relationship to alteration in nuclear factor-kappaB activity and inflammatory agents.

OBJECTIVE: This study determined the time course for inhibition of lipopolysaccharide-induced acute lung injury following a single dose of genistein. In addition, the study investigated whether a multiple dosing schedule with genistein retained the inhibitory effects on acute lung injury, nuclear factor-kappaB activation, and production of nuclear factor-kappaB-dependent inflammatory agents, such as matrix metalloproteinase-9 and nitric oxide. DESIGN: Prospective, randomized, laboratory study. SETTING: Experimental laboratory at a university. SUBJECTS: Rats weighing 280-300 g. INTERVENTIONS: Saline or lipopolysaccharide (6 mg/kg of body weight) administered intratracheally with a single dose of genistein (50 mg/kg) or a multiple dosing schedule with genistein (16 mg/kg every 6 hrs for 2 days with lipopolysaccharide treatment at 24 hrs after the first administration of genistein). MEASUREMENTS AND MAIN RESULTS: A 2-hr pretreatment with genistein (a single dose) inhibited biochemical lung injury variables as well as neutrophil infiltration with a maximal inhibition at 4 hrs after lipopolysaccharide treatment. These inhibitory effects of genistein declined with time and were no longer significant by 14-24 hrs after lipopolysaccharide treatment. The multiple dosing schedule with genistein retained significant inhibitory effects on biochemical lung injury variables and the number of neutrophils in the bronchoalveolar lavage fluid at 24 hrs after lipopolysaccharide treatment compared with a single pretreatment with genistein. The multiple dosing schedule with genistein also enhanced the inhibition of induced nuclear factor-kappaB activity as well as matrix metalloproteinase-9 activity and nitric oxide production at 24 hrs after lipopolysaccharide treatment. CONCLUSIONS: This study reports the time course of the inhibitory effects of a single genistein pretreatment on acute lung injury with the maximal effects at 4 hrs after lipopolysaccharide treatment. However, a multiple dosing schedule with genistein retained the inhibitory effect on acute lung injury at 24 hrs after lipopolysaccharide treatment. The mechanisms by which genistein exerts an inhibitory effect on acute lung injury may involve the suppression of nuclear factor-kappaB activation, matrix metalloproteinase-9 activity, and NO production.