Changes in Soybean Global Gene Expression after Application of Lipo-Chitooligosaccharide from Bradyrhizobium japonicum under Sub-Optimal Temperature

Lipo-chitooligosaccharides (LCOs), signal compounds produced by N2-fixing rhizobacteria after isoflavone induction, initiate nodule formation in host legumes. Given LCOs' structural similarity to pathogen-response-eliciting chitin oligomers, foliar application of LCOs was tested for ability to induce stress-related genes under optimal growth conditions. In order to study the effects of LCO foliar spray under stressed conditions, soybean (Glycine max) seedlings grown at optimal temperature were transferred to sub-optimal temperature. After a 5-day acclimation period, the first trifoliate leaves were sprayed with 10−7 M LCO (NodBj-V (C18∶1, MeFuc)) purified from genistein-induced Bradyrhizobium japonicum culture, and harvested at 0 and 48 h following treatment. Microarray analysis was performed using Affymetrix GeneChip® Soybean Genome Arrays. Compared to the control at 48 h after LCO treatment, a total of 147 genes were differentially expressed as a result of LCO treatment, including a number of stress-related genes and transcription factors. In addition, during the 48 h time period following foliar spray application, over a thousand genes exhibited differential expression, including hundreds of those specific to the LCO-treated plants. Our results indicated that the dynamic soybean foliar transcriptome was highly responsive to LCO treatment. Quantitative real-time PCR (qPCR) validated the microarray data

In Silico Analysis of Single Nucleotide Polymorphism (SNPs) in Human β-Globin Gene

Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies- the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT) server we have searched for the SNPs, which showed that 200 (80%) non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40%) non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K), HbD (E→Q), HbE (E→K) and HbS (E→V). Atomic Non-Local Environment Assessment (ANOLEA), Yet Another Scientific Artificial Reality Application (YASARA), CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref) of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on β-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid residues were determined and secondary structures were observed for solvent accessibility to confirm the protein stability. The functional study in this investigation may be a good model for additional future studies

Tasco®, a Product of Ascophyllum nodosum, Imparts Thermal Stress Tolerance in Caenorhabditis elegans

Tasco®, a commercial product manufactured from the brown alga Ascophyllum nodosum, has been shown to impart thermal stress tolerance in animals. We investigated the physiological, biochemical and molecular bases of this induced thermal stress tolerance using the invertebrate animal model, Caenorhabiditis elegans. Tasco® water extract (TWE) at 300 μg/mL significantly enhanced thermal stress tolerance as well as extended the life span of C. elegans. The mean survival rate of the model animals under thermal stress (35 °C) treated with 300 μg/mL and 600 μg/mL TWE, respectively, was 68% and 71% higher than the control animals. However, the TWE treatments did not affect the nematode body length, fertility or the cellular localization of daf-16. On the contrary, TWE under thermal stress significantly increased the pharyngeal pumping rate in treated animals compared to the control. Treatment with TWE also showed differential protein expression profiles over control following 2D gel-electrophoresis analysis. Furthermore, TWE significantly altered the expression of at least 40 proteins under thermal stress; among these proteins 34 were up-regulated while six were down-regulated. Mass spectroscopy analysis of the proteins altered by TWE treatment revealed that these proteins were related to heat stress tolerance, energy metabolism and a muscle structure related protein. Among them heat shock proteins, superoxide dismutase, glutathione peroxidase, aldehyde dehydrogenase, saposin-like proteins 20, myosin regulatory light chain 1, cytochrome c oxidase RAS-like, GTP-binding protein RHO A, OS were significantly up-regulated, while eukaryotic translation initiation factor 5A-1 OS, 60S ribosomal protein L18 OS, peroxiredoxin protein 2 were down regulated by TWE treatment. These results were further validated by gene expression and reporter gene expression analyses. Overall results indicate that the water soluble components of Tasco® imparted thermal stress tolerance in the C. elegans by altering stress related biochemical pathways

Tasco®: A Product of Ascophyllum nodosum Enhances Immune Response of Caenorhabditis elegans Against Pseudomonas aeruginosa Infection

The effects of Tasco®, a product made from the brown seaweed (Ascophyllum nodosum) were tested for the ability to protect Caenorhabditis elegans against Pseudomonas aeruginosa infection. A water extract of Tasco® (TWE) reduced P. aeruginosa inflicted mortality in the nematode. The TWE, at a concentration of 300 µg/mL, offered the maximum protection and induced the expression of innate immune response genes viz.; zk6.7 (Lypases), lys-1 (Lysozyme), spp-1 (Saponin like protein), f28d1.3 (Thaumatin like protein), t20g5.7 (Matridin SK domain protein), abf-1 (Antibacterial protein) and f38a1.5 (Lectin family protein). Further, TWE treatment also affected a number of virulence components of the P. aeuroginosa and reduced its secreted virulence factors such as lipase, proteases and toxic metabolites; hydrogen cyanide and pyocyanin. Decreased virulence factors were associated with a significant reduction in expression of regulatory genes involved in quorum sensing, lasI, lasR, rhlI and rhlR. In conclusion, the TWE-treatment protected the C. elegans against P. aeruginosa infection by a combination of effects on the innate immunity of the worms and direct effects on the bacterial quorum sensing and virulence factors

Signal compounds involved with plant perception and response to microbes alter plant physiological activities and growth of crop plants

Recent preliminary data have suggested that microbe-to-plant signals, and plant internal signals elicited by microbial signals, affect aspects of plant physiology, development and growth. The reported research investigated the responses of plants to signal compounds of microbial and plant origin, such as lipo-chitooligosaccharides (LCOs - signal molecules in rhizobia-legume associations), chitin and chitosan (present in fungal cell walls), and phenolic compounds (salicylic acid, acetylsalicylic acid and gentisic acid - internal signals in plants, often affected by signals from microbes). Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) are key enzymes of the phenylpropanoid pathway. Oligomers of chitin and chitosan increased the activities of both PAL and TAL in soybean leaves. The degree of increase was dependent on oligomer chain length and time after treatment. LCO [Nod Bj V (C18:1 , MeFuc)] was isolated from Bradyrhizobium japonicum strain 532C. When Arabidopsis thaliana plants were grown for two weeks on agar containing this LCO (10-8M) or chitin pentamer (10-4 M), they had greater root length, root diameter, root surface area and number of root tips than control plants. Chitosan (tetramer and pentamer) did not have this effect. Chitin and chitosan were also tested for effects on corn and soybean photosynthetic rates and growth. High molecular weight chitosan generally reduced photosynthetic rates, but did not reduce the growth of corn or soybean. However, foliar application of 10-6 M LCO to corn leaves increased photosynthetic rates (up to 36%). Foliar application of lumichrome (10-5 and 10-6 M), a breakdown product of riboflavin produced by some rhizosphere bacteria, to corn (C4 plant) and soybean (C3 plant) increased photosynthetic rates (up to 6%). Foliar application of lumichrome (10-5 M) increased soybean leaf area and shoot dry weight. Foliar application of SA, acetyl salicylic acid (ASA) and gentisic acid (G

Changes in Soybean Global Gene Expression after Application of Lipo-Chitooligosaccharide from Bradyrhizobium japonicum under Sub-Optimal Temperature

Lipo-chitooligosaccharides (LCOs), signal compounds produced by N₂-fixing rhizobacteria after isoflavone induction, initiate nodule formation in host legumes. Given LCOs' structural similarity to pathogen-response-eliciting chitin oligomers, foliar application of LCOs was tested for ability to induce stress-related genes under optimal growth conditions. In order to study the effects of LCO foliar spray under stressed conditions, soybean (Glycine max) seedlings grown at optimal temperature were transferred to sub-optimal temperature. After a 5-day acclimation period, the first trifoliate leaves were sprayed with 10⁻⁷ M LCO (NodBj-V (C[18∶1], MeFuc)) purified from genistein-induced Bradyrhizobium japonicum culture, and harvested at 0 and 48 h following treatment. Microarray analysis was performed using Affymetrix GeneChip® Soybean Genome Arrays. Compared to the control at 48 h after LCO treatment, a total of 147 genes were differentially expressed as a result of LCO treatment, including a number of stress-related genes and transcription factors. In addition, during the 48 h time period following foliar spray application, over a thousand genes exhibited differential expression, including hundreds of those specific to the LCO-treated plants. Our results indicated that the dynamic soybean foliar transcriptome was highly responsive to LCO treatment. Quantitative real-time PCR (qPCR) validated the microarray data

The four gene lists of interest in the microarray experiment, generated from four pair-wise comparisons (or contrasts) of gene expression profiles.

<p>1) gene list 1 is the collection of genes differentially expressed between the dH2O control and 10−7 M LCO-treated plants, for leaves harvested 0 h after foliar spray; 2) gene list 2 is a list of genes differentially expressed between the dH₂O control and 10⁻⁷ M LCO-treated plants, for leaves harvested 48 h after foliar spray; 3) gene list 3 includes all the differentially expressed genes of the dH₂O) control plants during the 48 h time period after foliar spray; 4) gene list 4 consists of genes in the 10⁻⁷ M LCO-treated plants that were differentially expressed during the 48 h time period after spray treatment.</p

Comparison and correlation between results obtained through qPCR and microarrray, in terms of fold change and p-value (or adjusted p-value).

<p>Correlation coefficients of logarithm-scaled fold change derived from qPCR and microarray were calculated in order to determine result repeatability by the two different quantification approaches. Statistically significant results at p<0.05 are marked with asterisk. As no differentially expressed gene was detected by microarray in contrast 1, nor did qPCR yield any statistically significant results existed between the fold change results derived from both methods. In contast 2, a high level of correlation (R² = 0.9) was found in the fold change values derived from qPCR and microarray, and 6 of the 7 genes (marked with asterisk) were regarded as statistically significant by both quantification methods.</p

Heat maps of known genes in gene list 2 showing up-regulated genes.

<p>A heat map depicts the gene expression data of all replicates in a color scheme: red color represents up-regulation and green represents down-regulation; higher color brightness indicates a greater magnitude of differential expression and vice versa. Ideally, the same group of samples should have similar colors.</p