Prior metabolite extraction fully preserves RNAseq quality and enables integrative multi-'omics analysis of the liver metabolic response to viral infection.

Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-'omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV versus Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-'omics approach.

Changes in nucleus accumbens gene expression accompany sex-specific suppression of spontaneous physical activity in aromatase knockout mice.

Aromatase catalyzes conversion of testosterone to estradiol and is expressed in a variety of tissues, including the brain. Suppression of aromatase adversely affects metabolism and physical activity behavior, but mechanisms remain uncertain. The hypothesis tested herein was that whole body aromatase deletion would cause gene expression changes in the nucleus accumbens (NAc), a brain regulating motivated behaviors such as physical activity, which is suppressed with loss of estradiol. Metabolic and behavioral assessments were performed in male and female wild-type (WT) and aromatase knockout (ArKO) mice. NAc-specific differentially expressed genes (DEGs) were identified with RNAseq, and associations between the measured phenotypic traits were determined. Female ArKO mice had greater percent body fat, reduced spontaneous physical activity (SPA), consumed less energy, and had lower relative resting energy expenditure (REE) than WT females. Such differences were not observed in ArKO males. However, in both sexes, a top DEG was Pts, a gene encoding an enzyme necessary for catecholamine (e.g., dopamine) biosynthesis. In comparing male and female WT mice, top DEGs were related to sexual development/fertility, immune regulation, obesity, dopamine signaling, and circadian regulation. SPA correlated strongly with Per3, a gene regulating circadian function, thermoregulation, and metabolism (r = -0.64, P = .002), which also correlated with adiposity (r = 0.54, P = .01). In conclusion, aromatase ablation leads to gene expression changes in NAc, which may in turn result in reduced SPA and related metabolic abnormalities. These findings may have significance to post-menopausal women and those treated with an aromatase inhibitor.

Sexual dimorphism in brain transcriptomes of Amami spiny rats (Tokudaia osimensis): a rodent species where males lack the Y chromosome.

BACKGROUND: Brain sexual differentiation is sculpted by precise coordination of steroid hormones during development. Programming of several brain regions in males depends upon aromatase conversion of testosterone to estrogen. However, it is not clear the direct contribution that Y chromosome associated genes, especially sex-determining region Y (Sry), might exert on brain sexual differentiation in therian mammals. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (T. tokunoshimensis) lack a Y chromosome/Sry, and these individuals possess an XO chromosome system in both sexes. Both Tokudaia species are highly endangered. To assess the neural transcriptome profile in male and female Amami spiny rats, RNA was isolated from brain samples of adult male and female spiny rats that had died accidentally and used for RNAseq analyses. RESULTS: RNAseq analyses confirmed that several genes and individual transcripts were differentially expressed between males and females. In males, seminal vesicle secretory protein 5 (Svs5) and cytochrome P450 1B1 (Cyp1b1) genes were significantly elevated compared to females, whereas serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) was upregulated in females. Many individual transcripts elevated in males included those encoding for zinc finger proteins, e.g. zinc finger protein X-linked (Zfx). CONCLUSIONS: This method successfully identified several genes and transcripts that showed expression differences in the brain of adult male and female Amami spiny rat. The functional significance of these findings, especially differential expression of transcripts encoding zinc finger proteins, in this unusual rodent species remains to be determined.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.

Transcriptomic alterations in the brain of painted turtles (Chrysemys picta) developmentally exposed to bisphenol A or ethinyl estradiol.

Developmental exposure of turtles and other reptiles to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), can stimulate partial to full gonadal sex-reversal in males. We have also recently shown that in ovo exposure to either EDC can induce similar sex-dependent behavioral changes typified by improved spatial learning and memory or possibly feminized brain responses. Observed behavioral changes are presumed to be due to BPA- and EE-induced brain transcriptomic alterations during development. To test this hypothesis, we treated painted turtles (Chrysemys picta) at developmental stage 17, incubated at 26°C (male-inducing temperature), with 1) BPA (1 ng/µl), 2) EE (4 ng/µl), or 3) vehicle ethanol (control group). Ten months after hatching and completion of the behavioral tests, juvenile turtles were euthanized, brains were collected and frozen in liquid nitrogen, and RNA was isolated for RNA-Seq analysis. Turtles exposed to BPA clustered separately from EE-exposed and control individuals. More transcripts and gene pathways were altered in BPA vs. EE individuals. The one transcript upregulated in both BPA- and EE-exposed individuals was the mitochondrial-associated gene, ND5, which is involved in oxidative phosphorylation. Early exposure of turtles to BPA increases transcripts linked with ribosomal and mitochondrial functions, especially bioenergetics, which has been previously linked with improved cognitive performance. In summary, even though both BPA and EE resulted in similar behavioral alterations, they diverge in the pattern of neural transcript alterations with early BPA significantly upregulating several genes involved in oxidative phosphorylation, mitochondrial activity, and ribosomal function, which could enhance cognitive performance.

Consumption of a high-fat diet alters the seminal fluid and gut microbiomes in male mice.

Our prior work showed that a novel microbiome resides in the seminal vesicles of wild-type and oestrogen receptor α (Esr1) knock-out mice and is impacted by the presence of functional Esr1 genes. The seminal fluid microbiome (SFM) may influence the health and reproductive status of the male, along with that of his partner and offspring. A high-fat diet (HFD) alters metabolites and other factors within seminal fluid and might affect the SFM. Adult (~15 weeks old) male mice were placed for 4 weeks on a control or high-fat diet and seminal fluid and fecal samples were collected, bacterial DNA isolated and subjected to 16s rRNA sequencing. Corynebacterium spp. were elevated in the seminal fluid of HFD males; however, Acinetobacter johnsonii, Streptophyta, Ammoniphilus spp., Bacillus spp. and Propionibacterium acnes were increased in control males. Rikenellaceae was more abundant in the fecal samples from HFD males. However, Bacteroides ovatus and another Bacteroides species, Bilophila, Sutterella spp., Parabacteroides, Bifidobacterium longum, Akkermansia muciniphila and Desulfovibrio spp. were greater in control males. Thus, short-term consumption of a HFD influences the seminal fluid and fecal microbiomes, which may have important health consequence for males and developmental origins of health and disease effects in resulting offspring.

Short-Stalked Prosthecomicrobium hirschii Cells Have a Caulobacter-Like Cell Cycle.

UNLABELLED: The dimorphic alphaproteobacterium Prosthecomicrobium hirschii has both short-stalked and long-stalked morphotypes. Notably, these morphologies do not arise from transitions in a cell cycle. Instead, the maternal cell morphology is typically reproduced in daughter cells, which results in microcolonies of a single cell type. In this work, we further characterized the short-stalked cells and found that these cells have a Caulobacter-like life cycle in which cell division leads to the generation of two morphologically distinct daughter cells. Using a microfluidic device and total internal reflection fluorescence (TIRF) microscopy, we observed that motile short-stalked cells attach to a surface by means of a polar adhesin. Cells attached at their poles elongate and ultimately release motile daughter cells. Robust biofilm growth occurs in the microfluidic device, enabling the collection of synchronous motile cells and downstream analysis of cell growth and attachment. Analysis of a draft P. hirschii genome sequence indicates the presence of CtrA-dependent cell cycle regulation. This characterization of P. hirschii will enable future studies on the mechanisms underlying complex morphologies and polymorphic cell cycles. IMPORTANCE: Bacterial cell shape plays a critical role in regulating important behaviors, such as attachment to surfaces, motility, predation, and cellular differentiation; however, most studies on these behaviors focus on bacteria with relatively simple morphologies, such as rods and spheres. Notably, complex morphologies abound throughout the bacteria, with striking examples, such as P. hirschii, found within the stalked Alphaproteobacteria. P. hirschii is an outstanding candidate for studies of complex morphology generation and polymorphic cell cycles. Here, the cell cycle and genome of P. hirschii are characterized. This work sets the stage for future studies of the impact of complex cell shapes on bacterial behaviors.

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.

Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

Characterization of the rat developmental liver transcriptome.

Gene regulation and transcriptome studies have been enabled by the development of RNA-Seq applications for high-throughput sequencing platforms. Next generation sequencing is remarkably efficient and avoids many issues inherent in hybridization-based microarray methodologies including the exon-specific dependence of probe design. Biologically relevant transcripts including messenger and regulatory RNAs may now be quantified and annotated regardless of whether they have previously been observed. We used RNA-Seq to investigate global patterns of gene expression in early developing rat liver. Liver samples from timed-pregnant Lewis rats were collected at six fetal and neonatal stages [embryonic day (E)14, E16, E18, E20, postnatal day (P)1, P7], transcripts were sequenced using an Illumina HiSeq 2000, and data analysis was performed with the Tuxedo software suite. Genes and isoforms differing in abundance were queried for enrichment within functionally related gene groups using the Functional Annotation Tool of the DAVID Bioinformatics Database. While hematopoietic gene expression is initiated by E14, hepatocyte maturation is a gradual process involving clusters of genes responsible for response to nutrients and enzymes responsible for glycolysis and fatty acid catabolism. Following birth, a large cluster of differentially abundant genes was enriched for mitochondrial gene expression and cholesterol synthesis indicating that by 1 wk of age, the liver is engaged in lipid sensing and bile production. Clustering results for differentially abundant genes and isoforms were similar with the greatest difference for the E14/E16 comparison. Finally, a bioinformatic approach was used to annotate 1,307 novel liver transcripts assembled from sequences that aligned to intergenic regions of the rat genome.

Genome-wide mapping of alternative splicing in Arabidopsis thaliana.

Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least approximately 42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC(+) isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC(+) isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC(+) and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression.

MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana.

MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (approximately 10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.

Transcriptome and DNA methylome analyses reveal underlying mechanisms for the racial disparity in uterine fibroids.

Uterine fibroids (leiomyomas) affect Black women disproportionately compared with women of other races and ethnicities in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids, and we examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified 4 clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, von Willibrands factor (VWF), was significantly hypomethylated in both myometrial samples from Black women and in all fibroids at 2 CpG probes that are near a putative enhancer site and that are correlated with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.

Computational and analytical framework for small RNA profiling by high-throughput sequencing.

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

Network discovery pipeline elucidates conserved time-of-day-specific cis-regulatory modules.

Correct daily phasing of transcription confers an adaptive advantage to almost all organisms, including higher plants. In this study, we describe a hypothesis-driven network discovery pipeline that identifies biologically relevant patterns in genome-scale data. To demonstrate its utility, we analyzed a comprehensive matrix of time courses interrogating the nuclear transcriptome of Arabidopsis thaliana plants grown under different thermocycles, photocycles, and circadian conditions. We show that 89% of Arabidopsis transcripts cycle in at least one condition and that most genes have peak expression at a particular time of day, which shifts depending on the environment. Thermocycles alone can drive at least half of all transcripts critical for synchronizing internal processes such as cell cycle and protein synthesis. We identified at least three distinct transcription modules controlling phase-specific expression, including a new midnight specific module, PBX/TBX/SBX. We validated the network discovery pipeline, as well as the midnight specific module, by demonstrating that the PBX element was sufficient to drive diurnal and circadian condition-dependent expression. Moreover, we show that the three transcription modules are conserved across Arabidopsis, poplar, and rice. These results confirm the complex interplay between thermocycles, photocycles, and the circadian clock on the daily transcription program, and provide a comprehensive view of the conserved genomic targets for a transcriptional network key to successful adaptation.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6.

BACKGROUND: Pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. RESULTS: We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system. CONCLUSIONS: The improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

Genome-wide profiling and analysis of Arabidopsis siRNAs.

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences.

Update of ASRP: the Arabidopsis Small RNA Project database.

Development of the Arabidopsis Small RNA Project (ASRP) Database, which provides information and tools for the analysis of microRNA, endogenous siRNA and other small RNA-related features, has been driven by the introduction of high-throughput sequencing technology. To accommodate the demands of increased data, numerous improvements and updates have been made to ASRP, including new ways to access data, more efficient algorithms for handling data, and increased integration with community-wide resources. New search and visualization tools have also been developed to improve access to small RNA classes and their targets. ASRP is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu/db/.

Draft Genome Sequence of Bradyrhizobium sp. Strain LVM 105, a Nitrogen-Fixing Symbiont of Chamaecrista fasciculata (Michx.) Greene.

Here, we present the draft genome sequence of Bradyrhizobium sp. strain LVM 105, a soil bacterium that forms nitrogen-fixing nodules on the roots of partridge pea. The genome size was 8,386,213 bp, with a GC content of 63.8%. The draft assembly contained 97 scaffolds with an N 50 value of 415,026 bp.

A seed germination transcriptomic study contrasting two soybean genotypes that differ in terms of their tolerance to the deleterious impacts of elevated temperatures during seed fill.

OBJECTIVE: Soybean seed development is negatively impacted by elevated temperatures during seed fill, which can decrease seed quality and economic value. Prior germplasm screens identified an exotic landrace able to maintain ~ 95% seed germination under stress conditions that reduce germination dramatically (> 50%) for typical soybean seeds. Seed transcriptomic analysis was performed for two soybean lines (a heat-tolerant landrace and a typical high-yielding adapted line) for dry, mature seed, 6-h imbibed seed and germinated seed. Seeds were produced in two environments: a typical Midwestern field and a heat stressed field located in the Midsouth soybean production region. RESULTS: Transcriptomic analysis revealed 23-30K expressed genes in each seed tissue sample, and differentially expressed genes (DEGs) with ≥ twofold gene expression differences (at q-value < 0.05) comprised ~ 5-44% of expressed genes. Gene ontology (GO) enrichment analysis on DEGs revealed enrichment in heat-tolerant seeds for genes annotated for general and temperature-specific stress, as well as protein-refolding. DEGs were also clustered in modules using weighted co-expressed gene network analysis, which were examined for enrichment of GO biological process terms. Collectively, our results provide new and valuable insights into this unique form of genetic abiotic stress tolerance and to soybean seed physiological responses to elevated temperatures.

The small genome of an abundant coastal ocean methylotroph.

OM43 is a clade of uncultured beta-proteobacteria that is commonly found in environmental nucleic acid sequences from productive coastal ocean ecosystems, and some freshwater environments, but is rarely detected in ocean gyres. Ecological studies associate OM43 with phytoplankton blooms, and evolutionary relationships indicate that they might be methylotrophs. Here we report on the genome sequence and metabolic properties of the first axenic isolate of the OM43 clade, strain HTCC2181, which was obtained using new procedures for culturing cells in natural seawater. We found that this strain is an obligate methylotroph that cannot oxidize methane but can use the oxidized C1 compounds methanol and formaldehyde as sources of carbon and energy. Its complete genome is 1304 428 bp in length, the smallest yet reported for a free-living cell. The HTCC2181 genome includes genes for xanthorhodopsin and retinal biosynthesis, an auxiliary system for producing transmembrane electrochemical potentials from light. The discovery that HTCC2181 is an extremely simple specialist in C1 metabolism suggests an unanticipated, important role for oxidized C1 compounds as substrates for bacterioplankton productivity in coastal ecosystems.