Flux of transcript patterns during soybean seed development

<p>Abstract</p> <p>Background</p> <p>To understand gene expression networks leading to functional properties of the soybean seed, we have undertaken a detailed examination of soybean seed development during the stages of major accumulation of oils, proteins, and starches, as well as the desiccating and mature stages, using microarrays consisting of up to 27,000 soybean cDNAs. A subset of these genes on a highly-repetitive 70-mer oligonucleotide microarray was also used to support the results.</p> <p>Results</p> <p>It was discovered that genes related to cell growth and maintenance processes, as well as energy processes like photosynthesis, decreased in expression levels as the cotyledons approached the mature, dry stage. Genes involved with some storage proteins had their highest expression levels at the stage of highest fresh weight. However, genes encoding many transcription factors and DNA binding proteins showed higher expression levels in the desiccating and dry seeds than in most of the green stages.</p> <p>Conclusions</p> <p>Data on 27,000 cDNAs have been obtained over five stages of soybean development, including the stages of major accumulation of agronomically-important products, using two different types of microarrays. Of particular interest are the genes found to peak in expression at the desiccating and dry seed stages, such as those annotated as transcription factors, which may indicate the preparation of pathways that will be needed later in the early stages of imbibition and germination.</p

Molecular approaches to malaria and babesiosis diagnosis.

The development of additional methods for detecting and identifying Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. The preliminary evaluate synthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK) was selected from the published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzyme-linked synthetic DNA assay for P. falciparum detected 30 parasites/mm3 from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species

Using RNA-Seq to profile soybean seed development from fertilization to maturity.

To understand gene expression networks leading to functional properties and compositional traits of the soybean seed, we have undertaken a detailed examination of soybean seed development from a few days post-fertilization to the mature seed using Illumina high-throughput transcriptome sequencing (RNA-Seq). RNA was sequenced from seven different stages of seed development, yielding between 12 million and 78 million sequenced transcripts. These have been aligned to the 79,000 gene models predicted from the soybean genome recently sequenced by the Department of Energy Joint Genome Institute. Over one hundred gene models were identified with high expression exclusively in young seed stages, starting at just four days after fertilization. These were annotated as being related to many basic components and processes such as histones and proline-rich proteins. Genes encoding storage proteins such as glycinin and beta-conglycinin had their highest expression levels at the stages of largest fresh weight, confirming previous knowledge that these storage products are being rapidly accumulated before the seed begins the desiccation process. Other gene models showed high expression in the dry, mature seeds, perhaps indicating the preparation of pathways needed later, in the early stages of imbibition. Many highly-expressed gene models at the dry seed stage are, as expected, annotated as hydrophilic proteins associated with low water conditions, such as late embryogenesis abundant (LEA) proteins and dehydrins, which help preserve the cellular structures and nutrients within the seed during desiccation. More significantly, the power of RNA-Seq to detect genes expressed at low levels revealed hundreds of transcription factors with notable expression in at least one stage of seed development. Results from a second biological replicate demonstrate high reproducibility of these data revealing a comprehensive view of the transciptome of seed development in the cultivar Williams, the reference cultivar for the first soybean genome sequence

An embryo lethal transgenic line manifests global expression changes and elevated protein/oil ratios in heterozygous soybean plants.

Understanding the molecular processes of seed development is important especially in agronomic crops that produce large amounts of nutrient reserves. Because soybean is a vital source of vegetable protein worldwide, producers are concerned about increasing the total amount of protein in the seed without substantially lowering the amount of oil, another economically important product. Here we describe a transgenic soybean line with increased protein and protein/oil ratio, containing an average of 42.2% protein vs. 38.5% in controls and with a protein/oil ratio of 2.02 vs. 1.76 in controls over several generations of greenhouse growth. Other phenotypic data show that the seeds are heavier, although there are overall lower yields per plant. We postulate these effects result from insertion site mutagenesis by the transgenic construct. As this line never achieves homozygosity and appears to be embryo lethal when homozygous, one functional copy of the gene is most likely essential for normal seed development. Global transcript analyses using RNA-Seq for 88,000 gene models over two stages of cotyledon development revealed that more genes are over-expressed in the transgenic line including ribosomal protein related genes and those in the membrane protein and transporters families. Localization of the insertion site should reveal the genes and developmental program that has been perturbed by the transgenic construct, resulting in this economically interesting increase in protein and the protein/oil ratio

Storage protein gene models grouped by annotation.

<p>Annotations were derived from PFAM and/or the NCBI non-redundant database. Clusters were created from Biological Replicate 1 data (A, C, E) and the same gene models were also graphed with Biological Replicate 2 data (B, D, F). A shows glycinin (pink) and beta-conglycinin (black) storage proteins with RPKM≥5 in at least one of seven stages; C is the same graph using log scale. (An RPKM of 0 will not display on a log scale and may appear as a break in the line.) E shows omega-3-fatty acid desaturase (pink) and omega-6-fatty acid desaturase (black) with RPKM≥5 in at least one of seven stages, log scale. Stages are numbered in order on the x axis: 4 DAF whole seed, 12–14 DAF whole seed, 22–24 DAF whole seed, 5–6 mg whole seed, 100–200 mg cotyledon, 400–500 mg cotyledon, dry whole seed.</p

Gene models with no significant difference between biological replicates.

<p>Number and percentage (of 78,773) of gene models with no significant difference in expression between two biological replicates (adjusted p-value >0.05), for each stage of soybean seed development.</p

Stages of soybean seed development and RNA-Seq data.

<p>Shown are the seven stages of soybean seed development used for RNA-Seq. Days after flowering (DAF) or fresh weight in milligrams (mg) is shown along with tissue and the number of processed reads obtained from two biological replicates of RNA high-throughput sequencing (in millions, M). Key events in development are noted.</p

Expression patterns of gene models shown in Figure 1C.

<p>The 20 gene models in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059270#pone-0059270-g001" target="_blank">Figure 1C</a> (genes with high RPKM at 5–6 mg whole seed) divided into groups based on their expression patterns in the 5–6 mg separated seed coat and cotyledon samples. Data from 5–6 mg combined whole seed sample also shown. RPKMs have been averaged for each group of gene models. The predominant annotation of each expression pattern group is also shown.</p

A histogram dividing 5608 transcription factor gene models into bins based on their RPKMs.

<p>Nine different libraries are shown, including 5–6 mg separated seed coat and cotyledon. Gene models must have RPKM≥5 at a stage to be displayed. Gene models with RPKM≥51 are compiled together at the top (orange). 5–6 mgWS = 5–6 mg whole seed.</p