Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Profiling the transcriptome of Thalassiosira pseudonana under environmentally relevant growth conditions

ABSTRACT: T. pseudonana gene-specific arrays were used for differential gene expression. Seven hundred nine genes were differentially expressed by more than 2-fold (Bayesian t-test, p< 0.001) under at least one growth-limiting condition relative to nutrient-replete conditions. A striking result of the hierarchical cluster analysis was the identification of a common set of genes that were upregulated by both iron and silicon limitation, but no other treatment. Together, these two treatments accounted for about one fourth of all differentially expressed genes but almost two thirds of the differentially expressed novel genes, further emphasizing the distinctive aspects of silicon manipulation in diatoms. We analyzed 6 different sets of arrays from 6 growth conditions. Four biological replicates (=4 arrays) were used for each growth condition.

ORGANISM(S): Thalassiosira pseudonana CCMP1335

SUBMITTER: Michael Sussman

PROVIDER: E-GEOD-9660 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Whole-genome expression profiling of the marine diatom Thalassiosira pseudonana identifies genes involved in silicon bioprocesses.

Mock Thomas T Samanta Manoj Pratim MP Iverson Vaughn V Berthiaume Chris C Robison Matthew M Holtermann Karie K Durkin Colleen C Bondurant Sandra Splinter SS Richmond Kathryn K Rodesch Matthew M Kallas Toivo T Huttlin Edward L EL Cerrina Francesco F Sussman Michael R MR Armbrust E Virginia EV

Proceedings of the National Academy of Sciences of the United States of America 20080122 5

Formation of complex inorganic structures is widespread in nature. Diatoms create intricately patterned cell walls of inorganic silicon that are a biomimetic model for design and generation of three-dimensional silica nanostructures. To date, only relatively simple silica structures can be generated in vitro through manipulation of known diatom phosphoproteins (silaffins) and long-chain polyamines. Here, we report the use of genome-wide transcriptome analyses of the marine diatom Thalassiosira p ...[more]

PMID: 18212125

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Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Transcriptomes were collected for diatom cultures harvested at the onset of stationary phase in low nitrate media (55 M-NM-<M NaNO3, 212 M-NM-<M Na2SiO3, 72.4 M-NM-<M NaH2PO4) or during mid-exponential growth in nutrient-replete media (882 M-NM-<M NaNO3, 106 M-NM-<M Na2SiO3, 36.2 M-NM-<M NaH2PO4) in artificial seawater, maintaining three biological replicates per condition and per diatom (N=18). The SOLiD sequencer (version 4) was used to generate the transcriptomes and the SEAStAR software package was used to process the SOLiD reads and to calculate gene counts. Pooled counts for the nitrate-limited treatment were normalized to pooled counts for the nutrient-replete M-bM-^@M-^\controlM-bM-^@M-^] treatment to generate log fold changes in gene transcription using the R software package edgeR from Bioconductor.

Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. Axenic T. pseudonana CCMP 1335 was grown at 14Â°C under 24 hour light (120 Âµmol photons m-2 s-1) after Dyhrman et al. (2012) in f/2 plus silica chelated media made from surface Sargasso Sea water. Nitrate, silica, vitamins, and trace metals were at f/2 concentrations (Guillard and Ryther 1962), while iron and phosphate were modified across treatments. In brief, triplicate cultures of replete (36 ÂµM PO4, 400 nM Fe), P-limited (0.4 ÂµM PO4, 400 nM Fe), Fe-limited (36 ÂµM PO4, 40 nM Fe), and Co-limited (0.4 ÂµM PO4, 40 nM Fe) treatments were harvested when growth deviated from the replete control. Growth was monitored by cell counts. Biomass was harvested onto 0.2 Âµm filters and flash frozen in liquid nitrogen and total RNA was extracted as described in Dyhrman et al. (2012). Tag-seq sequencing of the transcriptome was performed by Illumina with a polyA selection and NlaIII digestion, resulting in 21 bp sequence reads or tags (Dyhrman et al., 2012). Libraries were of varied sizes as follows: replete (~12 million), P-limited (~13 million), Fe-limited (~23 million), and Co-limited (~26 million). Tags were mapped to gene models (predicted protein coding regions) with a pipeline designed by Genesifter Inc., requiring 100% identity and covering 9759 genes. Tag counts within a gene were pooled and normalized to the size of the library, with resulting data expressed in tags per million (tpm). Genes with normalized tag counts less than 2.5 tpm for each of the four treatments were excluded (Figure S1) , leaving 7380 genes in the analysis.

Dataset Information

Profiling the transcriptome of Thalassiosira pseudonana under environmentally relevant growth conditions

Publications

Whole-genome expression profiling of the marine diatom Thalassiosira pseudonana identifies genes involved in silicon bioprocesses.

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