Project description:The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is currently a contentious issue that warrants investigation into the nutrient physiology of this dinoflagellate species. The molecular mechanisms by which dinoflagellates respond to nutrient availability are essentially unexplored. The current study sought to determine if the transcriptome of K. brevis is responsive to phosphorus and informative of nutrient status. Stationary phase P-limited cultures did not exhibit up-regulation of transcripts considered to be hallmark of P depletion relative to nutrient-replete stationary phase cultures, despite rapid growth upon addition of the limiting nutrient. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p<10-4. By far, the earliest responding genes were dominated by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. Transcriptomic responses to the addition of P reflected primarily chloroplast functions. Even the earliest responding transcripts, those encoding PPR proteins, possess a 5’ trans-spliced leader sequence, suggesting that the observed rapid response of the transcriptome may be achieved, in part, through post-transcriptional mechanisms.
Project description:The role of coastal nutrient sources in the persistence of Karenia brevis red tides in coastal waters of Florida is currently a contentious issue that warrants investigation into the nutrient physiology of this dinoflagellate species. The molecular mechanisms by which dinoflagellates respond to nutrient availability are essentially unexplored. The current study sought to determine if the transcriptome of K. brevis is responsive to nitrogen and informative of nutrient status. Stationary phase N-limited cultures did not exhibit up-regulation of transcripts considered to be hallmark of N depletion relative to nutrient-replete stationary phase cultures, despite rapid growth upon addition of the limiting nutrient. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. N-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p<10-4. By far, the earliest responding genes were dominated by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 4 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes. Transcriptomic responses to the addition of N reflected primarily chloroplast functions. Even the earliest responding transcripts, those encoding PPR proteins, possess a 5’ trans-spliced leader sequence, suggesting that the observed rapid response of the transcriptome may be achieved, in part, through post-transcriptional mechanisms.
Project description:Dinoflagellates have evolved a nuclear organization unlike that of any other eukaryotic group. Recent studies find a predominance of post-transcriptional control of dinoflagellate gene expression. This study investigated regulation of the environmental stress response in the red tide dinoflagellate, Karenia brevis using an Agilent custom oligonucleotide microarray. K. brevis cultures were exposed to 5°C or 10°C heat shock, or three different sources of oxidative stress: 60 µM H2O2, 10 mM NaNO2, or 12 µM PbCl2 over acute time courses. Ribosomal genes, genes involved in RNA processing, translation, and chaperones were among the classes of genes consistently downregulated across treatments, although within these functional classes the same genes did not always respond to different stressors. Genes involved in the photosystem and mitochondrial and chloroplast ATP generation dominated the down-regulated genes. Heat shock and oxidative stress response genes were not induced under any treatment, even under conditions that resulted in decreased viability. We subsequently identified the presence of a trans-spliced leader sequence on many stress response gene transcripts, which suggests that they may be transcribed constitutively and their expression regulated at the level of translation.
Project description:Dinoflagellates have evolved a nuclear organization unlike that of any other eukaryotic group. Recent studies find a predominance of post-transcriptional control of dinoflagellate gene expression. This study investigated regulation of the environmental stress response in the red tide dinoflagellate, Karenia brevis using an Agilent custom oligonucleotide microarray. K. brevis cultures were exposed to 5°C or 10°C heat shock, or three different sources of oxidative stress: 60 µM H2O2, 10 mM NaNO2, or 12 µM PbCl2 over acute time courses. Ribosomal genes, genes involved in RNA processing, translation, and chaperones were among the classes of genes consistently downregulated across treatments, although within these functional classes the same genes did not always respond to different stressors. Genes involved in the photosystem and mitochondrial and chloroplast ATP generation dominated the down-regulated genes. Heat shock and oxidative stress response genes were not induced under any treatment, even under conditions that resulted in decreased viability. We subsequently identified the presence of a trans-spliced leader sequence on many stress response gene transcripts, which suggests that they may be transcribed constitutively and their expression regulated at the level of translation. Cultures of were grown to mid-log phase. For each treatment, five replicate untreated control cultures and five replicate treated cultures were harvested at several time points following treatment. The following time courses were used: 5°C heat shock - 5, 15, 30, 60, and 240 min; 10°C heat shock - 60 min; 60 µM H2O2 - 5, 15, 30, 60, and 240 min; 10 mM NaNO2 -1, 4, and 7 hours; 12 µM PbCl2 -1, 4, and 7 hours. For each treatment, RNA was pooled from the controls and treated cultures at each timepoint. Two color arrays were then run comparing each the transcriptome at timepoint with the pooled control for that treatment. A technical dye swap array was run at each timepoint.
Project description:To gain an understanding of processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out to identify changes in the global transcriptome that accompany the entry and maintenance of stationary phase up to the onset of cell death. The transcriptome of K. brevis was assayed using a custom 10,263 feature oligonucleotide microarray from mid-logarithmic growth to the onset of culture demise. A total of 2,958 (29%) features were differentially expressed, with the mid-stationary phase timepoint demonstrating peak changes in expression. Gene ontology enrichment analyses identified a significant shift in transcripts involved in energy acquisition, ribosome biogenesis, gene expression, stress adaptation, calcium signaling, and putative brevetoxin biosynthesis. The extensive remodeling of the transcriptome observed in the transition into a quiescent non-dividing phase appears to be indicative of a global shift in the metabolic and signaling requirements and provides the basis from which to understand the process of chronological aging in a dinoflagellate.
Project description:To gain an understanding of processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out to identify changes in the global transcriptome that accompany the entry and maintenance of stationary phase up to the onset of cell death. The transcriptome of K. brevis was assayed using a custom 10,263 feature oligonucleotide microarray from mid-logarithmic growth to the onset of culture demise. A total of 2,958 (29%) features were differentially expressed, with the mid-stationary phase timepoint demonstrating peak changes in expression. Gene ontology enrichment analyses identified a significant shift in transcripts involved in energy acquisition, ribosome biogenesis, gene expression, stress adaptation, calcium signaling, and putative brevetoxin biosynthesis. The extensive remodeling of the transcriptome observed in the transition into a quiescent non-dividing phase appears to be indicative of a global shift in the metabolic and signaling requirements and provides the basis from which to understand the process of chronological aging in a dinoflagellate. Twenty seven 900ml batch cultures of K. brevis were inoculated at a starting concentration of approximately 1000 cells/ml from mid-logarithmic stage starter cultures on day 0. Triplicate cultures were harvested every other day from day 2 to 18 and total RNA was extracted. One color arrays were then run on all biological replicates (n=3 at each timepoint) for days 4, 6, 10, 14 and 18.
Project description:Allelopathy, or the release of compounds that inhibit competitors, is a form of interference competition that is common among bloom-forming phytoplankton. Allelopathy is hypothesized to play a role in bloom propagation and maintenance and is well established in the red tide dinoflagellate Karenia brevis. K. brevis typically suppresses competitor growth through unknown mechanisms over the course of many days. When we investigated the effects of allelopathy on the lipidomes of two competing phytoplankton, Asterionellopsis glacialis and Thalassiosira pseudonana using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS)- based metabolomics, we found that the lipidomes of both species were significantly altered, however A. glacialis maintained a more robust response whereas T. pseudonana saw significant alterations in fatty acid synthesis, cell membrane integrity, and a decrease in photosynthetic efficiency. Membrane- associated lipids were significantly suppressed for T. pseudonana exposed to allelopathy to the point of permeabilizing the cell membrane of living cells. The dominant mechanisms of K. brevis allelopathy appear to target lipid biosynthesis affecting multiple physiological pathways suggesting that exuded compounds have the ability to significantly alter competitor physiology and give K. brevis a competitive edge over sensitive species.