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Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum.

ABSTRACT: Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

SUBMITTER: Kadono T

PROVIDER: S-EPMC4686930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Gene silencing in the marine diatom Phaeodactylum tricornutum.

Project description:Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.

| S-EPMC2724275 | biostudies-literature

Histone modifications in the marine diatom Phaeodactylum tricornutum

Project description:Genome wide mapping of five histone marks (H3K4me2, H3K9me2, H3K9me3, H3K27me3 and H3AcK9/14) as well as nucleosome occupancy was generated by chromatin immunoprecipitation followed by deep sequencing. To gain insights into the dynamic nature of the P. tricornutum epigenome in response to an environmental cue, we analyzed the impact of nitrate depletion. We specifically examined three histone modifications (H3K4me2, H3K9/14Ac and H3K9me3) using Chip-seq.

2015-05-05 | GSE68513 | GEO

Histone modifications in the marine diatom Phaeodactylum tricornutum

2015-05-05 | E-GEOD-68513 | biostudies-arrayexpress

Apoptosis-inducing galactolipids from a cultured marine diatom, Phaeodactylum tricornutum.

Project description:Two monogalactosyl diacylglycerols, 1 and 2, were isolated from the marine diatom Phaeodactylum tricornutum, using the patented ApopScreen cell-based screen for apoptosis-inducing, potential anticancer compounds. The molecular structures of the galactolipids were determined using a combination of NMR, mass spectrometry, and chemical degradation. The bioactivities were confirmed using a specific apoptosis induction assay based on genetically engineered mammalian cell lines with differential, defined capacities for apoptosis. The galactolipids induce apoptosis in micromolar concentrations. This is the first report of apoptosis induction by galactolipids.

| S-EPMC2866075 | biostudies-literature

Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

Project description:Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum, using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, provision of glycerol mimics typical responses of nitrogen limitation on lipid metabolism at the level of triacylglycerol accumulation and fatty acid composition. The presence of glycerol, despite provoking features reminiscent of nutrient limitation, neither diminishes photosynthetic activity nor cell growth, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

| S-EPMC5516113 | biostudies-literature

Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum.

Project description:The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

| S-EPMC4710849 | biostudies-other

Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum.

Project description:A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

| S-EPMC6717305 | biostudies-literature

Transcriptional response of the marine pennate diatom Phaeodactylum tricornutum to Fe limitation

Project description:Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom

2008-01-01 | GSE8675 | GEO

The retrograde signals responsible for photoacclimation in the marine diatom Phaeodactylum tricornutum

Project description:Photoacclimation of unicellular algae allows for reversible changes in the number and/or effective absorption cross section of photosynthetic units on time scales of hours to days in response to changes in irradiance. The process involves an enigmatic signaling pathway from the plastid to the nucleus.Our results reveal, for the first time, a fundamental pathway of retrograde signal transduction in a eukaryotic photosynthetic alga.

2019-11-01 | GSE133301 | GEO

Characterization and engineering of a dual-function diacylglycerol acyltransferase in the oleaginous marine diatom <i>Phaeodactylum tricornutum</i>.

Project description:BackgroundPhotosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA.ResultsA dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions.ConclusionsPtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.

| S-EPMC5806285 | biostudies-literature

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