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Background CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism.

After the Deepwater Horizon oil spill in 2010, toxicity tests were conducted using 4 microalgae (Dunaliella tertiolecta, Skeletonema costatum, Isochrysis galbana, and Thalassiosira pseudonana) and one macroalga (Ectocarpus siliculosus) to study potential impacts on phytoplankton and other primary producers in the Gulf of Mexico and characterize species sensitivity. Tests were performed with Corexit 9500 and fresh source oil and weathered oil samples collected from the field during the Deepwater Horizon oil spill. Because crude oils are mixtures of poorly water-soluble hydrocarbons, dosing was performed using water-accommodated fractions (WAFs) and chemically enhanced (CE) WAFs with the addition of dispersant at a 1:20 dispersant:oil ratio using standard toxicity testing protocols. Exposure media were analyzed for volatile organic compounds, parent and alkylated polycyclic aromatic hydrocarbons, and saturated hydrocarbon compounds. Toxicity was reported as no-observable effect concentration and median effect concentration (EC50) values for average specific growth rate based on nominal percent dilution of stock solution WAFs and sum of dissolved oil toxic units for WAF/CEWAF tests. The macroalga and green alga D. tertiolecta were largely unaffected by any WAF or CEWAFs tested. Isochrysis galbana was found to be the most sensitive species overall with significant growth rate inhibitions for dispersant and all the WAFs/CEWAFs tested. Physically dispersed source oils were generally more toxic than weathered oils. The protectiveness of the chronic toxic units was effective at identifying observed algal growth rate inhibitions across algal species and oil types despite the impact of dispersants. Environ Toxicol Chem 2021;40:2742-2754. © 2021 SETAC.

Diatoms are unicellular phototrophic organisms with huge ecological impact. Characteristic for these organisms is their peculiar cell wall, which is composed of inorganic and organic components. Cell wall formation is a highly complex and orchestrated process, and in the last years has been studied intensively, also on the molecular level. Here, we review on the cell wall proteins of diatoms, with a focus on the species Thalassiosira pseudonana. We report on the expression patterns of these proteins in synchronized cultures, as well as their modifications and intracellular targeting.

Anthropogenic activities release effluents containing nutrients and pathogenic bacteria that change the characteristics of coastal ecosystems. An important type of marine pollution which has occurred in 3 different states in India during 2019 is sea foam. Sea foam was found on Hole beach, Goa (Lat: 15.404° N, Long: 73.787° E), where nutrients (NO3- = 137 μM and organic nitrogen = 121 μM) from a garbage dumpyard are released directly via streams/gutters to coastal waters. This resulted in a bloom of the diatom Thalassiosira pseudonana, associated with high concentration of total organic carbon and fucoxanthin. Decay of this bloom along with strong agitation due to rocks and wave action resulted in sea foam. We isolated foam-associated bacteria and identified pathogenic bacteria including Enterobacter cancerogenus through 16S rRNA gene sequencing. Such foam-associated pathogenic bacteria, could be antibiotic resistant, and may have adverse effects on human health. This can also hamper the tourism industry of a small state like Goa that relies heavily on tourism.

The mixotrophic cultivation of microalgae often couples high biomass production with lipid accumulation, and thus it is considered a useful approach to obtain high quantities of added value microalgal biomass. In this study, the marine diatom Thalassiosira pseudonana was supplied with glycerol to determine the potential of the mixotrophic alga for the accumulation of lipids. In preliminarily experiments, T. pseudonana was cultivated in the presence of increasing concentrations of glucose, acetate and glycerol in order to select the best organic carbon source for growth promotion, and glycerol was selected for further experiments. The best glycerol dose for both biomass productivity and lipid production was 2.5 g L−1. To overcome the costs of the pure glycerol supply, the crude glycerol derived from a biodiesel production plant was also tested, as a low-cost alternative. Beside growth and photosynthetic parameters useful to monitor the algal conditions, lipid amount and profile were compared between autotrophic and pure/crude glycerol-treated algae. Interestingly, an almost doubled lipid content was accumulated in mixotrophic algae compared to the autotrophic ones. Moreover, an evident increase in a relatively short-chain fatty acid (C14:0, myristic acid), potentially useful for bio-jet fuels, and in a long-chain omega3-polyunsaturated fatty acid (C18:4; stearidonic acid), interesting for nutritional purposes, was highlighted in mixotrophic samples, especially when cultivated with crude glycerol. Differently, the fatty acid profile obtained from the autotrophic cultures suggested a preferable employment of the algal biomass for biodiesel production, owing to the prevalence of palmitic and palmitoleic acids. This study highlights that the mixotrophic cultivation of T. pseudonana drives the algal metabolism towards the production of different lipids, suitable for diversified applications. Moreover, the use of biodiesel-derived crude glycerol could make mixotrophy economically advantageous, in particular considering a bio-refinery approach.

This study investigated the biosorption efficiency of Mn (II) by algae (Thalassiosira pseudonana) and membrane performance in a hybrid algae-membrane system. The presence of Mn (II) at 2–10 mM could influence (i.e., at lower algal inoculation ratio of OD = 0.1) or showed negligible effect (i.e., at higher inoculation ratio of OD = 0.17), on algal growth, cell morphology, pigment content, and extracellular organic matter production. The Mn (II) removal was mainly attributed to the algal uptake mechanisms; and higher removal efficiency (>66%) was achieved at higher algal inoculation ratio. Although increasing the Mn (II) concentration could reduce the algal uptake ratio of Mn (II), the membrane performance was improved. The optical coherence tomography (OCT) analysis revealed that the specific volume of cake layer increased gradually over time, however, the irreversible fouling was predominant and associated the presence of building blocks in soluble organics. Economic Development Board (EDB) The Economic Development Board (EDB) of Singapore is acknowledged for funding the Singapore Membrane Technology Centre (SMTC), Nanyang Environment & Water Research Institute, Nanyang Technological University.

Background CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is often involved in the redox metabolic on/off switch of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological predominance and their complex and still enigmatic evolutionary background. Methods A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance and small X ray scattering spectroscopy, was used to characterize a diatom CP12. Results Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 behaves abnormally under gel electrophoresis, is heat resistant and lacks a structural core, all features of intrinsically disorder family similarly to its homologues in other species. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as shown by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering showed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12, though it has features of disordered proteins, has a high content of α-helices. Nuclear magnetic resonance spectroscopy showed that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled-coil and disordered regions. Conclusions These findings bring new insights into the large family of intrinsically disordered proteins increasing the diversity of known CP12 proteins. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle.

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes, however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24&rsquo, )-dien-3&beta, ol was observed in all P. tricornutum overexpression lines, and campesterol increased three-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

Acidification of the ocean due to high atmospheric CO2 levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatom Thalassiosira pseudonana under elevated CO2. Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na+(K+)/H+ antiporter that is significantly upregulated at > 800 ppm CO2, with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO2 conditions. In future acidified oceanic conditions, dramatic oscillations of >10-fold change between nighttime (high) and daytime (low) in transcript abundances of the antiporter gene were associated with increased resilience of T. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO2 conditions in marine environments.

Background The nitrogen-containing polysaccharide chitin is the second most abundant biopolymer on earth and is found in the cell walls of diatoms, where it serves as a scaffold for biosilica deposition. Diatom chitin is an important source of carbon and nitrogen in the marine environment, but surprisingly little is known about basic chitinase metabolism in diatoms. Results Here, we identify and fully characterize 24 chitinase genes from the model centric diatom Thalassiosira pseudonana. We demonstrate that their expression is broadly upregulated under abiotic stresses, despite the fact that chitinase activity itself remains unchanged, and we discuss several explanations for this result. We also examine the potential transcriptional complexity of the intron-rich T. pseudonana chitinase genes and provide evidence for two separate tandem duplication events during their evolution. Conclusions Given the many applications of chitin and chitin derivatives in suture production, wound healing, drug delivery, and other processes, new insight into diatom chitin metabolism has both theoretical and practical value.

As oceans warm, the depth of the upper mixed layer is predicted to decrease, resulting in insufficient nutrient supply and higher solar radiation for phytoplankton. In order to understand the photophysiological responses of the key eukaryotic phytoplankton diatoms to high light and nutrient limitation, we grew two diatoms, Thalassiosira weissflogii and Thalassiosira pseudonana under N starvation conditions and exposed them to high visible light. It showed that the large-sized diatom T. weissflogii can maintain photosynthetic activity for a longer period of time under nitrogen starvation as compared with the small-sized diatom T. pseudonana. The electron transfer reaction was inhibited in both diatoms and the fast closing of reaction centers promoted the development of QB non-reducing PSII centers, thus facilitated the rapid induction of NPQ, however, the induction of NPQ depended on the degree of N starvation. N starvation exacerbated the photoinhibition caused by high light. The smaller-sized T. pseudonana had a higher σi value and was more sensitive to high-light, but its PSII repair rate was also higher. In contrast, T. weissflogii was more tolerant to high light with a lower σi value, but the tolerance was severely reduced under N-starvation. This study provides helpful insight into how climate change variables impact diatom's photosynthetic physiology.

Diatoms are unicellular photosynthetic algae known to secrete organic matter that fuels secondary production in the ocean, though our knowledge of how their physiology impacts the character of dissolved organic matter remains limited. Like all photosynthetic organisms, their use of light for energy and reducing power creates the challenge of avoiding cellular damage. To better understand the interplay between redox balance and organic matter secretion, we reconstructed a genome-scale metabolic model of Thalassiosira pseudonana strain CCMP 1335, a model for diatom molecular biology and physiology, with a 60-year history of studies. The model simulates the metabolic activities of 1,432 genes via a network of 2,792 metabolites produced through 6,079 reactions distributed across six subcellular compartments. Growth was simulated under different steady-state light conditions (5-200 μmol photons m−2 s−1) and in a batch culture progressing from exponential growth to nitrate-limitation and nitrogen-starvation. We used the model to examine the dissipation of reductants generated through light-dependent processes and found that when available, nitrate assimilation is an important means of dissipating reductants in the plastid; under nitrate-limiting conditions, sulfate assimilation plays a similar role. The use of either nitrate or sulfate uptake to balance redox reactions leads to the secretion of distinct organic nitrogen and sulfur compounds. Such compounds can be accessed by bacteria in the surface ocean. The model of the diatom Thalassiosira pseudonana provides a mechanistic explanation for the production of ecologically and climatologically relevant compounds that may serve as the basis for intricate, cross-kingdom microbial networks. Diatom metabolism has an important influence on global biogeochemistry; metabolic models of marine microorganisms link genes to ecosystems and may be key to integrating molecular data with models of ocean biogeochemistry.

Ferredoxins are iron–sulfur proteins essential for a wide range of organisms because they are an electron transfer mediator involved in multiple metabolic pathways. In phytoplankton, these proteins are active in the mature chloroplasts, but the petF gene, encoding for ferredoxin, has been found either to be in the chloroplast genome or transferred to the nuclear genome as observed in the green algae and higher plant lineage. We experimentally determined the location of the petF gene in 12 strains of Thalassiosira covering three species using DNA sequencing and qPCR assays. The results showed that petF gene is located in the nuclear genome of all confirmed Thalassiosira oceanica strains (CCMP0999, 1001, 1005, and 1006) tested. In contrast, all Thalassiosira pseudonana (CCMP1012, 1013, 1014, and 1335) and Thalassiosira weissflogii (CCMP1010, 1049, and 1052) strains studied retained the gene in the chloroplast genome, as generally observed for Bacillariophyceae. Our evolutionary analyses further extend the dataset on the localization of the petF gene in the Thalassiosirales. The realization that the petF gene is nuclear-encoded in the Skeletonema genus allowed us to trace the petF gene transfer back to a single event that occurred within the paraphyletic genus Thalassiosira. Phylogenetic analyses revealed the need to reassess the taxonomic assignment of the Thalassiosira strain CCMP1616, since the genes used in our study did not cluster within the T. oceanica lineage. Our results suggest that this strains’ diversification occurred prior to the ferredoxin gene transfer event. The functional transfer of petF genes provides insight into the evolutionary processes leading to chloroplast genome reduction and suggests ecological adaptation as a driving force for such chloroplast to nuclear gene transfer.

Simple Summary One of the challenges in the emerging field of synthetic biology is engineering organelle genomes. Creating synthetic organelle genomes can open the door to a wide range of applications, such as improving crop yields, treating mitochondrial diseases, or manufacturing high-value chemicals in an environmentally sustainable way. Organelles are tiny biological machines that work inside of living cells. Mitochondria, for example, are responsible for harvesting sugar to create energy for the cell. In previous work, we demonstrated a method to make copies of an alga mitochondrial genome using yeast and bacteria. Algae are of industrial interest for their potential to produce and store large quantities of biofuels and nutritional ingredients. Here, we applied the same approach to copy the mitochondrial genome of a related alga. Although the cloning of this mitochondrial genome in yeast using the previously developed method was possible, the properties of this genome may make it more susceptible to mutations during propagation in bacteria. This work expands our understanding of potential hurdles that can be encountered when cloning and propagating synthetic organelle genomes in host organisms. Abstract Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.

Thalassiosira pseudonana is a transformable and biotechnologically promising model diatom with an ability to synthesise nutraceuticals such as fucoxanthin and store a significant amount of polyglucans and lipids including omega-3 fatty acids. While it was the first diatom to be sequenced, a systems-level analysis of its metabolism has not been done yet. This work presents first comprehensive, compartmentalized, and functional genome-scale metabolic model of the marine diatom Thalassiosira pseudonana CCMP 1335, which we have termed iThaps987. The model includes 987 genes, 2477 reactions, and 2456 metabolites. Comparison with the model of another diatom Phaeodactylum tricornutum revealed presence of 183 unique enzymes (belonging primarily to amino acid, carbohydrate, and lipid metabolism) in iThaps987. Model simulations showed a typical C3-type photosynthetic carbon fixation and suggested a preference of violaxanthin&ndash, diadinoxanthin pathway over violaxanthin&ndash, neoxanthin pathway for the production of fucoxanthin. Linear electron flow was found be active and cyclic electron flow was inactive under normal phototrophic conditions (unlike green algae and plants), validating the model predictions with previous reports. Investigation of the model for the potential of Thalassiosira pseudonana CCMP 1335 to produce other industrially useful compounds suggest iso-butanol as a foreign compound that can be synthesized by a single-gene addition. This work provides novel insights about the metabolism and potential of the organism and will be helpful to further investigate its metabolism and devise metabolic engineering strategies for the production of various compounds.

Chitin constitutes an abundant component in many biologically formed minerals (biominerals). While the role of chitin for the formation and properties of calcium-based biominerals has been extensively studied, little is known about its role in silica-based biominerals. Furthermore, there is hardly any information about the enzyme machinery for chitin biosynthesis in biomineral-forming organisms. Here we have identified a chitin synthase, chs7305, in the diatom Thalassiosira pseudonana. In stationary cells, chs7305 is located specifically in a ring pattern in the region of the silicified girdle bands. The expression pattern of the chs7305 gene and the co-localization of the encoded enzyme with chitin provides evidence for the importance of chitin synthesis for cell wall function under nutrient limited conditions. Chs7305 is the first chitin synthase that has been localized in a diatom.

Stressful environmental conditions can induce many different acclimation mechanisms in marine phytoplankton, resulting in a range of changes in their photophysiology. Here we characterize the common photophysiological stress response of the model diatom Thalassiosira pseudonana to ten environmental stressors and identify diagnostic responses to particular stressors. We quantify the magnitude and temporal trajectory of physiological parameters including the functional absorption cross-section of PSII (σPSII ), quantum efficiency of PSII, non-photochemical quenching (NPQ), cell volume, Chl a, and carotenoid (Car) content in response to nutrient starvation (nitrogen (N), phosphorus (P), silicon (Si), and iron (Fe)), changes in temperature, irradiance, pH, and reactive oxygen species (ROS) over 5 time points (0, 2, 6, 24, 72 h). We find changes in conditions: temperature, irradiance, and ROS, often result in the most rapid changes in photophysiological parameters (

Diatom cells utilize a variety of metabolic pathways to cope with internal energy imbalances caused by stressful environmental conditions. In this study, the model diatom species, Thalassiosira pseudonana, was grown in nutrient replete and nitrate (NO3−)- and dissolved silicate (Si)-depleted media at three growth temperatures (4, 17, 28 °C) to determine how nutrient enrichment and temperature affects diatom growth, photosynthetic efficiency, nitrate reductase (NR) enzyme activity, biogenic silica (bSiO2) deposition, and NR gene expression. Growth rates for nutrient-replete cultures were highest at 17 °C. Across all nutrient treatments, the cells grown at 17 °C had an average Fv/Fm of 0.44 ± 0.006, while the cells were grown at 4 °C and 28 °C had an average Fv/Fm of 0.37 ± 0.004 and 0.38 ± 0.01, respectively. Activity of NR was variable across treatments with no significant effect of temperature. The relative expression of the targeted NR gene was, on average, ~ 10 times higher in the 4 °C cultures and ~ 4 times higher in the 28 °C than in the 17 °C cultures, while the activity of the NR enzyme was generally highest in the cultures grown at 17 °C that were enriched with NO3−. Cells grown under nutrient-replete conditions had significantly higher bSiO2 deposition rates at 4 °C than cells grown at 17 and 28 °C. These data support the notion that cold, nutrient-replete conditions lead to increases in diatom silicification and that NR activity may be regulated downstream of mRNA transcription under specific environmental conditions.

Solid-state NMR spectroscopy represents a powerful method for the investigation of diatom biosilica but detailed studies regarding its chemical composition and structural organization can be prohibited by insufficient spectroscopic sensitivity. Here, we used two-dimensional (2D) Dynamic Nuclear Polarization (DNP)-supported solid-state NMR experiments to obtain information about the molecular composition and supramolecular organization of proteins and carbohydrates in 13C,15N,29Si-labeled biosilica of C. cryptica. As a reference, we conducted DNP experiments on isotope-labeled biosilica of T. pseudonana. DNP-enhancement factors for different NMR signals, and thus, for different organic compounds, provide information about the supramolecular architecture of the biosilica. In addition, DNP-supported heteronuclear nitrogen-carbon correlation experiments allowed us to prove the presence of different structural elements of long chain polyamines (LCPAs) and revealed the occurrence of amine-nitrogen moieties exhibiting a correlation with carbonyl carbons that may indicate cross-linking of LCPAs to proteins as previously seen in studies on proteins extracted from other diatoms.

Alexandrium pacificum is a toxin-producing dinoflagellate with allelopathic effects. The elucidation of allelopathic mechanism of A. pacificum is of great significance for understanding A. pacificum blooms. To this end, using the model diatom Thalassiosira pseudonana as a target species, we observed changes in physiological, biochemical and gene transcription of T. pseudonana upon being co-cultured with A. pacificum. We found reciprocal effects between A. pacificum and T. pseudonana, and corroborated A. pacificum's allelopathy on T. pseudonana by observing inhibitory effects of filtrate from A. pacificum culture on the growth of T. pseudonana. We also found that co-culturing with A. pacificum, the expression of T. pseudonana genes related to photosynthesis, oxidative phosphorylation, antioxidant system, nutrient absorption and energy metabolism were drastically influenced. Coupled with the alterations in Fv/Fm (the variable/maximum fluorescence ratio), activity of superoxide dismutase, contents of malondialdehyde, neutral lipid and total protein in T. pseudonana co-cultured with A. pacificum, we propose that A. pacificum allelopathy could reduce the efficiency of photosynthesis and energy metabolism of T. pseudonana and caused the oxidative stress, while the nutrient absorption was also affected by allelopathic effects. The resultant data potentially uncovered the allelopathic molecular mechanism of A. pacificum to model alga T. pseudonana. The changes in nutrient uptake and even energy metabolism in T. pseudonana, as an adaptation to environmental conditions, may prevent it from stress-related injuries. Our finding might advance the understanding of allelopathic mechanism of A. pacificum.

International audience; Intertidal benthic diatoms experience a highly variable light regime, which especially challenges these organisms to cope with excess light energy during low tide. Non-photochemical quenching of chlorophyll fluorescence (NPQ) is one of the most rapid mechanisms diatoms possess to dissipate excess energy. Its capacity is mainly defined by the xanthophyll cycle (XC) and Light-Harvesting Complex X (LHCX) proteins. Whereas the XC and its relation to NPQ have been relatively well-studied in both planktonic and benthic diatoms, our current knowledge about LHCX proteins and their potential involvement in NPQ regulation is largely restricted to planktonic diatoms. While recent studies using immuno-blotting have revealed the presence of light regulated LHCX proteins in benthic diatom communities and isolates, nothing is as yet known about the diversity, identity and transcriptional regulation or function of these proteins. We identified LHCX genes in the draft genome of the model benthic diatom Seminavis robusta and followed their transcriptional regulation during a day/night cycle and during exposure to high light conditions. The S. robusta genome contains 17 LHCX sequences, which is much more than in the sequenced planktonic model diatoms (4-5), but similar to the number of LHCX genes in the sea ice associated diatom Fragilariopsis cylindrus. LHCX diversification in both species, however, appears to have occurred independently. Interestingly, the S. robusta genome contains LHCX genes that are related to the LHCX6 of the model centric diatom Thalassiosira pseudonana, which are lacking in the well-studied pennate model diatom Phaeodactylum tricornutum. All investigated LHCX genes, with exception of SrLHCX6, were upregulated during the daily dark-light transition. Exposure to 2,000 µmol photons m −2 s −1 , furthermore, increased transcription of all investigated LHCX genes. Our data suggest that the diversification and involvement of several light regulated LHCX genes in the photophysiology of S. robusta may represent an adaptation to the complex and highly variable light environment this benthic diatom species can be exposed to.

International audience; Diatoms are the world's most diverse group of algae, comprising at least 100,000 species. Contributing ;20% of annual global carbon fixation, they underpin major aquatic food webs and drive global biogeochemical cycles. Over the past two decades, Thalassiosira pseudonana and Phaeodactylum tricornutum have become the most important model systems for diatom molecular research, ranging from cell biology to ecophysiology, due to their rapid growth rates, small genomes, and the cumulative wealth of associated genetic resources. To explore the evolutionary divergence of diatoms, additional model species are emerging, such as Fragilariopsis cylindrus and Pseudo-nitzschia multistriata. Here, we describe how functional genomics and reverse genetics have contributed to our understanding of this important class of microalgae in the context of evolution, cell biology, and metabolic adaptations. Our review will also highlight promising areas of investigation into the diversity of these photosynthetic organisms, including the discovery of new molecular pathways governing the life of secondary plastid-bearing organisms in aquatic environments.

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms., Available genomics studies have mostly focused on planktonic centric diatom. Here, the authors report the genome assembly of the marine biofilm-forming diatom Seminavis robusta and the resequencing data of a panel of accessions to reveal their evolutionary adaptations.

Diatoms are a broadly distributed and evolutionarily diversified group of microalgae that produce a diverse range of sterol compounds. Sterols are triterpenoids that play essential roles in membrane-related processes in eukaryotic cells. Some sterol compounds possess bioactivities that promote human health and are currently used as nutraceuticals. The relationship between sterol diversity in diatoms and their acclimation to different environments is not well understood. In this study, we investigated the occurrence of different sterol types across twelve diatom species, as well as the effect of temperature reduction and changes in salinity on the sterol contents of three model diatom species. In the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum and Chaetoceros muelleri, we found that changes in the relative contents of minor sterols accompanied shifts in temperature and salinity. This may be indicative of acquired adaptive traits in diatom metabolism.

The distribution of marine phytoplankton will shift alongside changes in marine environments, leading to altered species frequencies and community composition. An understanding of the response of mixed populations to abiotic changes is required to adequately predict how environmental change may affect the future composition of phytoplankton communities. This study investigated the growth and competitive ability of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, along a temperature gradient (9–35°C) spanning the thermal niches of both species under both high‐nitrogen nutrient‐replete and low‐nitrogen nutrient‐limited conditions. Across this temperature gradient, the competitive outcome under both nutrient conditions at any assay temperature, and the critical temperature at which competitive advantage shifted from one species to the other, was well predicted by the temperature dependencies of the growth rates of the two species measured in monocultures. The temperature at which the competitive advantage switched from P. tricornutum to T. pseudonana increased from 18.8°C under replete conditions to 25.3°C under nutrient‐limited conditions. Thus, P. tricornutum was a better competitor over a wider temperature range in a low N environment. Being able to determine the competitive outcomes from physiological responses of single species to environmental changes has the potential to significantly improve the predictive power of phytoplankton spatial distribution and community composition models., An understanding of the response of mixed populations to abiotic changes is required in order to adequately predict the future composition of phytoplankton communities. This study investigated the growth and competitive ability of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, along a temperature gradient spanning the thermal niches of both species (from 9°C to 35°C) under high‐nitrogen and low‐nitrogen conditions. Competitive outcomes between the two diatoms were highly predictable based on their monoculture growth performances, indicating that their isolated performance can be used to infer competitive ability and resulting community structure.

Microalgal productivity in mass cultures is limited by the inefficiency with which available light energy is utilized. In dense cultures, cells closest to the light source absorb more light energy than they can use and dissipate the excess, while light penetrance into the culture is steeply attenuated. Reducing microalgal light harvesting and/or dissipating capacity per cell may improve total light utilization efficiency in mass cultures. In this study, two transgenic lines of the diatom Thalassiosira pseudonana with altered photosynthetic pigment content are evaluated with respect to photosynthetic parameters, growth, and macromolecule accumulation. In one line, violaxanthin de-epoxidase-like 2 (VDL2) is overexpressed (OE), resulting in a reduction of the diadinoxanthin cycle pigments, which are involved in light energy dissipation (non-photochemical quenching, NPQ), accompanied by a stoichiometric increase in the light-harvesting pigment fucoxanthin. No differences in the maximum potential quantum yield of photosystem II (Fv/Fm) or light-limited photosynthetic rate (α) were found. However, when adapted to 30 µmol photons m−2 sec−1, the VDL2 OE maximum relative electron transport rate (rETRmax) upon exposure to saturating light intensities was 86-95% of wild type (WT). When adapted to 300 µmol photons m−2 sec−1, VDL2 OE saturated photosynthesis at 62-71% of the light intensity needed to saturate WT (Ek). NPQ was substantially lower at and below 300 µmol photons m−2 sec−1. VDL2 OE accumulated up to 3.4 times as much triacylglycerol (TAG) as WT during exponential growth, and up to twice as much protein. Growth in terms of culture density was up to 7% slower. TAG and protein accumulation inversely correlated with NPQ. The second line evaluated was obtained by using antisense RNA to simultaneously silence or knock down (KD) both LUT1-like (LTL) genes, hypothesized to catalyze an intermediate carotenoid biosynthesis step of converting β-carotene to zeaxanthin. Overall reduction of photosynthetic pigment content without altering the relative abundance of individual pigments resulted. No significant differences in photosynthetic parameters compared to WT were found. LTL KD grew at a rate comparable to WT and accumulated up to 40% more TAG during exponential growth, while protein content was reduced by 11-19%. LTL KD cells were elongated and 5-10% smaller than WT, and cultures contained auxospores, indicating stress that may relate to a cell cycle progression defect.

Despite the ubiquity and ecological importance of diatoms, much remains to be understood about their physiology and metabolism, including their carotenoid biosynthesis pathway. Early carotenoid biosynthesis steps are well-conserved, while the identity of the enzymes that catalyze the later steps and their order remain unclear. Those steps lead to the biosynthesis of the final pathway products: the main accessory light-harvesting pigment fucoxanthin (Fx) and the main photoprotective pigment pool comprised of diadinoxanthin (Ddx) and its reversibly de-epoxidized form diatoxanthin (Dtx). We used sequence comparison to known carotenoid biosynthesis enzymes to identify novel candidates in the diatomThalassiosira pseudonana. Microarray and RNA-seq data was used to select candidates with transcriptomic responses similar to known carotenoid biosynthesis genes and to create full-length gene models, and we focused on those that encode proteins predicted to be chloroplast-localized. We identified a violaxanthin de-epoxidase-like gene (Thaps3_11707, VDL2) that when overexpressed results in increased Fx abundance while stoichiometrically reducing Ddx+Dtx. Based on transcriptomics, we hypothesize that Thaps3_10233 may also contribute to Fx biosynthesis, in addition to VDL2. Separately using antisense RNA to target VDL2, VDL1, and both LUT1-like copies (hypothesized to catalyze an earlier step in the pathway) simultaneously, reduced the overall cellular photosynthetic pigment content, including chlorophylls, suggesting destabilization of light-harvesting complexes by Fx deficiency. Based on transcriptomic and physiological data, we hypothesize that the two predictedT. pseudonanazeaxanthin epoxidases have distinct functions and that different copies of phytoene synthase and phytoene desaturase may serve to initiate carotenoid biosynthesis in response to different cellular needs. Finally, nine carotene cis/trans isomerase (CRTISO) candidates identified based on sequence identity to known CRTISO proteins were narrowed to two most likely to be part of theT. pseudonanacarotenoid biosynthesis pathway based on transcriptomic responses and predicted chloroplast targeting.

Diatoms play a key role in the marine carbon cycle with their high primary productivity and release of exudates such as extracellular polymeric substances (EPS) and transparent exopolymeric particles (TEP). These exudates contribute to aggregates (marine snow) that rapidly transport organic material to the seafloor, potentially capturing contaminants like petroleum components. Ocean acidification (OA) impacts marine organisms, especially those that utilize inorganic carbon for photosynthesis and EPS production. Here we investigated the response of the diatom Thalassiosira pseudonana grown to present day and future ocean conditions in the presence of a water accommodated fraction (WAF and OAWAF) of oil and a diluted chemically enhanced WAF (DCEWAF and OADCEWAF). T. pseudonana responded to WAF/DCEWAF but not OA and no multiplicative effect of the two factors (i.e., OA and oil/dispersant) was observed. T. pseudonana released more colloidal EPS (< 0.7 μm to > 3 kDa) in the presence of WAF/DCEWAF/OAWAF/OADCEWAF than in the corresponding Controls. Colloidal EPS and particulate EPS in the oil/dispersant treatments have higher protein-to-carbohydrate ratios than those in the control treatments, and thus are likely stickier and have a greater potential to form aggregates of marine oil snow. More TEP was produced in response to WAF than in Controls; OA did not influence its production. Polyaromatic hydrocarbon (PAH) concentrations and distributions were significantly impacted by the presence of dispersants but not OA. PAHs especially Phenanthrenes, Anthracenes, Chrysenes, Fluorenes, Fluoranthenes, Pyrenes, Dibenzothiophenes and 1-Methylphenanthrene show major variations in the aggregate and surrounding seawater fraction of oil and oil plus dispersant treatments. Studies like this add to the current knowledge of the combined effects of aggregation, marine snow formation, and the potential impacts of oil spills under ocean acidification scenarios.

Diatoms are a diverse and globally important phytoplankton group, responsible for an estimated 20% of carbon fixation on Earth. They frequently form spatially extensive phytoplankton blooms, responding rapidly to increased availability of nutrients, including phosphorus (P) and nitrogen (N). Although it is well established that diatoms are common first responders to nutrient influxes in aquatic ecosystems, little is known of the sensory mechanisms that they employ for nutrient perception. Here, we show that P-limited diatoms use a Ca2+-dependent signaling pathway, not previously described in eukaryotes, to sense and respond to the critical macronutrient P. We demonstrate that P-Ca2+ signaling is conserved between a representative pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom. Moreover, this pathway is ecologically relevant, being sensitive to sub-micromolar concentrations of inorganic phosphate and a range of environmentally abundant P forms. Notably, we show that diatom recovery from P limitation requires rapid and substantial increases in N assimilation and demonstrate that this process is dependent on P-Ca2+ signaling. P-Ca2+ signaling thus governs the capacity of diatoms to rapidly sense and respond to P resupply, mediating fundamental cross-talk between the vital nutrients P and N and maximizing diatom resource competition in regions of pulsed nutrient supply.

This study investigated the effects of microalgae addition on the water quality, accumulation of Vibrios in the water and sediment, and growth performance of whiteleg shrimp (Litopenaeus vannamei) in the intensive culture system. To evaluate the effects of two monospecific microalgae in whiteleg shrimp culture, an 84-day experiment was performed in concrete tanks seeded with Nannochloropsis oculata and Thalassiosira pseudonana at a density of 10 × 104~80 × 104 cells/mL. The results showed that adding T. pseudonana treatment significantly decreased the levels of nitrite-N, nitrate-N, orthophosphate-P, total ammonia nitrogen and suspended solids during the intermediate and the later culture tank. Additionally, the two added microalgae kept pH steady and increased dissolved oxygen (at daytime) in the culture water. We also found that the concentration of Vibrios in water and sediment from adding microalgae treatments was significantly reduced compared to control groups. Moreover, the highest mean weight gain (1.49 ± 0.056 g/week), the growth rate (1.50 ± 0.067 g/week) and the lowest feed conversion rate (1.42 ± 0.023) were observed in T. pseudonana treatment, which were significantly better than that of the control group (F (2, 6) = 13.77, P = 0.006). Productivity in the treatment of T. pseudonana was 3.65 ± 0.095 kg/m3, which was 25% higher than in the control group (2.92 ± 0.065 kg/m3, F (1, 4) = 143.58, P

The neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) are produced by cyanobacteria, diatoms and dinoflagellates and have been detected in seafood worldwide. Ou...

Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering. © Springer-Verlag GmbH Germany, part of Springer Nature 2018. This is a post-peer-review, pre-copyedit version of an article published in Plant Cell Reports. The final authenticated version is available online at: http://dx.doi.org/10.1007/s00299-018-2334-1. Locked until 23.08.2019 due to the copyright restrictions.

Rapid evolution in response to environmental change will likely be a driving force determining the distribution of species across the biosphere in coming decades. This is especially true of microorganisms, many of which may evolve in step with warming, including phytoplankton, the diverse photosynthetic microbes forming the foundation of most aquatic food webs. Here we tested the capacity of a globally important, model marine diatom Thalassiosira pseudonana, for rapid evolution in response to temperature. Selection at 16 and 31°C for 350 generations led to significant divergence in several temperature response traits, demonstrating local adaptation and the existence of trade-offs associated with adaptation to different temperatures. In contrast, competitive ability for nitrogen (commonly limiting in marine systems), measured after 450 generations of temperature selection, did not diverge in a systematic way between temperatures. This study shows how rapid thermal adaptation affects key temperature and nutrient traits and, thus, a population's long-term physiological, ecological, and biogeographic response to climate change.

Sexual reproduction roots the eukaryotic tree of life, although its loss occurs across diverse taxa. Asexual reproduction and clonal lineages persist in these taxa despite theoretical arguments suggesting that individual clones should be evolutionarily short-lived due to limited phenotypic diversity. Here, we present quantitative evidence that an obligate asexual lineage emerged from a sexual population of the marine diatom Thalassiosira pseudonana and rapidly expanded throughout the world’s oceans. Whole genome comparisons identified two lineages with characteristics expected of sexually reproducing strains in Hardy-Weinberg equilibrium. A third lineage displays genomic signatures for the functional loss of sexual reproduction followed by a recent global colonization by a single ancestral genotype. Extant members of this lineage are genetically differentiated and phenotypically plastic, potentially allowing for rapid adaptation when they are challenged by natural selection. Such mechanisms may be expected to generate new clones within marginal populations of additional unicellular species, facilitating the exploration and colonization of novel environments, aided by exponential growth and ease of dispersal.

The regulation of carbon partitioning in diatoms is key to understanding mechanisms related to their high productivity, and the mechanisms controlling carbon flux towards different metabolic fates are currently not well understood. A unique variant of 6-phosphofructo-2-kinase/fructose 2,6 bisphosphatase (PFK2/F2BP), a regulator of glycolytic flux previously uncharacterized in single-cell photosynthetic eukaryotes, was identified in diatom genomes and overexpressed in Thalassiosira pseudonana. Overexpression resulted in increased activity of the glycolytic rate-determining enzyme phosphofructokinase (PFK) and altered carbon partitioning among intracellular carbohydrate, lipid, and protein levels. Higher PFK activity in transformant lines correlated with reduced growth rate and an extension of the G1 phase of the cell cycle, demonstrating a link between carbon metabolism and the control of cell cycle processes. While the relationship between growth and carbon flux under environmental stress (such as nutrient limitation) is well documented in diatoms, our data demonstrate that manipulation of carbon metabolism, independent of an environmental trigger (such as nutrient limitation), is sufficient to delay cell cycle progression. The inverse relationship between growth and PFK activity (as an indicator of glycolytic flux) differs from those described for other unicellular eukaryotes and supports an alternate organization and regulation of central carbon metabolism in diatoms, including a prominent regulatory role for PFK2/F2BP.