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At present, the issue of obtaining a complex of biologically active substances with antioxidant activity from psychrophilic Psychrophilic microalgae and cyanobacteria are a prospective source of biologically active antioxidant substances. New antioxidant complexes could expand the scope of their industrial application. The research objective was to identify psychrophilic microalgae and cyanobacteria from the Baltic Sea in order to study their antioxidant properties. The research featured psychrophilic microalgae and cyanobacteria obtained from the Curonian Lagoon and the Baltic Bay in the Baltic Sea, Kaliningrad Region, Russia, in March – May 2022. The authors used the Pasteur method and the flow cytometry method to isolate pure cultures of psychrophilic microalgae and cyanobacteria. The method of spectrophotometry made it possible to study the antioxidant activity by analyzing radical scavenging, reducing ability, and chelating. The psychrophilic microalga Skeletonema pseudocostatum demonstrated the highest antioxidant activity in all three methods: 17.62 μmol/g trolox equivalents according to the ABTS method, 58.16 μmol/g trolox equivalents according to the DPPH method, and 3.91 μmol/g trolox equivalents according to the FRAP method. The psychrophilic microalga Thalassiosira pseudonana exhibited the following values of antioxidant activity: 12.08, 12.42, and 3.13 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic microalgae Fragilariopsis kerguelensis was 13.53, 11.84, and 1.09 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic cyanobacterium Aphanizomenon gracile was 15.73, 19.89, and 2.47 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic cyanobacterium Anabaena cylindrica was 12.62, 13.16, and 2.16 μmol/g trolox equivalents, respectively. The samples of psychrophilic microalgae and cyanobacteria obtained from natural environment in the Russian Baltic Sea demonstrated good antioxidant properties, which makes them a potential raw material for new pharmaceutical substances.

Thalassiosira pseudonana, a fast-growing and nutritionally rich planktonic diatom, has been cultivated on a large scale as high-quality plankton feed. Despite this, studies exploring the impact of light quality on the growth and compositional attributes of this algal species are scarce. This study investigated the effects of five different light qualities—white, red, yellow, green, and blue—on the growth, biomass, fucoxanthin accumulation, and fatty acid composition and content of T. pseudonana. The results indicated that T. pseudonana achieved the highest growth rate and greatest biomass under white and blue light, with the poorest performance observed under red light. Moreover, fucoxanthin accumulation was most pronounced under blue light, reaching 4.729 ± 0.182 mg g-1, followed in descending order by yellow, red, white, and green light. The total lipid content was highest under yellow light at 34%, significantly surpassing the levels observed under other conditions, with the order of effectiveness being blue light > green light ≥ red light ≥ white light. Analysis of fatty acid composition revealed a greater diversity under blue light, with a total of 15 types, whereas green light yielded only 10 types. The proportion of unsaturated fatty acids was highest under blue light at 69.71%, followed by yellow, white, green, and red light. Specifically, the performance of polyunsaturated fatty acids (PUFAs) varied as follows: yellow light (36.7%) > blue light (34.09%) > white light (33.29%) > green light (23.67%) > red light (17%). The concentration of eicosapentaenoic acid (EPA) was significantly greater under blue (17.03%) and yellow (16.31%) light compared to other treatments, with the lowest concentration (8.84%) observed under red light. In conclusion, blue light significantly enhances the growth rate of T. pseudonana and fosters fucoxanthin accumulation. Both yellow and blue light are favorable for the accumulation of PUFAs and EPA. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein–protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Perfluoroalkyl substances (PFAS) are widely dispersed persistent organic pollutants (POPs) throughout marine ecosystems. Due to ban of traditional long-chain PFAS, the emerging short-chain ones showed increased environmental detection as substitutes. As the foundation of aquatic food webs, microalgae play a pivotal role in the stability of marine environments. However, the toxicity of those short-chain PFAS was lack of investigation. Therefore, we chose 4C PFAS perfluorobutanoic acid (PFBA) and the marine model diatom Thalassiosira pseudonana as research targets, comprehensively studied the toxicity of PFBA to T. pseudonana in terms of the population growth, photosynthetic physiology and oxidative stress. Our results characterized the inhibited growth, inhibited photosynthetic parameters, increased reactive oxygen species (ROS) levels and activated antioxidant system under PFBA exposure. Further transcriptome analysis revealed the underlying molecular mechanisms: photosynthetic genes were slightly down-regulated and the expression of oxidative stress-related genes was enhanced; significant up-regulation of genes related to the DNA excision repair and replication-coupled DNA repair pathways; the expression of carbon metabolisms-related genes was increased, including the Calvin cycle, glycolysis, pentose phosphate pathway, tricarboxylic acid (TCA) cycle and fatty acid biosynthesis, that could provide sufficient energy for the recovery processes of microalgal cells. This study elucidated the underlying toxic mechanisms of PFBA on phytoplankton, and provided novel insights for assessing the environmental risks of PFAS.

This research aims to assess the influence of varying concentrations of caffeine (CA), one of the pharmaceutically active compounds, and its metabolites (i.e., paraxanthine (PX), theophylline (TP), and theobromine (TB)) on the growth of common marine phytoplankton (Isochrysis galbana and Thalassiosira pseudonana) under controlled laboratory conditions. Additionally, the study estimates the daily discharge of CA into marine ecosystems utilizing a developed model. The most effective metabolite inhibiting the proliferation of I. galbana was determined as TP (68.5%) at a concentration of 500 mg/L, while it was CA (65.8%) at the same concentration for T. pseudonana. After exposure to CA, the IC50 values for I. galbana and T. pseudonana were calculated as 107.3 mg/L and 136.8 mg/L, respectively after 96 hours. Furthermore, the lowest IC50 concentration was 7.4 mg/L in TP treatment in I. galbana. Based on a computational model developed in the paper, in a province with about 2 million inhabitants, where all wastewater is treated in wastewater treatment plants (WWTP), the quantity of CA discharged into the sea was computed as 8.11 kg/day. The CA amounts reaching the sea were potentially increased in regions devoid of wastewater treatment infrastructure. Consequently, WWTPs should undergo expansion and enhancement of their capacity, including advanced treatment for chemical removal for sustainable marine life. Additionally, further studies are needed to elucidate the mechanisms underlying the inhibition effects of CA on phytoplankton. [ABSTRACT FROM AUTHOR]

The effects of turbulence on phytoplankton growth have received considerable attentions. However, the complexity of turbulence poses a significant challenge to its systematic characterization in the laboratory, resulting in relatively limited data on the effects of turbulence on several algal species. Here, a laboratory turbulence simulation system was set up to systematically investigate the growth of three common diatom species (Thalassiosira pseudonana, Skeletonema costatum, and Phaeodactylum tricornutum) under stationary and turbulent conditions (at 60, 120, 180 rpm), and measurements were taken for the algal biomass, algal photosynthetic activity, and nutrients consumption. By comparing the growth of three algae species under different turbulence exposure intensities, this study found that different algae exhibit varying sensitivities to turbulence, and therefore have different shear thresholds. Meanwhile, cell morphology is the key factor influencing the different shear threshold values observed in the three diatom species. Additionally, turbulence could impact algal aggregation and light availability, and dramatically improve nutrient uptake by phytoplankton. Our study will provides theoretical support for future endeavors in using turbulence to cultivate phytoplankton or combat algal blooms. [ABSTRACT FROM AUTHOR]

Microalgal cells are the major contributors of extracellular polymeric substances (EPS) in the marine environment, which could be influenced by exogenous pollutants exposure. As the most dominant brominated flame retardant worldwide, studies on the influence of tetrabromobisphenol A (TBBPA) on microalgal cells and EPS production are rare. Therefore, we systemically investigated the physiological-biochemical responses of the marine diatom Thalassiosira pseudonana to TBBPA exposure and the roles of EPS. Our results indicated that the half effective concentrations (EC50) of TBBPA on T. pseudonana were 1.67–3.22 mg L−1 from 24 to 96 h; Photosynthetic damage, cellular materials increase, cellular oxidative stress and cell death were found under the toxicity of TBBPA, in which cellular materials increase and oxidative stress were positively correlated with the increase of EPS contents. The increase of soluble and bound EPS contents, protein/carbohydrate ratios together with the EPS addition assays indicated the protective effects of EPS to alleviate the toxicity of TBBPA. Increased microalgal EPS secretions and protein/carbohydrate ratios also happened under the environmental TBBPA concentration (2 μg L−1), which might accelerate the organic matters aggregation process especially in coastal areas. [ABSTRACT FROM AUTHOR]

Diatoms form a major component of phytoplankton. These eukaryotic organisms are responsible for approximately 40% of primary productivity in the oceans and contribute significantly to the food web. Here, the influences of ultraviolet radiation (UVR) and ocean warming on diatom photosynthesis were investigated in Thalassiosira pseudonana. The organism was grown at two temperatures, namely, 18°C, the present surface water temperature in summer, and 24°C, an estimate of surface temperature in the year 2,100, under conditions of high photosynthetically active radiation (P, 400–700 nm) alone or in combination with UVR (P + UVR, 295–700 nm). It was found that the maximum photochemical yield of PSII (Fv/Fm) in T. pseudonana was significantly decreased by the radiation exposure with UVR at low temperature, while the rise of temperature alleviated the inhibition induced by UVR. The analysis of PSII subunits turnover showed that high temperature alone or worked synergistically with UVR provoking fast removal of PsbA protein (KPsbA), and also could maintain high PsbD pool in T. pseudonana cells. With the facilitation of PSII repair process, less non-photochemical quenching (NPQ) occurred at high temperature when cells were exposed to P or P + UVR. In addition, irrespective of radiation treatments, high temperature stimulated the induction of SOD activity, which partly contributed to the higher PSII repair rate constant (Krec) as compared to KPsbA. Our findings suggest that the rise in temperature could benefit the photosynthetic performance of T. pseudonana via modulation of its PSII repair cycle and protective capacity, affecting its abundance in phytoplankton in the future warming ocean. [ABSTRACT FROM AUTHOR]

Summary: Marine planktonic diatoms are among the most important contributors to phytoplankton blooms and marine net primary production. Their ecological success has been attributed to their ability to rapidly respond to changing environmental conditions.Here, we report common molecular mechanisms used by the model marine diatom Thalassiosira pseudonana to respond to 10 diverse environmental stressors using RNA‐Seq analysis.We identify a specific subset of 1076 genes that are differentially expressed in response to stressors that induce an imbalance between energy or resource supply and metabolic capacity, which we termed the diatom environmental stress response (d‐ESR). The d‐ESR is primarily composed of genes that maintain proteome homeostasis and primary metabolism. Photosynthesis is strongly regulated in response to environmental stressors but chloroplast‐encoded genes were predominantly upregulated while the nuclear‐encoded genes were mostly downregulated in response to low light and high temperature.In aggregate, these results provide insight into the molecular mechanisms used by diatoms to respond to a range of environmental perturbations and the unique role of the chloroplast in managing environmental stress in diatoms. This study facilitates our understanding of the molecular mechanisms underpinning the ecological success of diatoms in the ocean. [ABSTRACT FROM AUTHOR]

Introduction: In their natural environment, microalgae can be transiently exposed to hypoxic or anoxic environments. Whereas fermentative pathways and their interactions with photosynthesis are relatively well characterized in the green alga model Chlamydomonas reinhardtii, little information is available in other groups of photosynthetic micro-eukaryotes. In C. reinhardtii cyclic electron flow (CEF) around photosystem (PS) I, and light-dependent oxygensensitive hydrogenase activity both contribute to restoring photosynthetic linear electron flow (LEF) in anoxic conditions. Methods: Here we analyzed photosynthetic electron transfer after incubation in dark anoxic conditions (up to 24 h) in two secondary microalgae: the marine diatom Thalassiosira pseudonana and the excavate Euglena gracilis. Results: Both species showed sustained abilities to prevent over-reduction of photosynthetic electron carriers and to restore LEF. A high and transient CEF around PSI was also observed specifically in anoxic conditions at light onset in both species. In contrast, at variance with C. reinhardtii, no sustained hydrogenase activity was detected in anoxic conditions in both species. Discussion: Altogether our results suggest that another fermentative pathway might contribute, along with CEF around PSI, to restore photosynthetic activity in anoxic conditions in E. gracilis and T. pseudonana. We discuss the possible implication of the dissimilatory nitrate reduction to ammonium (DNRA) in T. pseudonana and the wax ester fermentation in E. gracilis. [ABSTRACT FROM AUTHOR]

The projected ocean acidification (OA) associated with increasing atmospheric CO2 alters seawater chemistry and hence the bio-toxicity of metal ions. However, it is still unclear how OA might affect the long-term resilience of globally important marine microalgae to anthropogenic metal stress. To explore the effect of increasing pCO2 on copper metabolism in the diatom Thalassiosira pseudonana (CCMP 1335), we employed an integrated eco-physiological, analytical chemistry, and transcriptomic approach to clarify the effect of increasing pCO2 on copper metabolism of Thalassiosira pseudonana across different temporal (short-term vs. longterm) and spatial (indoor laboratory experiments vs. outdoor mesocosms experiments) scales. We found that increasing pCO2 (1,000 and 2,000 μatm) promoted growth and photosynthesis, but decreased copper accumulation and alleviated its bio-toxicity to T. pseudonana. Transcriptomics results indicated that T. pseudonana altered the copper detoxification strategy under OA by decreasing copper uptake and enhancing copper-thiol complexation and copper efflux. Biochemical analysis further showed that the activities of the antioxidant enzymes glutathione peroxidase (GPX), catalase (CAT), and phytochelatin synthetase (PCS) were enhanced to mitigate oxidative damage of copper stress under elevated CO2. Our results provide a basis for a better understanding of the bioremediation capacity of marine primary producers, which may have profound effect on the security of seafood quality and marine ecosystem sustainability under further climate change. [ABSTRACT FROM AUTHOR]

Diatoms are unicellular algae and occur ubiquitously in almost every marine and freshwater habitat on earth. They produce intricately structured cell walls, which mainly consist of amorphous silica. To synthesize their cell walls, diatoms take up monosilicic acid from the environment and store it. These silicon storage pools (SSPs) can exceed the solubility of silicic acid by one to two orders of magnitude, as observed in various diatom species. However, their chemical composition and cellular localization has not yet been elucidated. It is suggested that SSPs may consist of stabilized aggregates such as pre-condensed silica particles or silica-containing vesicles. Isolation protocols for SSPs without significant chemical modification are required to prove such hypotheses. A critical issue is the efficient separation of components of the SSPs from cell wall fragments or artefacts, which may interfere with analytical methods targeting silicon. To this end, a comparative study was performed on exponentially grown cells and extracted, purified cell walls (biosilica) to observe the sedimentation behavior after lysis. Cell cultures were lysed by bead beating and then fractionated by differential centrifugation. The obtained fractions were analyzed for total silicon content (tSi) using molybdenum blue assay (MBA) after alkaline treatment. It was revealed that cell wall fragments are almost absent in fractions above 1000 × g. Compared with biosilica, a significantly higher silicon concentration is found in lysed cell pellets after centrifugation at moderately high forces. The differences correspond to a few percent of total cellular silicon, which are assumed to be part of SSPs. Only relatively low amounts of silica/silicic acid remain in the supernatant at high centrifugal forces. This indicates that SSPs are mainly present in larger aggregates that sediment at lower centrifugal forces. According to Stokes' law, only silica particles below ca. 25 nm radius would remain in the final supernatant. This leads to the conclusion that SSPs must mainly consist of larger silica particles and/or are associated with larger compartments/aggregates. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave a silent phenotype in T. pseudonana under LC–HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles. [ABSTRACT FROM AUTHOR]

Summary: CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life‐cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism.Homologous recombination was induced in T. pseudonana using sequence‐specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT).Up to c. 85% of NAT‐resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell‐size regulation in centric diatoms.Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms. [ABSTRACT FROM AUTHOR]

IntroductionIn their natural environment, microalgae can be transiently exposed to hypoxic or anoxic environments. Whereas fermentative pathways and their interactions with photosynthesis are relatively well characterized in the green alga model Chlamydomonas reinhardtii, little information is available in other groups of photosynthetic micro-eukaryotes. In C. reinhardtii cyclic electron flow (CEF) around photosystem (PS) I, and light-dependent oxygen-sensitive hydrogenase activity both contribute to restoring photosynthetic linear electron flow (LEF) in anoxic conditions.MethodsHere we analyzed photosynthetic electron transfer after incubation in dark anoxic conditions (up to 24 h) in two secondary microalgae: the marine diatom Thalassiosira pseudonana and the excavate Euglena gracilis.ResultsBoth species showed sustained abilities to prevent over-reduction of photosynthetic electron carriers and to restore LEF. A high and transient CEF around PSI was also observed specifically in anoxic conditions at light onset in both species. In contrast, at variance with C. reinhardtii, no sustained hydrogenase activity was detected in anoxic conditions in both species.DiscussionAltogether our results suggest that another fermentative pathway might contribute, along with CEF around PSI, to restore photosynthetic activity in anoxic conditions in E. gracilis and T. pseudonana. We discuss the possible implication of the dissimilatory nitrate reduction to ammonium (DNRA) in T. pseudonana and the wax ester fermentation in E. gracilis.

Environmental variability is an inherent feature of natural systems which complicates predictions of species interactions. Primarily, the complexity in predicting the response of organisms to environmental fluctuations is in part because species' responses to abiotic factors are non‐linear, even in stable conditions. Temperature exerts a major control over phytoplankton growth and physiology, yet the influence of thermal fluctuations on growth and competition dynamics is largely unknown. To investigate the limits of coexistence in variable environments, stable mixed cultures with constant species abundance ratios of the marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were exposed to different temperature fluctuation regimes (n = 17) under high and low nitrogen (N) conditions. Here we demonstrate that phytoplankton exhibit substantial resilience to temperature variability. The time required to observe a shift in the species abundance ratio decreased with increasing fluctuations, but coexistence of the two model species under high N conditions was disrupted only when amplitudes of temperature fluctuation were high (±8.2°C). N limitation caused the thermal amplitude for disruption of species coexistence to become lower (±5.9°C). Furthermore, once stable conditions were reinstated, the two species differed in their ability to recover from temperature fluctuations. Our findings suggest that despite the expectation of unequal effect of fluctuations on different competitors, cycles in environmental conditions may reduce the rate of species replacement when amplitudes remain below a certain threshold. Beyond these thresholds, competitive exclusion could, however, be accelerated, suggesting that aquatic heatwaves and N availability status are likely to lead to abrupt and unpredictable restructuring of phytoplankton community composition. [ABSTRACT FROM AUTHOR]

Perfluoroalkyl substances (PFAS) are widely dispersed persistent organic pollutants (POPs) throughout marine ecosystems. Due to ban of traditional long-chain PFAS, the emerging short-chain ones showed increased environmental detection as substitutes. As the foundation of aquatic food webs, microalgae play a pivotal role in the stability of marine environments. However, the toxicity of those short-chain PFAS was lack of investigation. Therefore, we chose 4C PFAS perfluorobutanoic acid (PFBA) and the marine model diatom Thalassiosira pseudonana as research targets, comprehensively studied the toxicity of PFBA to T. pseudonana in terms of the population growth, photosynthetic physiology and oxidative stress. Our results characterized the inhibited growth, inhibited photosynthetic parameters, increased reactive oxygen species (ROS) levels and activated antioxidant system under PFBA exposure. Further transcriptome analysis revealed the underlying molecular mechanisms: photosynthetic genes were slightly down-regulated and the expression of oxidative stress-related genes was enhanced; significant up-regulation of genes related to the DNA excision repair and replication-coupled DNA repair pathways; the expression of carbon metabolisms-related genes was increased, including the Calvin cycle, glycolysis, pentose phosphate pathway, tricarboxylic acid (TCA) cycle and fatty acid biosynthesis, that could provide sufficient energy for the recovery processes of microalgal cells. This study elucidated the underlying toxic mechanisms of PFBA on phytoplankton, and provided novel insights for assessing the environmental risks of PFAS. • PFBA exhibited toxic effects on T. pseudonana in terms of population growth and physiological-biochemical parameters. • Oxidative stress was induced in T. pseudonana under PFBA exposure. • Various DNA repair pathways were activated in response to PFBA stress. • Enhanced carbon metabolisms provided sufficient energy for T. pseudonana to restore physiological activities. [ABSTRACT FROM AUTHOR]

The projected ocean acidification (OA) associated with increasing atmospheric CO2 alters seawater chemistry and hence the bio-toxicity of metal ions. However, it is still unclear how OA might affect the long-term resilience of globally important marine microalgae to anthropogenic metal stress. To explore the effect of increasing pCO2 on copper metabolism in the diatom Thalassiosira pseudonana (CCMP 1335), we employed an integrated eco-physiological, analytical chemistry, and transcriptomic approach to clarify the effect of increasing pCO2 on copper metabolism of Thalassiosira pseudonana across different temporal (short-term vs. long-term) and spatial (indoor laboratory experiments vs. outdoor mesocosms experiments) scales. We found that increasing pCO2 (1,000 and 2,000 μatm) promoted growth and photosynthesis, but decreased copper accumulation and alleviated its bio-toxicity to T. pseudonana. Transcriptomics results indicated that T. pseudonana altered the copper detoxification strategy under OA by decreasing copper uptake and enhancing copper-thiol complexation and copper efflux. Biochemical analysis further showed that the activities of the antioxidant enzymes glutathione peroxidase (GPX), catalase (CAT), and phytochelatin synthetase (PCS) were enhanced to mitigate oxidative damage of copper stress under elevated CO2. Our results provide a basis for a better understanding of the bioremediation capacity of marine primary producers, which may have profound effect on the security of seafood quality and marine ecosystem sustainability under further climate change.

Diatoms can provide important paleoenvironmental information about seasonal sea ice extent, productivity, sea surface temperature and ocean circulation variability, yet there are relatively few studies analysing the last glacial cycle near the Antarctic continent. This study examines diatom assemblages over the last glacial cycle from core TAN1302-44, from off Adélie Land, East Antarctica. Four distinct diatom assemblages were identified using principal components analyses. The PC 1 assemblage is associated with the interglacial, sedimentary facies, Facies 1, and comprises Thalassiosira lentiginosa, Actinocyclus actinochilus, Eucampia antarctica, Azpeitia tabularis and Asteromphalus hyalinus , suggesting that MIS 5e and Holocene interglacial time periods were characterised by seasonal sea ice environments with similar ocean temperature and circulation. The PC 2 assemblage is associated with the glacial, Facies 2, and comprises Fragilariopsis obliquecostata, Asteromphalus parvulus, Rhizosolenia styliformis, Thalassiosira tumida, Chaetoceros dichaeta , and a Eucampia antarctica terminal/intercalary ratio. This indicates that, during the MIS 4-2 glacial there was an increase in the length of the sea ice season compared with the interglacial period, yet still no permanent sea ice cover. The PC 2 assemblage is also associated with the glaciation and deglacial facies. There is an initial increase of PC 2 at the start of MIS 5d-a glaciation stage and then a gradual increase throughout late MIS 4-2, suggests that sea ice cover steadily increased reaching a maximum at the end of MIS 2. The PC 3 assemblage is associated with all four facies and comprises Actinocyclus ingens, Actinocyclus actinochilus, Thalassiosira oliverana and Fragilariopsis kerguelensis , suggesting that reworking of sediments and an influx of older sediments occurred throughout the last glacial cycle. Finally, the PC 4 assemblage is associated with the deglacial, glaciation, and glacial facies and comprises Fragilariopsis kerguelensis , Thalassiothrix antarctica, Chaetoceros bulbosum and Eucampia antarctica , suggesting that during the last glaciation, the last two deglacials, and the early glacial, there was a period of enhanced upwelling of nutrient-rich, warmer water, which is inferred to reflect an increase in Circumpolar Deep Water. Interestingly, the diatom data suggest the onset of increased Circumpolar Deep Water during the last deglacial occurred after the rapid loss of a prolonged sea ice season at the end of last glacial. Together, these results suggest changes in ocean circulation and sea ice season were important factors during climate transitions. The results fill a gap in our understanding of the sea ice extent and ocean circulation changes proximal to East Antarctica over the last glacial cycle. [ABSTRACT FROM AUTHOR]

The mutual influence of the rafidophyte microalgae Heterosigma akashiwo, which causes algal blooms, and the diatom Thalassiosira pseudonana, which is regularly present in sea waters, was studied under experimental conditions. Experiments were carried out with monocultures, monocultural filtrates, and mixed cultures of different initial concentrations. The growth and physiological state of the microalgae were assessed by flow cytometry. Both algae displayed inhibited growth in co-culture and on filtrates, but growth was suppressed to a greater extent in T. pseudonana. The fluorescence of chlorophyll a and the contents of reactive oxygen species and neutral lipids decreased in H. akashiwo cultures grown on T. pseudonana filtrate. Similar changes were noted in a T. pseudonana culture grown on H. akashiwo filtrate. It is concluded that these algae exhibit allelopathic activity in relation to each other. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a / c -binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein-protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis , underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms., Competing Interests: KK, YN, JX, MK, HO, JS, KI, RN No competing interests declared, (© 2024, Kato, Nakajima, Xing et al.)

Increasing marine microplastics (MPs) pollution potentially threatens the stability of phytoplankton community structures in marine environments. MPs toxicities to microalgae are largely determined by particle size, while the size-dependent mechanisms are still not fully understood. In this study, two sizes (0.1 µm and 1 µm) of polystyrene (PS) MPs were used as experimental targets to systemically compare their different effecting mechanisms on the marine model diatom Thalassiosira pseudonana with respect to oxidative stress and photosynthesis. The results indicated the toxicity of 1 µm sized MPs was higher than 0.1 µm sized MPs regarding to population growth. In condition of similar microalgal population inhibition rates, we found more enhanced cellular oxidative stress and cell death happened in the 1 µm MPs treatments, which could be linked to higher zeta potential of 1 µm MPs and more severe cell surface damage; microalgal surface light shading and cellular pigments decline were more obvious in the 0.1 µm MPs treatment, which could be linked to high aggregation abilities of 0.1 µm MPs. Gene expressions supported the morphological and physiological findings on the transcriptional level. Environmental related MPs concentrations (5 μg L−1) also aroused gene expression changes of T. pseudonana while more changing genes were found under 0.1 µm MPs than 1 µm MPs. These results provide novel insights into the size-dependent mechanisms of MPs toxicity on marine microalgae, as well as their potential influence on the marine environment.

Abstract Background The marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds. Results We have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes. Conclusion Despite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is ‘better’ than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.

SUMMARY: Morphogenesis of the intricate patterns of diatom silica cell walls is a protein‐guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species‐specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In‐depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall. Significance Statement: This comprehensive analysis of silica‐associated diatom proteins employs a combination of proteomics, bioinformatics, and protein localization studies to identify candidate proteins involved in silica biogenesis. Through in‐depth statistical analysis and phylogenomics, we show that these proteins are dominated by intrinsically disordered domains, are enriched in unconventional sequence motifs, and identify potential members of the basal silica biomineralization machinery as well as proteins that probably exert species‐specific functions in this process. [ABSTRACT FROM AUTHOR]

Summary: Housekeeping genes (HKGs) are constitutively expressed with low variation across tissues/conditions. They are thought to be highly conserved and fundamental to cellular maintenance, with distinctive genomic features.Here, we identify 1505 HKGs in the unicellular marine diatom Thalassiosira pseudonana based on an RNA‐seq analysis of 232 samples taken under 12 experimental conditions over 0–72 h. We identify promising internal reference genes (IRGs) for T. pseudonana from the most stably expressed HKGs.A comparative analysis indicates < 18% of HKGs in T. pseudonana have orthologs in other eukaryotes, including other diatom species. Contrary to work on human tissues, T. pseudonana HKGs are longer than non‐HKGs, due to elongated introns. More ancient HKGs tend to be shorter than more recent HKGs, and expression levels of HKGs decrease more rapidly with gene length relative to non‐HKGs.Our results indicate that HKGs are highly variable across the tree of life and thus unlikely to be universally fundamental for cellular maintenance. We hypothesize that the distinct genomic features of HKGs of T. pseudonana may be a consequence of selection pressures associated with high expression and low variance across conditions. [ABSTRACT FROM AUTHOR]

Abstract 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.

Phloroglucinol improves shoot formation and somatic embryogenesis in several horticultural and grain crops, but its function in microalgae remains unclear. Here, we found that sufficiently high concentrations of phloroglucinol significantly increased fucoxanthin synthesis, growth, and photosynthetic efficiency in the microalga Thalassiosira pseudonana. These results suggested that the role of phloroglucinol is conserved across higher plants and microalgae. Further analysis showed that, after phloroglucinol treatment, the contents of cis-zeatin and brassinolide in T. pseudonana increased significantly, while the contents of trans-zeatin, N6-isopentenyladenine (iP), auxin, and gibberellin were unaffected. Indeed, functional studies showed that the effects of cis-zeatin and brassinolide in T. pseudonana were similar to those of phloroglucinol. Knockout of key enzyme genes in the ciszeatin synthesis pathway of T. pseudonana or treatment of T. pseudonana with a brassinolide synthesis inhibitor (brassinazole) significantly reduced growth and fucoxanthin content in T. pseudonana, and phloroglucinol treatment partially alleviated these inhibitory effects. However, phloroglucinol treatment was ineffective when the cis-zeatin and brassinolide pathways were simultaneously inhibited. These results suggested that the cis-zeatin and brassinolide signaling pathways are independent regulators of fucoxanthin synthesis in T. pseudonana and that phloroglucinol affects both pathways. Thus, this study not only characterizes the mechanism by which phloroglucinol promotes fucoxanthin synthesis but also demonstrates the roles of cis-zeatin and brassinolide in T. pseudonana. [ABSTRACT FROM AUTHOR]

Biomass and lipid production by the marine centric diatom Thalassiosira pseudonana were characterized in media based on palm oil mill effluent (POME) as a source of key nutrients. The optimal medium comprised 20% by volume POME, 80 µM Na2SiO3, and 35 g NaCl L−1 in water at pH ~7.7. In 15-day batch cultures (16:8 h/h light–dark cycle; 200 µmol photons m−2 s−1, 26 ± 1 °C) bubbled continuously with air mixed with CO2 (2.5% by vol), the peak concentration of dry biomass was 869 ± 14 mg L−1 corresponding to a productivity of ~58 mg L−1 day−1. The neutral lipid content of the biomass was 46.2 ± 1.1% by dry weight. The main components of the esterified lipids were palmitoleic acid methyl ester (31.6% w/w) and myristic acid methyl ester (16.8% w/w). The final biomass concentration and the lipid content were affected by the light–dark cycle. Continuous (24 h light) illumination at the above-specified irradiance reduced biomass productivity to ~54 mg L−1 day−1 and lipid content to 38.1%. [ABSTRACT FROM AUTHOR]

Temperature is one of the environmental factors that most strongly influence the life of microalgae, from the enzymatic level up to the modification of biosynthetic and degradation potential and, thus, cellular composition. When exposed to a change in environmental milieu two main responses can be attained by cells: acclimation or homoeostasis. Recent studies testing the impact of other environmental factors indicated that the choice between these strategies is related to growth rates, i.e. cells dividing at a faster rate showed inclination to acclimation while cells dividing at a slower rate were more prone to homoeostasis. These findings motivated us to further explore the extent to which these compositional response modes are applied under varying temperature. Two representative marine diatoms, one centric, Thalassiosira pseudonana, and one pennate, Cylindrotheca fusiformis were investigated. Species were cultured at 15°C, 20°C and 30°C and at two growth rates. Compositional response was analysed with respect to elemental stoichiometry (carbon, nitrogen, silica, phosphorus, sulphur, manganese, iron, zinc and copper) and the major organic pool ratios (carbohydrate:lipid, carbohydrate:protein and protein:lipid). While T. pseudonana exhibited an acclimation response to temperature, C. fusiformis proved to be strongly homoeostatic. In the case of T. pseudonana, our data showed that the variations in composition were consistent with the growth rate dependence hypothesis for many micronutrients, but not for carbon and nitrogen contents, and C:N ratio. These species-specific differences in response mode highlight the diversity in ecophysiological strategies of diatoms, which needs to be considered when predicting climate change responses. [ABSTRACT FROM AUTHOR]

As emerging pollutants in the aquatic environments, micro- and nano-plastics (MNPs) aroused widespread environmental concerns for their potential threats to the ecological health. Previous research has proved that microalgae growth could recover from the MNPs toxicities, in which the extracellular polymeric substances (EPS) might play the key role. In order to comprehensively investigate the recovery process of microalgae from MNPs stress and the effecting mechanisms of EPS therein, this study conducted a series of experiments by employing two sizes (0.1 and 1 μm) of polystyrene (PS) MNPs and the marine model diatom Thalassiosira pseudonana during 14 days. The results indicated: the pigments accumulations and photosynthetic recovery of T. pseudonana under MPs exposure showed in the early stage (4–5 days), while the elevation of reactive oxygen species (ROS) and EPS contents lasted longer time period (7–8 days). EPS was aggregated with MNPs particles and microalgal cells, corresponding to the increased settlement rates. More increase of soluble (SL)-EPS contents was found than bound (B)-EPS under MNPs exposure, in which the increase of the protein proportion and humic acid-like substances in SL-EPS was found, thus facilitating aggregates formation. ROS was the signaling molecule mediating the overproduction of EPS. The transcriptional results further proved the enhanced EPS biosynthesis on the molecular level. Therefore, this study elucidated the recovery pattern of microalgae from MNPs stress and linked "ROS-EPS production changes-aggregation formation" together during the growth recovery process, with important scientific and environmental significance. [Display omitted] • Microalgae recovered time orderly under MNPs stress. • Increased EPS contents and changes of EPS compositions were stimulated by MNPs. • Aggregations of MNPs particles and microalgal cells were caused by EPS. • ROS was the signaling molecule for mediating EPS overproduction. [ABSTRACT FROM AUTHOR]

Hypoxic zones and oceanic deoxygenation are spreading worldwide due to anthropogenic activities and climate change, greatly affecting marine organisms exposed to lowered O 2. Yet, the effects of the lowered O 2 on phytoplankton are often neglected when studying O 2 effects as they are the O 2 producers. Here we showed that low O 2 (dissolved O 2 , 150 ± 10 μmol L−1) enhanced the growth of the marine diatom Thalassiosira pseudonana in limited light but reduced it in moderate to inhibitory light and that hypoxia (40 ± 7.5 μmol L−1) reduced its growth at any growth lights. Low O 2 and hypoxia decreased cellular chlorophyll a and carotenoid content under growth light from 30 to 400 µmol photons m−2 s−1, as well as photosynthetic efficiency (F V /F M). Such a decrease effect by low O 2 or hypoxia also occurred in the O 2 production by photosynthesis and the O 2 consumption by dark respiration, especially under moderate to inhibitory growth light. Malondialdehyde, a product of lipid peroxidation, increased by up to 200% under low O 2 , and by 400% under hypoxia at the highest light. The lowered O 2 mechanistically down-regulated the expression of proteins related to chlorophyll synthesis, light energy conversion and transfer, and carbon metabolism. In addition, we found that the lowered O 2 generally decreased the fatty acid content by more than 30%, along with the down-regulation of proteins related to the synthesis of these fatty acids and the elongation and desaturation of carbon chains. This suggests the change in the nutrition of marine grazers' food and thus the potential impacts on the food chains in increasingly deoxygenated/hypoxic coastal waters. [Display omitted] • Growth response of diatom Thalassiosira pseudonana to low O 2 is light-dependent, but not to hypoxia. • Lowered O 2 decreases photosynthesis and respiration and increases cell membrane damage. • Lowered O 2 decreases cellular fatty acid content, accompanied by a downregulation of proteins related to its synthesis. [ABSTRACT FROM AUTHOR]

Abstract Temperature effects on the fatty acid (FA) profiles of phytoplankton, major primary producers in the ocean, have been widely studied due to their importance as industrial feedstocks and to their indispensable role as global producers of long‐chain, polyunsaturated FA (PUFA), including omega‐3 (ω3) FA required by organisms at higher trophic levels. The latter is of global ecological concern for marine food webs, as some evidence suggests an ongoing decline in global marine‐derived ω3 FA due to both a global decline in phytoplankton abundance and to a physiological reduction in ω3 production by phytoplankton as temperatures rise. Here, we examined both short‐term (physiological) and long‐term (evolutionary) responses of FA profiles to temperature by comparing FA thermal reaction norms of the marine diatom Thalassiosira pseudonana after ~500 generations (ca. 2.5 years) of experimental evolution at low (16°C) and high (31°C) temperatures. We showed that thermal reaction norms for some key FA classes evolved rapidly in response to temperature selection, often in ways contrary to our predictions based on prior research. Notably, 31°C‐selected populations showed higher PUFA percentages (including ω3 FA) than 16°C‐selected populations at the highest assay temperature (31°C, above T. pseudonana's optimum temperature for population growth), suggesting that high‐temperature selection led to an evolved ability to sustain high PUFA production at high temperatures. Rapid evolution may therefore mitigate some of the decline in global phytoplankton‐derived ω3 FA production predicted by recent studies. Beyond its implications for marine food webs, knowledge of the effects of temperature on fatty acid profiles is of fundamental importance to our understanding of the mechanistic causes and consequences of thermal adaptation.

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.

Background: The marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds. Results: We have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes. Conclusion: Despite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is 'better' than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diatoms belong to a major, diverse and species-rich eukaryotic clade, the Heterokonta, within the polyphyletic chromalveolates. They evolved as a result of secondary endosymbiosis with one or more Plantae ancestors, but their precise evolutionary history is enigmatic. Nevertheless, this has conferred them with unique structural and biochemical properties that have allowed them to flourish in a wide range of different environments and cope with highly variable conditions. We review the effect of pH, light and dark, and CO2 concentration on the regulation of carbon uptake and assimilation. We discuss the regulation of the Calvin-Benson-Bassham cycle, glycolysis, lipid synthesis, and carbohydrate synthesis at the level of gene transcripts (transcriptomics), proteins (proteomics) and enzyme activity. In contrast to Viridiplantae where redox regulation of metabolic enzymes is important, it appears to be less common in diatoms, based on the current evidence, but regulation at the transcriptional level seems to be widespread. The role of post-translational modifications such as phosphorylation, glutathionylation, etc., and of protein-protein interactions, has been overlooked and should be investigated further. Diatoms and other chromalveolates are understudied compared to the Viridiplantae, especially given their ecological importance, but we believe that the ever-growing number of sequenced genomes combined with proteomics, metabolomics, enzyme measurements, and the application of novel techniques will provide a better understanding of how this important group of algae maintain their productivity under changing conditions.

Biomass and lipid production by the marine centric diatom Thalassiosira pseudonana were characterized in media based on palm oil mill effluent (POME) as a source of key nutrients. The optimal medium comprised 20% by volume POME, 80 µM Na2SiO3, and 35 g NaCl L−1 in water at pH ~7.7. In 15-day batch cultures (16:8 h/h light–dark cycle; 200 µmol photons m−2 s−1, 26 ± 1 °C) bubbled continuously with air mixed with CO2 (2.5% by vol), the peak concentration of dry biomass was 869 ± 14 mg L−1 corresponding to a productivity of ~58 mg L−1 day−1. The neutral lipid content of the biomass was 46.2 ± 1.1% by dry weight. The main components of the esterified lipids were palmitoleic acid methyl ester (31.6% w/w) and myristic acid methyl ester (16.8% w/w). The final biomass concentration and the lipid content were affected by the light–dark cycle. Continuous (24 h light) illumination at the above-specified irradiance reduced biomass productivity to ~54 mg L−1 day−1 and lipid content to 38.1%.

Diatoms belong to a major, diverse and species-rich eukaryotic clade, the Heterokonta, within the polyphyletic chromalveolates. They evolved as a result of secondary endosymbiosis with one or more Plantae ancestors, but their precise evolutionary history is enigmatic. Nevertheless, this has conferred them with unique structural and biochemical properties that have allowed them to flourish in a wide range of different environments and cope with highly variable conditions. We review the effect of pH, light and dark, and CO2 concentration on the regulation of carbon uptake and assimilation. We discuss the regulation of the Calvin-Benson-Bassham cycle, glycolysis, lipid synthesis, and carbohydrate synthesis at the level of gene transcripts (transcriptomics), proteins (proteomics) and enzyme activity. In contrast to Viridiplantae where redox regulation of metabolic enzymes is important, it appears to be less common in diatoms, based on the current evidence, but regulation at the transcriptional level seems to be widespread. The role of post-translational modifications such as phosphorylation, glutathionylation, etc., and of protein-protein interactions, has been overlooked and should be investigated further. Diatoms and other chromalveolates are understudied compared to the Viridiplantae, especially given their ecological importance, but we believe that the ever-growing number of sequenced genomes combined with proteomics, metabolomics, enzyme measurements, and the application of novel techniques will provide a better understanding of how this important group of algae maintain their productivity under changing conditions. [ABSTRACT FROM AUTHOR]

Increasing consumption of metal‐oxide nanoparticles (NPs) and carbon‐based nanomaterials has caused significant concern about their potential hazards in aquatic environments. The release of NPs into aquatic environments could result in adsorption of NPs on microorganisms. While metal‐oxide NP‐conjugated carbon‐based nanohybrids (NHs) may exhibit enhanced toxicity toward microorganisms due to their large surface area and the generation of reactive oxygen species (ROS), there is a lack of information regarding the ecotoxicological effects of NHs on marine diatom algae, which are an indicator of marine pollution. Moreover, there is scant information on toxicity mechanisms of NHs on aquatic organisms. In this study, four NHs (ie, ZnO‐conjugated graphene oxide [GO], ZnO‐conjugated carbon nanotubes [CNTs], TiO2‐conjugated GO, and TiO2‐conjugated CNT) that were synthesized by a hydrothermal method were investigated for their toxicity effects on a Thalassiosira pseudonana marine diatom. The in vitro cellular viability and ROS formation employed at the concentration ranges of 50 and 100 mg/L of NHs over 72 hours revealed that ZnO‐GO had the most negative effect on T. pseudonana. The primary mechanism identified was the generation of ROS and GO‐induced dispersion that caused electrostatic repulsion, preventing aggregation, and an increase in surface areas of NHs. In contrast to GO‐induced dispersion, large aggregates were observed in ZnO/TiO2‐conjugated CNT‐based NHs. The scanning electron microscopy images suggest that NHs covered algae cells and interacted with them (shading effects); this reduced light availability for photosynthesis. Detailed in vitro toxicity effects and mechanisms that cause high adverse acute toxicity on T. pseudonana were unveiled; this implied concerns about potential hazards of these mechanisms in aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Exacerbating deoxygenation is extensively affecting marine organisms, with no exception for phytoplankton. To probe these effects, we comparably explored the growth, cell compositions, photosynthesis, and transcriptome of a diatom Thalassiosira pseudonana under a matrix of pO2 levels and Light:Dark cycles at an optimal growth light. The growth rate (μ) of T. pseudonana under a 8:16 L:D cycle was enhanced by 34% by low pO2 but reduced by 22% by hypoxia. Under a 16:8 L:D cycle, however, the μ decreased with decreasing pO2 level. The cellular Chl a content decreased with decreasing pO2 under a 8:16 L:D cycle, whereas the protein content decreased under a 16:8 L:D cycle. The prolonged photoperiod reduced the Chl a but enhanced the protein contents. The lowered pO2 reduced the maximal PSII photochemical quantum yield (FV/FM), photosynthetic oxygen evolution rate (Pn), and respiration rate (Rd) under the 8:16 or 16:8 L:D cycles. Cellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were higher under low pO2 than ambient pO2 or hypoxia. Moreover, the prolonged photoperiod reduced the FV/FM and Pn among all three pO2 levels but enhanced the Rd, MDA, and SOD activity. Transcriptome data showed that most of 26 differentially expressed genes (DEGs) that mainly relate to photosynthesis, respiration, and metabolism were down-regulated by hypoxia, with varying expression degrees between the 8:16 and 16:8 L:D cycles. In addition, our results demonstrated that the positive or negative effect of lowering pO2 upon the growth of diatoms depends on the pO2 level and is mediated by the photoperiod.

Abstract Background An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. Results In this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5′ fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Conclusions Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.

Summary: The post‐transcriptional regulation involved in the responses of diatoms to silicon is poorly understood. Using a poly(A)‐tag sequencing (PAT‐seq) technique that interrogates only the junctions of 3′‐untranslated region (UTR) and the poly(A) tails at the transcriptome level, a comprehensive comparison of alternative polyadenylation (APA) was performed to understand the role of post‐transcriptional regulation in various silicon‐related cellular responses for the marine diatom Thalassiosira pseudonana. In total, 23 701 poly(A) clusters and 6894 APA genes, treated with silicon starvation and replenishment, were identified at nine time points. Significant APA was found in numerous genes (e.g. five cingulin genes) closely associated with the silicon‐starvation response, girdle bands and valve synthesis, suggesting that many genes participated in the responses to silicon availability and biosilica formation through changes in transcript isoforms. The poly(A) site usage profiles were distinct during various stages of silicon biomineralization responses. Moreover, a correlation between APA and expression levels of APA switching genes was also discovered. This is an interesting study that presents a genome‐wide profile of transcript ends in diatoms, which is distinct from that of higher plants, animals and other microalgae. This work provides an important resource to understand a different aspect of cell‐wall synthesis. Significance Statement: Although silicon plays an important role in the successful survival of diatoms due to its requirement in silicified cell walls, the post‐transcriptional regulation associated with the responses of diatoms to silicon availability is poorly understood. This work presents the uniqueness of an alternative polyadenylation profile that dynamically responds to silicon starvation and replenishment, indicating its potential contribution to post‐transcriptional regulation in biosilicification. [ABSTRACT FROM AUTHOR]

Temperature effects on the fatty acid (FA) profiles of phytoplankton, major primary producers in the ocean, have been widely studied due to their importance as industrial feedstocks and to their indispensable role as global producers of long‐chain, polyunsaturated FA (PUFA), including omega‐3 (ω3) FA required by organisms at higher trophic levels. The latter is of global ecological concern for marine food webs, as some evidence suggests an ongoing decline in global marine‐derived ω3 FA due to both a global decline in phytoplankton abundance and to a physiological reduction in ω3 production by phytoplankton as temperatures rise. Here, we examined both short‐term (physiological) and long‐term (evolutionary) responses of FA profiles to temperature by comparing FA thermal reaction norms of the marine diatom Thalassiosira pseudonana after ~500 generations (ca. 2.5 years) of experimental evolution at low (16°C) and high (31°C) temperatures. We showed that thermal reaction norms for some key FA classes evolved rapidly in response to temperature selection, often in ways contrary to our predictions based on prior research. Notably, 31°C‐selected populations showed higher PUFA percentages (including ω3 FA) than 16°C‐selected populations at the highest assay temperature (31°C, above T. pseudonana's optimum temperature for population growth), suggesting that high‐temperature selection led to an evolved ability to sustain high PUFA production at high temperatures. Rapid evolution may therefore mitigate some of the decline in global phytoplankton‐derived ω3 FA production predicted by recent studies. Beyond its implications for marine food webs, knowledge of the effects of temperature on fatty acid profiles is of fundamental importance to our understanding of the mechanistic causes and consequences of thermal adaptation. [ABSTRACT FROM AUTHOR]

Summary: Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms.We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes. Chitin deacetylase (CDA) genes from each genome (PtCDA and TpCDA) were overexpressed in P. tricornutum. We performed comparative analysis of their sequence structure, phylogeny, transcriptional profiles, localization and enzymatic activities.The chitin relevant proteins show complex subcellular compartmentation. PtCDA was likely acquired by horizontal gene transfer from prokaryotes, whereas TpCDA has closer relationships with sequences in Opisthokonta. Using transgenic P. tricornutum lines expressing CDA‐green fluorescent protein (GFP) fusion proteins, PtCDA predominantly localizes to Golgi apparatus whereas TpCDA localizes to endoplasmic reticulum/chloroplast endoplasmic reticulum membrane. CDA‐GFP overexpression upregulated the transcription of chitin synthases and potentially enhanced the ability of chitin synthesis. Although both CDAs are active on GlcNAc5, TpCDA is more active on the highly acetylated chitin polymer DA60.We have addressed the ambiguous characters of CDAs from P. tricornutum and T. pseudonana. Differences in localization, evolution, expression and activities provide explanations underlying the greater potential of centric diatoms for chitin biosynthesis. This study paves the way for in vitro applications of novel CDAs. [ABSTRACT FROM AUTHOR]

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. Our present knowledge of their metabolism or biosynthesis is limited. In this study, the production of BMAA and DAB as a function of time was monitored in five strains representing four species of diatoms, i.e. Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira pseudonana and Navicula pelliculosa, previously identified as BMAA and DAB producers. Subsequently, three strains were selected and exposed to three nitrogen treatments - starvation, control (the standard concentration in f/2 medium) and enrichment, because BMAA metabolism has been suggested to be closely associated with cellular nitrogen metabolism in both cyanobacteria and diatoms. Chlorophyll a and total protein concentrations were also determined. Our results indicate that BMAA and DAB production in diatoms is species- and strain-specific. However, production might also be affected by stress, particularly as related to nitrogen starvation and cell density. Furthermore, this study shows a significant correlation between the production of the two neurotoxins which might further suggest common steps in the metabolic pathways. [ABSTRACT FROM AUTHOR]

Abstract Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg2+) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing. Graphical abstract Unlabelled Image Highlights • Mg2+ participates in the regulation of diatom thylakoid membrane distances. • Optimal cation concentrations are in the same range as in higher plants grana. • Higher salt concentrations lead to reversible increase of the interthylakoidal space. • The inner four core thylakoid membranes resemble grana in their adhesion behaviour. [ABSTRACT FROM AUTHOR]

Summary: The assimilation of inorganic compounds in foraminiferal metabolism compared to predation or organic matter assimilation is unknown. Here, we investigate possible inorganic‐compound assimilation in Nonionellina labradorica, a common kleptoplastidic benthic foraminifer from Arctic and North Atlantic sublittoral regions. The objectives were to identify the source of the foraminiferal kleptoplasts, assess their photosynthetic functionality in light and darkness and investigate inorganic nitrogen and sulfate assimilation. We used DNA barcoding of a ~ 830 bp fragment from the SSU rDNA to identify the kleptoplasts and correlated transmission electron microscopy and nanometre‐scale secondary ion mass spectrometry (TEM‐NanoSIMS) isotopic imaging to study 13C‐bicarbonate, 15N‐ammonium and 34S‐sulfate uptake. In addition, respiration rate measurements were determined to assess the response of N. labradorica to light. The DNA sequences established that over 80% of the kleptoplasts belonged to Thalassiosira (with 96%–99% identity), a cosmopolitan planktonic diatom. TEM‐NanoSIMS imaging revealed degraded cytoplasm and an absence of 13C assimilation in foraminifera exposed to light. Oxygen measurements showed higher respiration rates under light than dark conditions, and no O2 production was detected. These results indicate that the photosynthetic pathways in N. labradorica are not functional. Furthermore, N. labradorica assimilated both 15N‐ammonium and 34S‐sulfate into its cytoplasm, which suggests that foraminifera might have several ammonium or sulfate assimilation pathways, involving either the kleptoplasts or bona fide foraminiferal pathway(s) not yet identified. [ABSTRACT FROM AUTHOR]

Abstract Background CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

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.