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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]

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]

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]

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]

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]

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]

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 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]

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.

Phytoplankton cells living in the surface waters of oceans are experiencing alterations in environmental conditions associated with global change. Given their importance in global primary productivity, it is of considerable concern to know how these organisms will perform physiologically under the changing levels of pH, temperatures, and nutrients predicted for future oceanic ecosystems. Here we show that the model diatom, Thalassiosira pseudonana , when grown at different temperatures (20 or 24 °C), pCO2 (400 or 1000 µatm), and nitrate concentrations (2.5 or 102.5 µmol l−1), displayed contrasting performance in its physiology. Elevated pCO2 (and hence seawater acidification) under the nitrate-limited conditions led to decreases in specific growth rate, cell size, pigment content, photochemical quantum yield of PSII, and photosynthetic carbon fixation. Furthermore, increasing the temperature exacerbated the negative effects of the seawater acidification associated with elevated pCO2 on specific growth rate and chlorophyll content under the N-limited conditions. These results imply that a reduced upward transport of nutrients due to enhanced stratification associated with ocean warming might act synergistically to reduce growth and carbon fixation by diatoms under progressive ocean acidification, with important ramifications for ocean productivity and the strength of the biological CO2 pump. [ABSTRACT FROM AUTHOR]

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira . In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira. [ABSTRACT FROM AUTHOR]

Diatoms are one of the most important groups of phytoplankton in terms of abundance and ecological functionality in the ocean. They usually dominate the phytoplankton communities in coastal waters and experience frequent and large fluctuations in light. In order to evaluate the combined effects of ocean warming and acidification on the diatom's exploitation of variable light environments, we grew a globally abundant diatom Thalassiosira pseudonana under two levels of temperature (18, 24 °C) and pCO 2 (400, 1000 μatm) to examine its physiological performance after light challenge. It showed that the higher temperature increased the photoinactivation rate in T. pseudonana at 400 μatm pCO 2 , while the higher pCO 2 alleviated the negative effect of the higher temperature on PSII photoinactivation. Higher pCO 2 stimulated much faster PsbA removal, but it still lagged behind the photoinactivation of PSII under high light. Although the sustained phase of nonphotochemical quenching (NPQs) and activity of superoxide dismutase (SOD) were provoked during the high light exposure in T. pseudonana under the combined pCO 2 and temperature conditions, it could not offset the damage caused by these multiple environmental changes, leading to decreased maximum photochemical yield. [ABSTRACT FROM AUTHOR]

Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P-) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean. [ABSTRACT FROM AUTHOR]

The silicon transport and use inside cells are key processes for understanding how diatoms metabolize this element in the silica biogenic cycle in the ocean. A spin-probe electron paramagnetic resonance (EPR) study over time helped to investigate the interacting properties and the internalization mechanisms of silicic acid from different silicon sources into the cells. Diatom cells were grown in media containing biogenic amorphous substrates, such as diatomaceous earth and sponge spicules, and crystalline sodium metasilicate. It was found that the amorphous biogenic silicon slowed down the internalization process probably due to formation of colloidal particles at the cell surface after silicic acid condensation. Weaker interactions occurred with sponge spicules silicon source if compared to the other sources. The EPR results were explained by analyzing transcript level changes of silicon transporters (SITs) and silaffins (SILs) in synchronized Thalassiosira pseudonana cultures over time. The results indicated that the transport role of SITs is minor for silicic acid from both biogenic and crystalline substrates, and the role of SIT3 is linked to the transport of silicon inside the cells, mainly in the presence of sponge spicules. SIL3 transcripts were expressed in the presence of all silicon sources, while SIL1 transcripts only with sponge spicules. The data suggest that the transport of silicic acid from various silicon sources in diatoms is based on different physico-chemical interactions with the cell surface. [ABSTRACT FROM AUTHOR]

Background: Flagella have been lost in the vegetative phase of the diatom life cycle, but they are still present in male gametes of centric species, thereby representing a hallmark of sexual reproduction. This process, besides maintaining and creating new genetic diversity, in diatoms is also fundamental to restore the maximum cell size following its reduction during vegetative division. Nevertheless, sexual reproduction has been demonstrated in a limited number of diatom species, while our understanding of its different phases and of their genetic control is scarce. Results: In the transcriptome of Leptocylindrus danicus, a centric diatom widespread in the world's seas, we identified 22 transcripts related to the flagella development and confirmed synchronous overexpression of 6 flagellum-related genes during the male gamete formation process. These transcripts were mostly absent in the closely related species L. aporus, which does not have sexual reproduction. Among the 22 transcripts, L. danicus showed proteins that belong to the Intra Flagellar Transport (IFT) subcomplex B as well as IFT-A proteins, the latter previously thought to be absent in diatoms. The presence of flagellum-related proteins was also traced in the transcriptomes of several other centric species. Finally, phylogenetic reconstruction of the IFT172 and IFT88 proteins showed that their sequences are conserved across protist species and have evolved similarly to other phylogenetic marker genes. Conclusion: Our analysis describes for the first time the diatom flagellar gene set, which appears to be more complete and functional than previously reported based on the genome sequence of the model centric diatom, Thalassiosira pseudonana. This first recognition of the whole set of diatom flagellar genes and of their activation pattern paves the way to a wider recognition of the relevance of sexual reproduction in individual species and in the natural environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses research on the expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana. Topics include the development of expression systems in order to produce a vaccine against bovine respiratory disease, the efficiency of using unicellular microalgae as an expression platform, and modest immune response successfully exhibited in mice exposed to diatom-expressed DR2 in whole or sonicated cells.

Ocean acidification due to atmospheric CO rise is expected to influence marine phytoplankton. Diatoms are responsible for about 40% of the total primary production in the ocean. In order to investigate the physiological response of marine diatom Thalassiosira pseudonana to ocean acidification, we grew the cells under ambient CO level (380 µatm) versus the elevated CO level (800 µatm) at a light level of 180 µmol m s for 30 generations. Our results showed that the elevated CO concentration caused a decrease of the effective photochemical efficiency of PSII $$\left( {{{F_{{\text{v}}}^{\prime } } \mathord{\left/ {\vphantom {{F_{{\text{v}}}^{\prime } } {F_{{\text{m}}}^{\prime } }}} \right. \kern-\nulldelimiterspace} {F_{{\text{m}}}^{\prime } }}} \right)$$ and increase of the dark respiration in T. pseudonana. The intracellular carbonic anhydrase activity was suppressed and the photosynthetic affinity for CO was lowered in the high CO-grown cells, reflecting a downregulation of the CO concentrating mechanism (CCM). PSI activity was enhanced to support an increase in ATP synthesis by cyclic electron transfer as required for transport of inorganic carbon and regulation of intracellular pH. The energetic benefit from the downregulation of CCM to growth as reported in other diatom species was not observed here in T. pseudonana. [ABSTRACT FROM AUTHOR]

Light drives phytoplankton productivity, so phytoplankton must exploit variable intensities and durations of light exposure, depending upon season, latitude, and depth. We analyzed the growth, photophysiology and composition of small, Thalassiosira pseudonana, and large, Thalassiosira punctigera, centric diatoms from temperate, coastal marine habitats, responding to a matrix of photoperiods and growth light intensities. T. pseudonana showed fastest growth rates under long photoperiods and low to moderate light intensities, while the larger T. punctigera showed fastest growth rates under short photoperiods and higher light intensities. Photosystem II function and content responded primarily to instantaneous growth light intensities during the photoperiod, while diel carbon fixation and RUBISCO content responded more to photoperiod duration than to instantaneous light intensity. Changing photoperiods caused species-specific changes in the responses of photochemical yield (e−/photon) to growth light intensity. These photophysiological variables showed complex responses to photoperiod and to growth light intensity. Growth rate also showed complex responses to photoperiod and growth light intensity. But these complex responses resolved into a close relation between growth rate and the cumulative daily generation of reductant, across the matrix of photoperiods and light intensities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The hydrogen isotopic ( 2 H/ 1 H) composition of lipids in microalgae is significantly depleted relative to extracellular water. While a variety of growth conditions influence the magnitude of 2 H-depletion, the effect of salinity is of particular interest due to the paleohydrological applications of lipid 2 H/ 1 H. In previous studies, lipid–water 2 H/ 1 H fractionation was shown to decrease as salinity increased, a response largely independent of lipid type, species, or setting. The mechanism responsible for this response remains uncertain, primarily because salinity is rarely isolated as the sole variable in laboratory cultivation experiments investigating hydrogen isotope systematics in microalgae. Here we report the lipid–water 2 H/ 1 H fractionation response to salinity in nutrient-replete continuous cultures of the centric diatom Thalassiosira pseudonana . In six cultures with the same growth rate at salinities between 14–40 ppt, lipid–water 2 H/ 1 H fractionation decreased linearly as salinity increased by 1.3‰/ppt in fatty acids (C 14:0 , C 16:0 , C 16:1 ) and by 1.0‰/ppt in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol. A constant growth rate between cultures reveals that the fractionation response to salinity is independent of growth rate. Sensitivity tests using a simple hydrogen flux model indicated that at high salinity a greater proportion of metabolic NAD(P)H in lipids at the expense of photosynthetic NADPH can cause 2 H-enrichment. Additionally, increased exudate release or decreased hydrogen transport can enrich both lipids and cell–water in 2 H. The 1.0–1.3‰/ppt increase in lipid–water 2 H/ 1 H fractionation observed in T. pseudonana is within the 0.8–2‰/ppt range observed in field studies and culture studies, supporting the application of algal lipid 2 H/ 1 H as a paleosalinity proxy. [ABSTRACT FROM AUTHOR]

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity., To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation., Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes., Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels. [ABSTRACT FROM AUTHOR]

Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms ( CCMs) involving transporters and carbonic anhydrases ( CAs) are well known, but the contribution of a biochemical CCM involving C4 metabolism is contentious., The CCM(s) present in the marine-centric diatom, Thalassiosira pseudonana, were studied in cells exposed to high or low concentrations of CO2, using a range of approaches., At low CO2, cells possessed a CCM based on active uptake of CO2 (70% contribution) and bicarbonate, while at high CO2, cells were restricted to CO2. CA was highly and rapidly activated on transfer to low CO2 and played a key role because inhibition of external CA produced uptake kinetics similar to cells grown at high CO2. The activities of phosphoenolpyruvate ( PEP) carboxylase ( PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase ( PPDK), were lower in cells grown at low than at high CO2. The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, even at low CO2., Our data suggest that the kinetic properties of this species results from a biophysical CCM and not from C4 type metabolism. [ABSTRACT FROM AUTHOR]

Diatoms account for a large proportion of marine primary productivity, they tend to be the predominant species in the phytoplankton communities in the surface ocean with frequent and large light fluctuations. To understand the impacts of increased CO 2 on diatoms' capacity in exploitation of variable solar radiation, we cultured a model diatom Thalassiosira pseudonana with 400 or 1000ppmv CO 2 and exposed it to high photosynthetically active radiation (PAR) alone or PAR plus ultraviolet radiation (UVR) to examine its physiological performances. The results showed that the maximum photochemical efficiency (F v /fm) was significantly reduced by high PAR and PAR + UVR in T. pseudonana , UVR-induced inhibition on PSII activity was exacerbated by high CO 2. PSII activity drops coincide approximately with PsbA content in the cells exposed to high PAR or PAR + UVR, which was pronounced at high CO 2. The removal of PsbD in T. pseudonana cells declined under high CO 2 during UVR exposure, limiting the repair capacity of PSII. In addition, high CO 2 reversed the induction of energy-dependent form of NPQ by UVR to the increase of Y (No) , indicating the severe damage of the photoprotective reactions. Our findings suggest that the adverse impacts of UVR on PSII function of T. pseudonana were aggravated by the elevated CO 2 through modulating its capacity in repair and protection, which thereby would influence its abundance and competitiveness in phytoplankton communities. • High CO 2 exacerbated the UVR-induced inhibition on PSII activity. • UVR stimulated the removal rates of both PsbA and PsbD. • The removal of PsbD declined by high CO 2 under the exposure of UVR. • High CO 2 reversed the UVR-induced Y NPQ to Y No. [ABSTRACT FROM AUTHOR]

Three centric diatoms, Thalassiosira pseudonana (diameter ~ 4 μm), Thalassiosira weissflogii (~ 11 μm), and Thalassiosira punctigera (~ 47 μm), were exposed to low and high levels of UV radiation. UV-induced inhibition on photosystem II was correlated with cell size under high light levels, though it was insignificant under low light levels (PAR < 63 W m − 2 ). The highest inhibition (~ 15%) was observed for the smallest species. Several mechanisms may explain the observed relationship between cell size and response to UV. All three species counteracted UV-related photosystem damage via protein synthesis within the chloroplast. Non-photochemical quenching (NPQ) was induced when that process was blocked with an inhibitor in T. pseudonana and T. weissflogii , but not T. punctigera , as neither radiation nor the inhibitor had a significant effect on NPQ in this species. Moreover, UV-induced inhibition for cells treated with lincomycin was highest for T. weissflogii , which was in accordance with the highest UV exposure within the cell. The intracellular UV distribution was not associated with cell size, indicating that the package effect was not the only determinant of cell-size dependent UV sensitivity in phytoplankton. [ABSTRACT FROM AUTHOR]

Energy transfer dynamics in dimeric photosystem II (PSII) complexes isolated from four diatoms, Chaetoceros gracilis , Cyclotella meneghiniana , Thalassiosira pseudonana , and Phaeodactylum tricornutum , are examined. Time-resolved fluorescence measurements were conducted in the range of 0–80 ns. Delayed fluorescence spectra showed a clear difference between PSII monomer and PSII dimer isolated from the four diatoms. The difference can be interpreted as reflecting suppressed energy transfer between PSII monomers in the PSII dimer for efficient energy trapping at the reaction center. The observation was especially prominent in C. gracilis and T. pseudonana . The pathways seem to be suppressed under a low pH condition in isolated PSII complexes from C. gracilis , and excitation energy may be quenched with fucoxanthin chlorophyll a/c -binding protein (FCP) that was closely associated with PSII in C. gracilis . The energy transfer between PSII monomers in the PSII dimer may play a role in excitation energy regulation in diatoms. [ABSTRACT FROM AUTHOR]

Investigation of the production and structural characteristics of the extracellular polysaccharides (ECPS) released by diatoms according to nutrient status is urgently necessary for our understanding of the roles of ECPS in aquatic ecosystem. In this study, the effects of N and P depletion and variable nutrient sources on the production and structural characterization of the soluble ECPS released by the marine diatom Thalassiosira pseudonana in batch culture were examined. The abundance of the soluble ECPS produced under N (NO) depletion was higher than that obtained in the P (PO) depletion and control treatments. NH rather than NO and urea and sodium-glycerophosphate rather than PO were found to induce a higher abundance of soluble ECPS production at the end of the experiment, indicating that the abundance of soluble ECPS is also affected by the nutrient source. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the structural characteristics of the soluble ECPS samples showed that the degree of polymerization (d.p.) profiles and the distribution of the polymers with different molecular masses varied in response to nutrient depletion and different nutrient sources. An increase in the proportion of both low- and high-molecular-weight polymers under the N (NO) and P (PO) depletion treatments was found. Based on the different characteristics of high- and low-molecular-weight polymers, our results suggest that the production of soluble ECPS may be one of the strategies that T. pseudonana utilizes to survive under nutrient stress conditions. [ABSTRACT FROM AUTHOR]