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Marine microalgae are the primary producers of ω3 polyunsaturated fatty acids (PUFAs), such as octadecapentaenoic acid (OPA, 18:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) for food chains. However, the biosynthetic mechanisms of these PUFAs in the algae remain elusive. To study how these fatty acids are synthesized in microalgae, a series of radiolabeled precursors were used to trace the biosynthetic process of PUFAs in Emiliania huxleyi. Feeding the alga with 14C-labeled acetic acid in a time course showed that OPA was solely found in glycoglycerolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) synthesized plastidically by sequential desaturations while DHA was exclusively found in phospholipids synthesized extraplastidically. Feeding the alga with 14C-labeled α-linolenic acid (ALA), linoleic acid (LA), and oleic acid (OA) showed that DHA was synthesized extraplastidically from fed ALA and LA, but not from OA, implying that the aerobic pathway of DHA biosynthesis is incomplete with missing a Δ12 desaturation step. The in vitro enzymatic assays with 14C-labeled malonyl-CoA showed that DHA was synthesized from acetic acid by a PUFA synthase. These results provide the first and conclusive biochemistry evidence that OPA is synthesized by a plastidic aerobic pathway through sequential desaturations with the last step of Δ3 desaturation, while DHA is synthesized by an extraplastidic anaerobic pathway catalyzed by a PUFA synthase in the microalga.

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Emiliania huxleyi is a marine microalga playing a significant ecological and biogeochemical role in oceans. It can produce several polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and octadecapentaenoic acid (OPA, 18:5-3,6,9,12,15), providing a primary source for nutritionally important ω3 PUFAs in the marine food chain. However, the biosynthesis of these PUFAs in this organism is not well understood. In this study, a full length plastidial ω3 desaturase cDNA (EhN3) was cloned from this alga. Heterologous expression of EhN3 with and without the chloroplast targeting peptide (cTP) in cyanobacterium Synechococcus elongatus showed that it possessed high desaturation activity toward C18-ω6 PUFAs, linoleic acid (LA, 18:2-9,12), γ-linolenic acid (GLA, 18:3-6,9,12), and C20-ω6 PUFAs, dihomo-γ-linolenic acid (DGLA, 20:3-8,11,14) and arachidonic acid (ARA, 20:4-5,8,11,14) that were exogenously supplied. Desaturation efficiency could reach almost 100% in a time course. On the other hand, when expressed in Saccharomyces cerevisiae, EhN3 with and without cTP did not exhibit any activity. Lipid analysis of Synechococcus transformants expressing EhN3 showed that it utilized galactolipids as substrates. Transcriptional expression analysis revealed that the expression of the gene increased while the growth temperature decreased, which was correlated with the increased production of ω3-PUFAs, particularly OPA. This is the first report of a plastidial ω3 desaturase from microalgae that can effectively introduce an ω3 double bond into both C18-ω6 and C20-ω6 PUFAs. EhN3 might also be one of the key enzymes involved in the biosynthesis of OPA in E. huxleyi through the plastidial aerobic pathway.

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Algal blooms drive global biogeochemical cycles of key nutrients and serve as hotspots for biological interactions in the ocean. The massive blooms of the cosmopolitan coccolithophore Emiliania huxleyi are often infected by the lytic E. huxleyi virus, which is a major mortality agent triggering bloom demise. This multi-annual "boom and bust" pattern of E. huxleyi blooms suggests that coexistence is essential for these host-virus dynamics. To investigate host-virus coexistence, we developed a new model system from an E. huxleyi culture that recovered from viral infection. The recovered population coexists with the virus, as host cells continue to divide in parallel to viral production. By applying single-molecule fluorescence in situ hybridization (smFISH) to quantify the fraction of infected cells, and assessing infection-specific lipid biomarkers, we identified a small subpopulation of cells that were infected and produced new virions, whereas most of the host population could resist infection. To further assess population heterogeneity, we generated clonal strain collections using single-cell sorting and subsequently phenotyped their susceptibility to E. huxleyi virus infection. This unraveled substantial cell-to-cell heterogeneity across a continuum of susceptibility to resistance, highlighting that infection outcome may vary depending on the individual cell. These results add a new dimension to our understanding of the complexity of host-virus interactions that are commonly assessed in bulk and described by binary definitions of resistance or susceptibility. We propose that phenotypic heterogeneity drives the host-virus coexistence and demonstrate how the coexistence with a lytic virus provides an ecological advantage for the host by killing competing strains.

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Both temperature and nutrient levels are rising in worldwide ocean ecosystems, and they strongly influence biological responses of phytoplankton. However, few studies have addressed the interactive effects of temperature and nitrogen sources on physiological performance of the coccolithophore Emiliania huxleyi. In this study, we evaluated algal growth, photosynthesis and respiration, elemental composition, enzyme activity, and calcification under a matrix of two temperatures gradients (ambient temperature 20 °C and high temperature 24 °C) and two nitrogen sources (nitrate (NO3-) and ammonium (NH4+)). When the algae was cultured with NO3- medium, high temperature reduced algal photosynthesis and nitrate reductase activity, but it did not change other indicators significantly relative to ambient temperature. In addition, E. huxleyi preferred NO3- as the growth medium, whereas NH4+ had negative effects on physiological parameters. In the NH4+ medium, the growth rate, photosynthesis and photosynthetic rate, nitrate reductase activity, and particulate organic carbon and particulate organic nitrogen production rate of the algae decreased as temperature increased. Conversely, high temperature increased cellular particulate organic carbon, cellular particulate organic nitrogen, and particulate inorganic carbon levels. In summary, our findings indicate that the distribution and abundance of microalgae could be greatly affected under warming ocean temperature and different nutrient conditions.

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BACKGROUND: The particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability outside their host environment. METHOD: Here we report for the first time, the application of a viability qPCR (V-qPCR) method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the cell culture-based assay method that suggests that virus "infectivity" is lost in a matter of seconds (for EhV) and minutes (for ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the V-qPCR data for EhV. RESULTS: We observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the V-qPCR method, we confirm that no PRRSV particles were detectable after 20 min of exposure to temperatures up to 100 °C. We also show that the EhV particles that remain after 50 °C 20 min exposure was in fact still infectious only after the three blind passages in bioassay experiments. CONCLUSIONS: This study raises the possibility that ASFV is not always eliminated or contained after applying time and temperature inactivation treatments in current decontamination or biosecurity protocols. This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances.

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The effects of ocean acidification (OA) on coccolithophore's photosynthesis, calcification rates, and growth have been extensively studied. However, how the intracellular Ca2+, mechanical properties and chemical composition of the coccoliths are affected by OA have not yet been investigated. This study tries to fill these gaps using Emiliania huxleyi as a model coccolithophore. When the seawater pCO2 increased from 400 µatm to 1200 µatm, the intracellular Ca2+ and coccolith area were reduced by 66% and 36%, respectively. Single-cell mapping by atomic force microscopy revealed that the modulus and hardness of coccolith decreased from 23.6 ± 0.2 GPa to 12.0 ± 5.5 GPa and from 0.53 ± 0.15 GPa to 0.20 ± 0.06 GPa, respectively. Additionally, the proportional organic matter and silicon in the coccolith surfaces increased with pCO2. The copepods Acartia pacifica fed on more E. huxleyi grown at higher pCO2. Our study implies that OA could change coccolithophore's competitive interactions with other phytoplankton and ultimately influence carbon export to the deep ocean.

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The interactions of Emiliania huxleyi and its specific lytic virus (EhV) have a profound influence on marine biogeochemical carbon-sulfur cycles and play a prominent role in global climate change. MicroRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential due to their role in virus-host interactions. However, the application of miRNA signatures as diagnostic markers in marine viral infection has made limited progress. Based on our previous small-RNA sequencing data, one host miRNA biomarker that is upregulated in early infection and seven viral miRNA biomarkers that are upregulated in late infection were identified and verified using qRT-PCR and a receiver operating characteristic curve analysis in pure culture, mixed culture, and natural seawater culture. The host ehx-miR20-5p was able to significantly differentiate infection groups from the control in the middle (24 h post-infection, hpi) and late infection (48 hpi) phases, while seven virus-derived miRNA biomarkers could diagnose the early and late stages of EhV infection. Functional enrichment analysis showed that these miRNAs participated in numerous essential metabolic pathways, including gene transcription and translation, cell division-related pathways, protein-degradation-related processes, and lipid metabolism. Additionally, a dual-luciferase reporter assay confirmed the targeted relationship between a viral ehv-miR7-5p and the host dihydroceramide desaturase gene (hDCD). This finding suggests that the virus-derived miRNA has the ability to inhibit the host sphingolipid metabolism, which is a specific characteristic of EhV infection during the late stage. Our data revealed a cluster of potential miRNA biomarkers with significant regulatory functions that could be used to diagnose EhV infection, which has implications for assessing the infectious activity of EhV in a natural marine environment.

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Dissolved organic phosphorus (DOP) is a potential source of aquatic eutrophication and pollution because it can potentially stimulate growth in some species and inhibit growth in other species of algae, the foundation of the marine ecosystem. Inositol hexaphosphate (also named phytic acid or PA), an abundant organophosphate, is presumably ubiquitous in the marine environment, but how it affects marine primary producers is poorly understood. Here, we investigated the bioavailability of this DOP to the cosmopolitan coccolithophore Emiliania huxleyi. Our results showed that E. huxleyi cells can take up PA and dissolved inorganic phosphorus (DIP) simultaneously. Absorbed PA can efficiently support algal growth, producing cell yield between DIP and phosphorus (P)-depleted conditions. Accordingly, PA supply as the sole P source highly influences cellular metabolism and nutrient stoichiometry. Particularly, PA-grown cultures exhibited enhanced carbon fixation, increased lipid content, activated energy metabolism, and induced nitrogen assimilation. However, our data suggest that PA may also exert some levels of toxic effects on E. huxleyi. This study provides novel insights into the variable effects of a DOP on marine phytoplankton, which will inform new inquiries about how the complex DOP constituencies in the ocean will shape phytoplankton community structure and function. IMPORTANCE The dissolved organic phosphorus (DOP) utilization in phytoplankton plays vital roles in cellular P homeostasis, P-nutrient niche, and the dynamics of community structure in marine ecosystems, but its mechanisms, potentially varying with species, are far from clear. In this study, we investigated the utilization of a widespread DOP species, which is commonly produced by plants (land plants and marine macrophytes) and released into coastal areas, in a globally distributed bloom-forming coccolithophore species in various phosphorus environments. Using a combination of physiological and transcriptomic measurements and analyses, our experimental results revealed the complex mechanism and two-sided effects of DOP (major algal growth-supporting and minor toxic effects) in this species, providing a novel perspective on phytoplankton nutrient regulation.

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Haptophyte algae, including coccolithophores, play key roles in global carbon cycling and ecosystem. They exhibit exceptional morphological and functional diversity. However, their phylogeny is mostly based on short markers and genome researches are always limited to few species, hindering a better understanding about their evolution and diversification. In this study, by assembling 69 new plastid genomes, 65 new mitochondrial genomes, and 55 nuclear drafts, we systematically analyzed their genome variations and built the most comprehensive phylogenies in haptophytes and Noelaerhabdaceae, with the latter is the family of the model coccolithophore Emiliania huxleyi. The haptophyte genomes vary significantly in size, gene content, and structure. We detected phylogenetic incongruence of Prymnesiales between genome compartments. In Noelaerhabdaceae, by including Reticulofenestra sessilis and a proper outgroup, we found R. sessilis was not the basal taxon of this family. Noelaerhabdaceae strains have very similar genomic features and conserved sequences, but different gene content and dynamic structure. We speculate that was caused by DNA double-strand break repairs. Our results provide valuable genetic resources and new insights into the evolution of haptophytes, especially coccolithophores.

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Coccolithophore microalgae, such as Emiliania huxleyi (EHUX) and Chrysotila pseudoroscoffensis (CP), are composed of calcium carbonate (CaCO3) and contain bioactive compounds that can be explored to produce sustainable food packaging. In this study, for the first time, these microalgae were incorporated as fillers in starch-based films, envisioning the development of biodegradable and bioactive materials for food packaging applications. The films were obtained by solvent casting using different proportions of the filler (2.5, 5, 10, and 20%, w/w). For comparison, commercial CaCO3, used as filler in the plastic industry, was also tested. The incorporation of CaCO3 and microalgae (EHUX or CP) made the films significantly less rigid, decreasing Young's modulus up to 4.7-fold. Moreover, the incorporation of microalgae hydrophobic compounds as lipids turned the surface hydrophobic (water contact angles > 90°). Contrary to what was observed with commercial CaCO3, the films prepared with microalgae exhibited antioxidant activity, increasing from 0.9% (control) up to 60.4% (EHUX 20%) of ABTS radical inhibition. Overall, the introduction of microalgae biomass improved hydrophobicity and antioxidant capacity of starch-based films. These findings should be considered for further research using coccolithophores to produce active and sustainable food packaging material.

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Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in the biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we analyzed bacterial transcriptomes in response to exudates derived from algae in exponential growth and stationary phase, which supported the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. In pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and diverse transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles, supporting our previous findings. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7 and hindered the DMSP-induced lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological state of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during interaction.

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The coccolithophore Emiliania huxleyi shows a variety of responses to ocean acidification (OA) and to high-CO2 concentrations, but there is still controversy on differentiating between these two factors when using different strains and culture methods. A heavily calcified type A strain isolated from the Norwegian Sea was selected and batch cultured in order to understand whether acclimation to OA was mediated mainly by CO2 or H+, and how it impacted cell growth performance, calcification, and physiological stress management. Emiliania huxleyi responded differently to each acidification method. CO2-enriched aeration (1200 µatm, pH 7.62) induced a negative effect on the cells when compared to acidification caused by decreasing pH alone (pH 7.60). The growth rates of the coccolithophore were more negatively affected by high pCO2 than by low pH without CO2 enrichment with respect to the control (400 µatm, pH 8.1). High CO2 also affected cell viability and promoted the accumulation of reactive oxygen species (ROS), which was not observed under low pH. This suggests a possible metabolic imbalance induced by high CO2 alone. In contrast, the affinity for carbon uptake was negatively affected by both low pH and high CO2. Photochemistry was only marginally affected by either acidification method when analysed by PAM fluorometry. The POC and PIC cellular quotas and the PIC:POC ratio shifted along the different phases of the cultures; consequently, calcification did not follow the same pattern observed in cell stress and growth performance. Specifically, acidification by HCl addition caused a higher proportion of severely deformed coccoliths, than CO2 enrichment. These results highlight the capacity of CO2 rather than acidification itself to generate metabolic stress, not reducing calcification.

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Detailed information on the photo-generated triplet states of diatom and haptophyte Fucoxanthin Chlorophyll-binding Proteins (FCPs and E-FCPs, respectively) have been obtained from a combined spectroscopic investigation involving Transient Absorption and Time-Resolved Electron Paramagnetic Resonance. Pennate diatom Phaeodactylum tricornutum FCP shows identical photoprotective Triplet-Triplet Energy Transfer (TTET) pathways to the previously investigated centric diatom Cyclotella meneghiniana FCP, with the same two chlorophyll a-fucoxanthin pairs that involve the fucoxanthins in sites Fx301 and Fx302 contributing to TTET in both diatom groups. In the case of the haptophyte Emilianina huxleyi E-FCP, only one of the two chlorophyll a-fucoxanthins pairs observed in diatoms, the one involving chlorophyll a409 and Fx301, has been shown to be active in TTET. Furthermore, despite the marked change in the pigment content of E-FCP with growth light intensity, the TTET pathway is not affected. Thus, our comparative investigation of FCPs revealed a photoprotective TTET pathway shared within these classes involving the fucoxanthin in site Fx301, a site exposed to the exterior of the antenna monomer that has no equivalent in Light-Harvesting Complexes from the green lineage.

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Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. The structure and selection of prokaryotic communities associated with the most abundant coccolithophore and bloom-forming species, Emiliania huxleyi, are still poorly known. In this study, we assessed the diversity of bacterial communities associated with an E. huxleyi bloom in the Celtic Sea (Eastern North Atlantic), exposed axenic E. huxleyi cultures to prokaryotic communities derived from bloom and non-bloom conditions, and followed the dynamics of their microbiome composition over one year. Bloom-associated prokaryotic communities were dominated by SAR11, Marine group II Euryarchaeota and Rhodobacterales and contained substantial proportions of known indicators of phytoplankton bloom demises such as Flavobacteriaceae and Pseudoalteromonadaceae. The taxonomic richness of bacteria derived from natural communities associated with axenic E. huxleyi rapidly shifted and then stabilized over time. The succession of microorganisms recruited from the environment was consistently dependent on the composition of the initial bacterioplankton community. Phycosphere-associated communities derived from the E. huxleyi bloom were highly similar to one another, suggesting deterministic processes, whereas cultures from non-bloom conditions show an effect of stochasticity. Overall, this work sheds new light on the importance of the initial inoculum composition in microbiome recruitment and elucidates the temporal dynamics of its composition and long-term stability.

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Solar radiation varies quantitatively and qualitatively while penetrating through the seawater column and thus is one of the most important environmental factors shaping the vertical distribution pattern of phytoplankton. The haploid and diploid life-cycle phases of coccolithophores might have different vertical distribution preferences. Therefore, the two phases respond differently to high solar photosynthetically active radiation (PAR, 400-700 nm) and ultraviolet radiation (UVR, 280-400 nm). To test this, the haploid and diploid Emiliania huxleyi were exposed to oversaturating irradiance. In the presence of PAR alone, the effective quantum yield was reduced by 10% more due to the higher damage rate of photosystem II in haploid cells than in diploid cells. The addition of UVR resulted in further inhibition of the quantum yield for both haploid and diploid cells in the first 25 min, partly because of the increased damage of photosystem II. Intriguingly, this UVR-induced inhibition of the haploid cells completely recovered half an hour later. This recovery was confirmed by the comparable maximum quantum yields, maximum relative electron transport rates and yields of the haploid cells treated with PAR and PAR + UVR. Our data indicated that photosynthesis of the haploid phase was more sensitive to high visible light than the diploid phase but resistant to UVR-induced inhibition, reflecting the ecological niches to which this species adapts.

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African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and is stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of the NCLDVs, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV thereby making it a safe surrogate, with results that are applicable to ASFV and suitable for use in real-world experiments. Here we inoculated conventional soybean meal (SBMC), organic soybean meal (SBMO), and swine complete feed (CF) matrices with EhV strain 86 (EhV-86) at a concentration of 6.6 × 107 virus g-1, and then transported these samples in the trailer of a commercial transport vehicle for 23 days across 10,183 km covering 29 states in various regions of the United States. Upon return, samples were evaluated for virus presence and viability using a previously validated viability qPCR (V-qPCR) method. Results showed that EhV-86 was detected in all matrices and no degradation in EhV-86 viability was observed after the 23-day transportation event. Additionally, sampling sensitivity (we recorded unexpected increases, as high as 49% in one matrix, when virus was recovered at the end of the sampling period) rather than virus degradation best explains the variation of virus quantity observed after the 23-day transport simulation. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in swine feed matrices during long-term transport across the continental United States.

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The interactions established between marine microbes, namely phytoplankton-bacteria, are key to the balance of organic matter export to depth and recycling in the surface ocean. Still, their role in the response of phytoplankton to rising CO2 concentrations is poorly understood. Here, we show that the response of the cosmopolitan Emiliania huxleyi (E. huxleyi) to increasing CO2 is affected by the coexistence with bacteria. Specifically, decreased growth rate of E. huxleyi at enhanced CO2 concentrations was amplified in the bloom phase (potentially also related to nutrient concentrations) and with the coexistence with Idiomarina abyssalis (I. abyssalis) and Brachybacterium sp. In addition, enhanced CO2 concentrations also affected E. huxleyi's cellular content estimates, increasing organic and decreasing inorganic carbon, in the presence of I. abyssalis, but not Brachybacterium sp. At the same time, the bacterial isolates only survived in coexistence with E. huxleyi, but exclusively I. abyssalis at present CO2 concentrations. Bacterial species or group-specific responses to the projected CO2 rise, together with the concomitant effect on E. huxleyi, might impact the balance between the microbial loop and the export of organic matter, with consequences for atmospheric carbon dioxide.

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Changes in phytoplankton abundance and biomass during the period 1933-2020 were examined by statistical modeling using data from the Inner Oslofjorden phytoplankton database. The phytoplankton abundances increased with eutrophication from 1930s to 1970s, but with the implementation of sewage cleaning measures and a resulting reduction in nutrient releases, the phytoplankton abundance has since then decreased significantly. The onset of the seasonal blooms has started progressively later during the last 15 years, especially the spring bloom. The delayed spring bloom co-occurred with increasing temperature in winter and spring. The diatom biomass decreased more than that of dinoflagellates and other microeukaryotes. The diatom genus Skeletonema dominated the spring bloom and was found to be the key taxa in explaining these changes in abundance and phenology. Extensive summer blooms of the coccolithophore Emiliania huxleyi, which has been characteristic for the inner Oslofjorden, has also gradually decreased during the last decades, along with reducing eutrophication. Dinoflagellates have not had the same reduction in abundance as the other groups. Despite an increasing proportion of dinoflagellates compared with other taxa, there are no clear indications of increased occurrence of toxic algal blooms in inner Oslofjorden. However, the introduction of new "toxin-producing" species may cause concern.

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BACKGROUND: Polyunsaturated fatty acid (PUFA) synthase is a multi-domain mega-enzyme that effectively synthesizes a series of PUFAs in marine microorganisms. The dehydratase (DH) domain of a PUFA synthase plays a crucial role in double bond positioning in fatty acids. Sequencing results of the coccolithophore Emiliania huxleyi (E. huxleyi, Eh) indicated that this species contains a PUFA synthase with multiple DH domains. Therefore, the current study, sought to define the functions of these DH domains (EhDHs), by cloning and overexpressing the genes encoding FabA-like EhDHs in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana). RESULTS: A complementation test showed that the two FabA-like DH domains could restore DH function in a temperature-sensitive (Ts) mutant. Meanwhile, overexpression of FabA-like EhDH1 and EhDH2 domains increased the production of unsaturated fatty acids (UFAs) in recombinant E. coli by 43.5-32.9%, respectively. Site-directed mutagenesis analysis confirmed the authenticity of active-site residues in these domains. Moreover, the expression of tandem EhDH1-DH2 in A. thaliana altered the fatty acids content, seed weight, and germination rate. CONCLUSIONS: The two FabA-like DH domains in the E. huxleyi PUFA synthase function as 3-hydroxyacyl-acyl carrier protein dehydratase in E. coli. The expression of these domains in E. coli and A. thaliana can alter the fatty acid profile in E. coli and increase the seed lipid content and germination rate in A. thaliana. Hence, introduction of DH domains controlling the dehydration process of fatty acid biosynthesis in plants might offer a new strategy to increase oil production in oilseed plants.

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Nutrient conditions influence the physiology and stoichiometry of marine phytoplankton. While extensive studies have documented the effects of abundances and types of nutrients such as nitrogen (N) and phosphorus (P), the effect of phosphonates as a P source is less understood and underexplored. Here, with the cosmopolitan coccolithophorid Emiliania huxleyi as a model phytoplankter, we investigated the effect of the phosphonate type of herbicide glyphosate as the sole P source in comparison with the P-depleted and P-replete (with 36 µM dissolved inorganic phosphate [DIP]) cultures. We measured changes in cellular C (carbon):P and N:P ratios and physiological performance and documented the corresponding transcriptomic and miRNAomic responses in E. huxleyi to glyphosate treatment. We found that glyphosate supported population growth but not to the full scale relative to DIP, and this was under the concerted regulation of DNA replication and cell cycle arrest genes as well as the growth-regulating miRNA. Furthermore, our data suggest that E. huxleyi took up glyphosate directly, bypassing extracellular hydrolysis, and this involved ABC transporters. Meanwhile, glyphosate-grown cultures displayed marked increases in cellular particulate organic C (POC) and PON contents, cell size, and transcription of genes for CO2 fixation and citrate cycle, nitrate transport, and protein biosynthesis. However, compared to DIP, the maximum absorption rate of glyphosate was only 33%, and glyphosate-grown E. huxleyi cells exhibited a mild P-stress symptom and elevated cellular C:P and N:P ratios. Interestingly, glyphosate-grown cells showed an increased sinking rate, suggesting that glyphosate as the sole P source might enhance the efficiency of C export by E. huxleyi, which would compensate for the expected decline in primary productivity (and hence carbon efflux) in the future more nutrient-depleted ocean. This biogeochemical implication needs to be further studied and verified, however.

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