Pan genome of the phytoplankton Emiliania underpins its global distribution.

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

Overexpression of plastid transketolase in tobacco results in a thiamine auxotrophic phenotype.

To investigate the effect of increased plastid transketolase on photosynthetic capacity and growth, tobacco (Nicotiana tabacum) plants with increased levels of transketolase protein were produced. This was achieved using a cassette composed of a full-length Arabidopsis thaliana transketolase cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed a major and unexpected effect of plastid transketolase overexpression as the transgenic tobacco plants exhibited a slow-growth phenotype and chlorotic phenotype. These phenotypes were complemented by germinating the seeds of transketolase-overexpressing lines in media containing either thiamine pyrophosphate or thiamine. Thiamine levels in the seeds and cotyledons were lower in transketolase-overexpressing lines than in wild-type plants. When transketolase-overexpressing plants were supplemented with thiamine or thiamine pyrophosphate throughout the life cycle, they grew normally and the seed produced from these plants generated plants that did not have a growth or chlorotic phenotype. Our results reveal the crucial importance of the level of transketolase activity to provide the precursor for synthesis of intermediates and to enable plants to produce thiamine and thiamine pyrophosphate for growth and development. The mechanism determining transketolase protein levels remains to be elucidated, but the data presented provide evidence that this may contribute to the complex regulatory mechanisms maintaining thiamine homeostasis in plants.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

Plasticity in the proteome of Emiliania huxleyi CCMP 1516 to extremes of light is highly targeted.

Optimality principles are often applied in theoretical studies of microalgal ecophysiology to predict changes in allocation of resources to different metabolic pathways, and optimal acclimation is likely to involve changes in the proteome, which typically accounts for > 50% of cellular nitrogen (N). We tested the hypothesis that acclimation of the microalga Emiliania huxleyi CCMP 1516 to suboptimal vs supraoptimal light involves large changes in the proteome as cells rebalance the capacities to absorb light, fix CO2 , perform biosynthesis and resist photooxidative stress. Emiliania huxleyi was grown in nutrient-replete continuous culture at 30 (LL) and 1000 µmol photons m(-2) s(-1) (HL), and changes in the proteome were assessed by LC-MS/MS shotgun proteomics. Changes were most evident in proteins involved in the light reactions of photosynthesis; the relative abundance of photosystem I (PSI) and PSII proteins was 70% greater in LL, light-harvesting fucoxanthin-chlorophyll proteins (Lhcfs) were up to 500% greater in LL and photoprotective LI818 proteins were 300% greater in HL. The marked changes in the abundances of Lhcfs and LI818s, together with the limited plasticity in the bulk of the E. huxleyi proteome, probably reflect evolutionary pressures to provide energy to maintain metabolic capabilities in stochastic light environments encountered by this species in nature.

The trade-off between the light-harvesting and photoprotective functions of fucoxanthin-chlorophyll proteins dominates light acclimation in Emiliania huxleyi (clone CCMP 1516).

Mechanistic understanding of the costs and benefits of photoacclimation requires knowledge of how photophysiology is affected by changes in the molecular structure of the chloroplast. We tested the hypothesis that changes in the light dependencies of photosynthesis, nonphotochemical quenching and PSII photoinactivation arises from changes in the abundances of chloroplast proteins in Emiliania huxleyi strain CCMP 1516 grown at 30 (Low Light; LL) and 1000 (High Light; HL) µmol photons m(-2) s(-1) photon flux densities. Carbon-specific light-saturated gross photosynthesis rates were not significantly different between cells acclimated to LL and HL. Acclimation to LL benefited cells by increasing biomass-specific light absorption and gross photosynthesis rates under low light, whereas acclimation to HL benefited cells by reducing the rate of photoinactivation of PSII under high light. Differences in the relative abundances of proteins assigned to light-harvesting (Lhcf), photoprotection (LI818-like), and the photosystem II (PSII) core complex accompanied differences in photophysiology: specifically, Lhcf:PSII was greater under LL, whereas LI818:PSII was greater in HL. Thus, photoacclimation in E. huxleyi involved a trade-off amongst the characteristics of light absorption and photoprotection, which could be attributed to changes in the abundance and composition of proteins in the light-harvesting antenna of PSII.

Designer diatom episomes delivered by bacterial conjugation.

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

Emiliania huxleyi increases calcification but not expression of calcification-related genes in long-term exposure to elevated temperature and pCO2.

Increased atmospheric pCO2 is expected to render future oceans warmer and more acidic than they are at present. Calcifying organisms such as coccolithophores that fix and export carbon into the deep sea provide feedbacks to increasing atmospheric pCO2. Acclimation experiments suggest negative effects of warming and acidification on coccolithophore calcification, but the ability of these organisms to adapt to future environmental conditions is not well understood. Here, we tested the combined effect of pCO2 and temperature on the coccolithophore Emiliania huxleyi over more than 700 generations. Cells increased inorganic carbon content and calcification rate under warm and acidified conditions compared with ambient conditions, whereas organic carbon content and primary production did not show any change. In contrast to findings from short-term experiments, our results suggest that long-term acclimation or adaptation could change, or even reverse, negative calcification responses in E. huxleyi and its feedback to the global carbon cycle. Genome-wide profiles of gene expression using RNA-seq revealed that genes thought to be essential for calcification are not those that are most strongly differentially expressed under long-term exposure to future ocean conditions. Rather, differentially expressed genes observed here represent new targets to study responses to ocean acidification and warming.