Stimulating and toxic effect of chromium on growth and photosynthesis of a marine chlorophyte.

Marine phytoplankton can interchange trace metals in various biochemical functions, particularly under metal-limiting conditions. Here, we investigate the stimulating and toxicity effect of chromium (Cr) on a marine Chlorophyceae Osetreococcus tauri under Fe-replete and Fe-deficient conditions. We determined the growth, photosynthesis, and proteome expressions of Osetreococcus tauri cultured under different Cr and Fe concentrations. In Fe-replete conditions, the presence of Cr(VI) stimulated significantly the growth rate and the maximum yield of photochemistry of photosystem II (Fv /Fm ) of the phytoplankton, while the functional absorption cross-section of photosystem II (σPSII ) did not change. Minor additions of Cr(VI) partially rescued phytoplankton growth under Fe-limited conditions. Proteomic analysis of this alga grown in Fe-replete normal and Fe-replete with Cr addition media (10 µM Cr) showed that the presence of Cr significantly decreased the expression of phosphate-transporting proteins and photosynthetic proteins, while increasing the expression of proteins related to carbon assimilation. Cr can stimulate the growth and photosynthesis of O. tauri, but the effects are dependent on both the Cr(VI) concentration and the availability of Fe. The proteomic results further suggest that Cr(VI) addition might significantly increase starch production and carbon fixation.

Calcifying Coccolithophore: An Evolutionary Advantage Against Extracellular Oxidative Damage.

The evolutionary advantages afforded by phytoplankton calcification remain enigmatic. In this work, fluoroelectrochemical experiments reveal that the presence of a CaCO3 shell of a naturally calcifying coccolithophore, Coccolithus braarudii, offers protection against extracellular oxidants as measured by the time required for the switch-off in their chlorophyll signal, compared to the deshelled equivalents, suggesting the shift toward calcification offers some advantages for survival in the surface of radical-rich seawater.

AI facilitated fluoro-electrochemical phytoplankton classification.

Marine phytoplankton is extremely diverse. Counting and characterising phytoplankton is essential for understanding climate change and ocean health not least since phytoplankton extensively biomineralize carbon dioxide whilst generating 50% of the planet's oxygen. We report the use of fluoro-electrochemical microscopy to distinguish different taxonomies of phytoplankton by the quenching of their chlorophyll-a fluorescence using chemical species oxidatively electrogenerated in situ in seawater. The rate of chlorophyll-a quenching of each cell is characteristic of the species-specific structural composition and cellular content. But with increasing diversity and extent of phytoplankton species under study, human interpretation and distinction of the resulting fluorescence transients becomes increasingly and prohibitively difficult. Thus, we further report a neural network to analyse these fluorescence transients, with an accuracy >95% classifying 29 phytoplankton strains to their taxonomic orders. This method transcends the state-of-the-art. The success of the fluoro-electrochemical microscopy combined with AI provides a novel, flexible and highly granular solution to phytoplankton classification and is adaptable for autonomous ocean monitoring.

The cellular response to ocean warming in Emiliania huxleyi.

Marine phytoplankton contribute substantially to the global flux of carbon from the atmosphere to the deep ocean. Sea surface temperatures will inevitably increase in line with global climate change, altering the performance of marine phytoplankton. Differing sensitivities of photosynthesis and respiration to temperature, will likely shift the strength of the future oceanic carbon sink. To further clarify the molecular mechanisms driving these alterations in phytoplankton function, shotgun proteomic analysis was carried out on the globally-occurring coccolithophore Emiliania huxleyi exposed to moderate- (23°C) and elevated- (28°C) warming. Compared to the control (17°C), growth of E. huxleyi increased under elevated temperatures, with higher rates recorded under moderate- relative to elevated- warming. Proteomic analysis revealed a significant modification of the E. huxleyi cellular proteome as temperatures increased: at lower temperature, ribosomal proteins and photosynthetic machinery appeared abundant, as rates of protein translation and photosynthetic performance are restricted by low temperatures. As temperatures increased, evidence of heat stress was observed in the photosystem, characterized by a relative down-regulation of the Photosystem II oxygen evolving complex and ATP synthase. Acclimation to elevated warming (28°C) revealed a substantial alteration to carbon metabolism. Here, E. huxleyi made use of the glyoxylate cycle and succinate metabolism to optimize carbon use, maintain growth and maximize ATP production in heat-damaged mitochondria, enabling cultures to maintain growth at levels significantly higher than those recorded in the control (17°C). Based on the metabolic changes observed, we can predict that warming may benefit photosynthetic carbon fixation by E. huxleyi in the sub-optimal to optimal thermal range. Past the thermal optima, increasing rates of respiration and costs of repair will likely constrain growth, causing a possible decline in the contribution of this species to the oceanic carbon sink depending on the evolvability of these temperature thresholds.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Quantifying the Extent of Calcification of a Coccolithophore Using a Coulter Counter.

Although, in principle, the Coulter Counter technique yields an absolute measure of particle volume, in practice, calibration is near-universally employed. For regularly shaped and non-biological samples, the use of latex beads for calibration can provide sufficient accuracy. However, this is not the case with particles encased in biogenically formed calcite. To date, there has been no effective route by which a Coulter Counter can be calibrated to enable the calcification of coccolithophores─single cells encrusted with biogenic calcite─to be quantified. Consequently, herein, we seek to answer the following question: to what extent can a Coulter Counter be used to provide accurate information regarding the calcite content of a single-species coccolithophore population? Through the development of a new calibration methodology, based on the measurement and dynamic tracking of the acid-driven calcite dissolution reaction, a route by which the cellular calcite content can be determined is presented. This new method allows, for the first time, a Coulter Counter to be used to yield an absolute measurement of the amount of calcite per cell.

Calcium Carbonate Dissolution from the Laboratory to the Ocean: Kinetics and Mechanism.

The ultimate fate, over the course of millennia, of nearly all of the carbon dioxide formed by humankind is for it to react with calcium carbonate in the world's oceans. Although, this reaction is of global relevance, aspects of the calcite dissolution reaction remain poorly described with apparent contradictions present throughout the expansive literature. In this perspective we aim to evidence how a lack of appreciation of the role of mass-transport may have hampered developments in this area. These insights have important implications for both idealised experiments performed under laboratory conditions and for the measurement and modelling of oceanic calcite sediment dissolution.

Deuterium in marine organic biomarkers: toward a new tool for quantifying aquatic mixotrophy.

The traditional separation between primary producers (autotrophs) and consumers (heterotrophs) at the base of the marine food web is being increasingly replaced by the paradigm that mixoplankton, planktonic protists with the nutritional ability to use both phago(hetero)trophy and photo(auto)trophy to access energy are widespread globally. Thus, many 'phytoplankton' eat, while 50% of 'protozooplankton' also perform photosynthesis. Mixotrophy may enhance primary production, biomass transfer to higher trophic levels and the efficiency of the biological pump to sequester atmospheric CO2 into the deep ocean. Although this view is gaining traction, science lacks a tool to quantify the relative contributions of autotrophy and heterotrophy in planktonic protists. This hinders our understanding of their impacts on carbon cycling within marine pelagic ecosystems. It has been shown that the hydrogen (H) isotopic signature of lipids is uniquely sensitive to heterotrophy relative to autotrophy in plants and bacteria. Here, we explored whether it is also sensitive to the trophic status in protists. The new understanding of H isotope signature of lipid biomarkers suggests it offers great potential as a novel tool for quantifying the prevalence of mixotrophy in diverse marine microorganisms and thus for investigating the implications of the 'mixoplankton' paradigm.

Proteomic response of the marine ammonia-oxidising archaeon Nitrosopumilus maritimus to iron limitation reveals strategies to compensate for nutrient scarcity.

Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.

Rapid Opto-electrochemical Differentiation of Marine Phytoplankton.

The use of electro-generated oxidants in seawater facilitates the discrimination of different plankton groups via monitoring the decay in real time of their chlorophyll-a (chl-a) fluorescence signals following potentiostatic initiation of electrolysis in their vicinity (Yang M.Chem. Sci.2019, 10( (34), ), 7988-7993). In this paper, we explore the sensitivity of phytoplankton to different chemical species produced at various potentials in seawater. At low potentials, the oxidation of ca. millimolar bromide naturally present in seawater to hypobromous acid 'switches-off' the chl-a signal of individual Chlamydomonas concordia cells (green algae) located on the electrode surface within tens of seconds of the potential onset. At higher oxidative potentials, the oxidation of chloride and water produces oxidants (Cl2, OH, H2O2, etc.) that are also lethal to the plankton. To deconvolute the contributions to the response from the chemical identity of the oxidant and the amount of charge delivered to 'titrate' the individual living plankton using the loss of fluorescence as the 'end point', we introduce a ramped galvanostatic method. This approach enabled the controlled injection of charge applied to a bespoke electrochemical cell in which the plankton are immobilized on an electrode surface for rapid and sensitive measurement. It is shown that the number of moles (charge) of oxidants required to react leading to chl-a switch-off is independent of the chemical identity of the electro-generated oxidant(s) among hypobromous acid, chlorine, or water-derived oxidants. Comparative experiments between C. concordia and Emiliania huxleyi (where the latter are encapsulated by extracellular plates of calcium carbonate) show that significantly different amounts of absolute charge (moles of electro-generated oxidants) are required in each case to 'switch-off' the chl-a signal. The method provides the basis for a tool that could distinguish between different plankton cells within ca. 2 min including the setup time.

The mode of speciation during a recent radiation in open-ocean phytoplankton.

Despite the enormous ecological importance of marine phytoplankton, surprisingly little is known about how new phytoplankton species originate and evolve in the open ocean, in the absence of apparent geographic barriers that typically act as isolation mechanisms in speciation. To investigate the mechanism of open-ocean speciation, we combined fossil and climatic records from the late Quaternary with genome-wide evolutionary genetic analyses of speciation in the ubiquitous and abundant pelagic coccolithophore genus Gephyrocapsa (including G. huxleyi, formerly known as Emiliania huxleyi). Based on the analysis of 43 sequenced genomes, we report that the best-fitting scenario for all speciation events analyzed included an extended period of complete isolation followed by recent (Holocene) secondary contact, supporting the role of geographic or oceanographic barriers in population divergence and speciation. Consistent with this, fossil data reveal considerable diachroneity of species first occurrence. The timing of all speciation events coincided with glacial phases of glacial-interglacial cycles, suggesting that stronger isolation between the ocean basins and increased segregation of ecological niches during glaciations are important drivers of speciation in marine phytoplankton. The similarity across multiple speciation events implies the generality of this inferred speciation scenario for marine phytoplankton.

Opto-Electrochemical Dissolution Reveals Coccolith Calcium Carbonate Content.

Coccoliths are plates of biogenic calcium carbonate secreted by calcifying marine phytoplankton; annually these phytoplankton are responsible for exporting >1 billion tonnes (1015  g) of calcite to the deep ocean. Rapid and reliable methods for assessing the degree of calcification are technically challenging because the coccoliths are micron sized and contain picograms (pg) of calcite. Here we pioneer an opto-eletrochemical acid titration of individual coccoliths which allows 3D reconstruction of each individual coccolith via in situ optical imaging enabling direct inference of the coccolith mass. Coccolith mass ranging from 2 to 400 pg are reported herein, evidencing both inter- and intra-species variation over four different species. We foresee this scientific breakthrough, which is independent of knowledge regarding the species and calibration-free, will allow continuous monitoring and reporting of the degree of coccolith calcification in the changing marine environment.

The influence of elevated SiO2 (aq) on intracellular silica uptake and microbial metabolism.

Microbes are known to accumulate intracellular SiO2 (aq) up to 100s of mmol/l from modern seawater (SiO2 (aq) <100 µmol/l), despite having no known nutrient requirement for Si. Before the evolution of siliceous skeletons, marine silica concentrations were likely an order of magnitude higher than the modern ocean, raising the possibility that intracellular SiO2 (aq) accumulation interfered with normal cellular function in non-silicifying algae. Yet, because few culturing studies have isolated the effects of SiO2 (aq) at high concentration, the potential impact of elevated marine silica on early microbial evolution is unknown. Here, we test the influence of elevated SiO2 (aq) on eukaryotic algae, as well as a prokaryote species. Our results demonstrate that under SiO2 (aq) concentrations relevant to ancient seawater, intracellular Si accumulates to concentrations comparable to those found in siliceous algae such as diatoms. In addition, all eukaryotic algae showed a statistically significant response to the high-Si treatment, including reduced average cell sizes and/or a reduction in the maximum growth rate. In contrast, there was no consistent response to the high-Si treatment by the prokaryote species. Our results highlight the possibility that elevated marine SiO2 (aq) may have been an environmental stressor during early eukaryotic evolution.

Marine ammonia-oxidising archaea and bacteria occupy distinct iron and copper niches.

Ammonia oxidation by archaea and bacteria (AOA and AOB), is the first step of nitrification in the oceans. As AOA have an ammonium affinity 200-fold higher than AOB isolates, the chemical niche allowing AOB to persist in the oligotrophic ocean remains unclear. Here we show that marine isolates, Nitrosopumilus maritimus strain SCM1 (AOA) and Nitrosococcus oceani strain C-107 (AOB) have contrasting physiologies in response to the trace metals iron (Fe) and copper (Cu), holding potential implications for their niche separation in the oceans. A greater affinity for unchelated Fe may allow AOB to inhabit shallower, euphotic waters where ammonium supply is high, but competition for Fe is rife. In contrast to AOB, AOA isolates have a greater affinity and toxicity threshold for unchelated Cu providing additional explanation to the greater success of AOA in the marine environment where Cu availability can be highly variable. Using comparative genomics, we predict that the proteomic and metal transport basis giving rise to contrasting physiologies in isolates is widespread across phylogenetically diverse marine AOA and AOB that are not yet available in pure culture. Our results develop the testable hypothesis that ammonia oxidation may be limited by Cu in large tracts of the open ocean and suggest a relatively earlier emergence of AOB than AOA when considered in the context of evolving trace metal availabilities over geologic time.

A new method for isolating and analysing coccospheres within sediment.

Size is a fundamental cellular trait that is important in determining phytoplankton physiological and ecological processes. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by the phytoplankton coccolithophores, can provide a rare window into cell size in the past. Coccospheres are delicate however and are therefore poorly preserved in sediment. We demonstrate a novel technique combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the morphological information provided by microscopy with high sample numbers associated with flow cytometry. High throughput imaging overcomes the constraints of labour-intensive manual microscopy and allows statistically robust analysis of morphological features and coccosphere concentration despite low coccosphere concentrations in sediments. Applying this technique to the fine-fraction of sediments, hundreds of coccospheres can be visually isolated quickly with minimal sample preparation. This approach has the potential to enable rapid processing of down-core sediment records and/or high spatial coverage from surface sediments and may prove valuable in investigating the interplay between climate change and coccolithophore physiological/ecological response.

Biophysical analysis of the structural evolution of substrate specificity in RuBisCO.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO2, is competitively displaced by O2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO2 for O2 Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO2 and O2 Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO2 This paper identifies a key structural feature that enabled the enzyme to evolve improved CO2 sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.

Evolution of Mutation Rate in Astronomically Large Phytoplankton Populations.

Genetic diversity is expected to be proportional to population size, yet, there is a well-known, but unexplained lack of genetic diversity in large populations-the "Lewontin's paradox." Larger populations are expected to evolve lower mutation rates, which may help to explain this paradox. Here, we test this conjecture by measuring the spontaneous mutation rate in a ubiquitous unicellular marine phytoplankton species Emiliania huxleyi (Haptophyta) that has modest genetic diversity despite an astronomically large population size. Genome sequencing of E. huxleyi mutation accumulation lines revealed 455 mutations, with an unusual GC-biased mutation spectrum. This yielded an estimate of the per site mutation rate µ = 5.55×10-10 (CI 95%: 5.05×10-10 - 6.09×10-10), which corresponds to an effective population size Ne ∼ 2.7×106. Such a modest Ne is surprising for a ubiquitous and abundant species that accounts for up to 10% of global primary productivity in the oceans. Our results indicate that even exceptionally large populations do not evolve mutation rates lower than ∼10-10 per nucleotide per cell division. Consequently, the extreme disparity between modest genetic diversity and astronomically large population size in the plankton species cannot be explained by an unusually low mutation rate.

Antagonistic co-limitation through ion promiscuity - On the metal sensitivity of Thalassiosira oceanica under phosphorus stress.

Nutrient limitation of primary producers is a fundamental principle in biogeochemical oceanography and has been used with great success in prescribing understanding to patterns of marine primary productivity. In recent years the paradigm of nutrient limitation has expanded from single nutrient limitation towards concepts of co-limitation by multiple resources. Interactive effects between multiple limiting resources are now thought commonplace in marine microbial communities. Here we investigate the response exhibited by phosphate-limited Thalassiosira oceanica to elevated concentrations of the phosphate analogs vanadate, arsenate and molybdate. Enrichments in external arsenate and vanadate to phosphate-limited cultures act to suppress growth rates entirely, an effect not seen in phosphate replete conditions. Retardation of growth rates is attributed to mistaken uptake through ion promiscuity as evidenced by observations of significant intracellular accumulation of both arsenic and vanadium under phosphate limited conditions. We describe this novel co-limitation scenario as dependent antagonistic co-limitation (DAC), and suggest that this phenomenon of non-deliberate intracellular accumulation could be used as both a proxy of phosphate stress in the modern ocean and a possible marker of phosphate depletion limiting the duration of oceanic anoxic events.

Susceptibility of algae to Cr toxicity reveals contrasting metal management strategies.

At the Paleozoic-Mesozoic boundary, the dominance of marine eukaryotic algae shifted from the green (chlorophyll b) to the red (chlorophyll c) superfamily. Selection pressures caused by the bioavailability of trace metals associated with increasing oxygenation of the ocean may have played a key role in this algal revolution. From a scan of elemental compositions, a significant difference in the cellular Cr/P quota was found between the two superfamilies. Here, the different responses to high levels of Cr exposure reveal contrasting strategies for metal uptake and homeostasis in these algal lineages. At high Cr(VI) concentrations, red lineages experience growth inhibition through reduced photosynthetic capability, while green lineages are completely unaffected. Moreover, Cr(VI) has a more significant impact on the metallomes of red lineage algae, in which metal/P ratios increased with increasing Cr(VI) concentration for many trace elements. Green algae have higher specificity transporters to prevent Cr(VI) from entering the cell, and more specific intracellular stores of Cr within the membrane fraction than the red algae, which accumulate more Cr mistakenly in the cytosol fraction via lower affinity transport mechanisms. Green algal approaches require greater nutrient investments in the more numerous transport proteins required and management of specific metals, a strategy better adapted to the resource-rich coastal waters. By contrast, the red algae are nutrient-efficient with fewer and less discriminate metal transporters, which can be fast and better adapted in the oligotrophic, oxygenated open ocean, which has prevailed since the deepening of the oxygen minimum zones at the start of the Mesozoic era.