Publisher Correction: Glacial expansion of oxygen-depleted seawater in the eastern tropical Pacific.

In this Letter, 'δ18C' should have been 'δ13C' in Fig. 3b, and the x axis should extend to 50 kyr rather than 40 kyr. This figure has been corrected online.

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.

Glacial expansion of oxygen-depleted seawater in the eastern tropical Pacific.

Increased storage of carbon in the oceans has been proposed as a mechanism to explain lower concentrations of atmospheric carbon dioxide during ice ages; however, unequivocal signatures of this storage have not been found1. In seawater, the dissolved gases oxygen and carbon dioxide are linked via the production and decay of organic material, with reconstructions of low oxygen concentrations in the past indicating an increase in biologically mediated carbon storage. Marine sediment proxy records have suggested that oxygen concentrations in the deep ocean were indeed lower during the last ice age, but that near-surface and intermediate waters of the Pacific Ocean-a large fraction of which are poorly oxygenated at present-were generally better oxygenated during the glacial1-3. This vertical opposition could suggest a minimal net basin-integrated change in carbon storage. Here we apply a dual-proxy approach, incorporating qualitative upper-water-column and quantitative bottom-water oxygen reconstructions4,5, to constrain changes in the vertical extent of low-oxygen waters in the eastern tropical Pacific since the last ice age. Our tandem proxy reconstructions provide evidence of a downward expansion of oxygen depletion in the eastern Pacific during the last glacial, with no indication of greater oxygenation in the upper reaches of the water column. We extrapolate our quantitative deep-water oxygen reconstructions to show that the respired carbon reservoir of the glacial Pacific was substantially increased, establishing it as an important component of the coupled mechanism that led to low levels of atmospheric carbon dioxide during the glacial.

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.

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.

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.

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.

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.

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.

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.

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.

Direct measurement of multi-elements in high matrix samples with a flow injection ICP-MS: application to the extended Emiliania huxleyi Redfield ratio.

The quotas of a limited number of trace elements in the extended Redfield ratios have been determined before and thought to reflect the requirements of phytoplankton. However, these quotas are found to be quite variable under different environmental conditions, suggesting that the cellular trace metal quota is not an accurate measure of cellular trace metal requirement. Here we present a method that has been developed and optimised for direct analysis of 32 elements simultaneously in small volume of cell lysate in buffers with a high salt matrix (800 µL, up to 30% TDS). We then demonstrate the application of the method to resolve the extended Redfield ratio of cell requirement by measuring the intracellular trace element composition of six Emiliania huxleyi strains isolated from different locations. The method uses a quadrupole-ICP-MS with a collision/reaction cell to resolve polyatomic interferences. The ICP-MS is interfaced with an Elemental Scientific Flow Injection Automation System (FIAS). The accuracy of the analysis according to this new method is verified by measuring 2 certified reference materials, BCR 273 and BCR 414. This work presents a number of running parameters, optimised for multi-element analysis of samples with a high TDS sample matrix. This method allows direct measurement of protein samples in their native state: no alteration or digestion is needed, which simplifies the steps for sample preparation. In this study with 6 E. huxleyi strains isolated from the environment, our method reveals significant differences between whole cell and intracellular metal quotas for all strains. The intracellular metal composition, interpreted as a truer representation of organisms' metal requirements, shows an environmentally dependent signal. This suggests that, compared with whole cell metal quotas, the metalloproteins are a better indicator of metal requirements of phytoplankton under various environmental conditions.

The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae.

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.

Post-mortem oxygen isotope exchange within cultured diatom silica.

RATIONALE: Potential post-mortem alteration to the oxygen isotope composition of biogenic silica is critical to the validity of palaeoclimate reconstructions based on oxygen isotope ratios (δ18 O values) from sedimentary silica. We calculate the degree of oxygen isotope alteration within freshly cultured diatom biogenic silica in response to heating and storing in the laboratory. METHODS: The experiments used freshly cultured diatom silica. Silica samples were either stored in water or dried at temperatures between 20 °C and 80 °C. The mass of affected oxygen and the associated silica-water isotope fractionation during alteration were calculated by conducting parallel experiments using endmember waters with δ18 O values of -6.3 to -5.9 ‰ and -36.3 to -35.0 ‰. Dehydroxylation and subsequent oxygen liberation were achieved by stepwise fluorination with BrF5 . The 18 O/16 O ratios were measured using a ThermoFinnigan MAT 253 isotope ratio mass spectrometer. RESULTS: Significant alterations in silica δ18 O values were observed, most notably an increase in the δ18 O values following drying at 40-80 °C. Storage in water for 7 days between 20 and 80 °C also led to significant alteration in δ18 O values. Mass balance calculations suggest that the amount of affected oxygen is positively correlated with temperature. The estimated oxygen isotope fractionation during alteration is an inverse function of temperature, consistent with the extrapolation of models for high-temperature silica-water oxygen isotope fractionation. CONCLUSIONS: Routinely used preparatory methods may impart significant alterations to the δ18 O values of biogenic silica, particularly when dealing with modern cultured or field-collected material. The significance of such processes within natural aquatic environments is uncertain; however, there is potential that similar processes also affect sedimentary diatoms, with implications for the interpretation of biogenic silica-hosted δ18 O palaeoclimate records.

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms.

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23-68 µM), SC/O (57-116mol mol(-1)), and K O (413-2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat (c) for each diatom Rubisco showed less variation (2.1-3.7s(-1)), thus averting the canonical trade-off typically observed between K C and k cat (c) for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily.

Nonspecific uptake and homeostasis drive the oceanic cadmium cycle.

The global marine distributions of Cd and phosphate are closely correlated, which has led to Cd being considered as a marine micronutrient, despite its toxicity to life. The explanation for this nutrient-like behavior is unknown because there is only one identified biochemical function for Cd, an unusual Cd/Zn carbonic anhydrase. Recent developments in Cd isotope mass spectrometry have revealed that Cd uptake by phytoplankton causes isotopic fractionation in the open ocean and in culture. Here we investigate the physiochemical pathways that fractionate Cd isotopes by performing subcellular Cd isotope analysis on genetically modified microorganisms. We find that expression of the Cd/Zn carbonic anhydrase makes no difference to the Cd isotope composition of whole cells. Instead, a large proportion of the Cd is partitioned into cell membranes with a similar direction and magnitude of Cd isotopic fractionation to that seen in surface seawater. This observation is well explained if Cd is mistakenly imported with other divalent metals and subsequently managed by binding within the cell to avoid toxicity. This process may apply to other divalent metals, whereby nonspecific uptake and subsequent homeostasis may contribute to elemental and isotopic distributions in seawater, even for elements commonly considered as micronutrients.

Migration of the subtropical front as a modulator of glacial climate.

Ice cores extracted from the Antarctic ice sheet suggest that glacial conditions, and the relationship between isotopically derived temperatures and atmospheric PCO(2) have been constant over the last 800,000 years of the Late Pleistocene epoch. But independent lines of evidence, such as the extent of Northern Hemisphere ice sheets, sea level and other temperature records, point towards a fluctuating severity of glacial periods, particularly during the more extreme glacial stadials centred around 340,000 and 420,000 years ago (marine isotope stages 10 and 12). Previously unidentified mechanisms therefore appear to have mediated the relationship between insolation, CO(2) and climate. Here we test whether northward migration of the subtropical front (STF) off the southeastern coast of South Africa acts as a gatekeeper for the Agulhas current, which controls the transport of heat and salt from the Indo-Pacific Ocean to the Atlantic Ocean. Using a new 800,000-year record of sea surface temperature and ocean productivity from ocean sediment core MD962077, we demonstrate that during cold stadials (particularly marine isotope stages 10 and 12), productivity peaked and sea surface temperature was up to 6 degrees C cooler than modern temperatures. This suggests that during these cooler stadials, the STF moved northward by up to 7 degrees latitude, nearly shutting off the Agulhas current. Our results, combined with faunal assemblages from the south Atlantic show that variable northwards migration of the Southern Hemisphere STF can modulate the severity of each glacial period by altering the strength of the Agulhas current carrying heat and salt to the Atlantic meridional overturning circulation. We show hence that the degree of northwards migration of the STF can partially decouple global climate from atmospheric partial pressure of carbon dioxide, P CO(2), and help to resolve the long-standing puzzle of differing glacial amplitudes within a consistent range of atmospheric PCO(2).

Phanerozoic co-evolution of O2-CO2 and ocean habitability.

This perspective reviews how atmospheric compositions, animals and marine algae evolved together to determine global ocean habitability during the past 500 million years.