Regulation and integration of membrane transport in marine diatoms.

Diatoms represent one of the most successful groups of marine phytoplankton and are major contributors to ocean biogeochemical cycling. They have colonized marine, freshwater and ice environments and inhabit all regions of the World's oceans, from poles to tropics. Their success is underpinned by a remarkable ability to regulate their growth and metabolism during nutrient limitation and to respond rapidly when nutrients are available. This requires precise regulation of membrane transport and nutrient acquisition mechanisms, integration of nutrient sensing mechanisms and coordination of different transport pathways. This review outlines transport mechanisms involved in acquisition of key nutrients (N, C, P, Si, Fe) by marine diatoms, illustrating their complexity, sophistication and multiple levels of control.

Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH.

Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH.

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon.

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO2 in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO2 to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO2 availability, little is known about how eCA contributes to CO2 supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO2 supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O2 demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake.

Ca2+ elevations disrupt interactions between intraflagellar transport and the flagella membrane in Chlamydomonas.

The movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using retrograde IFT motors to move adhesive glycoproteins in the flagella membrane. Ca2+ signalling contributes directly to the gliding process, although uncertainty remains over the mechanism through which it acts. Here, we show that flagella Ca2+ elevations initiate the movement of paused retrograde IFT trains, which accumulate at the distal end of adherent flagella, but do not influence other IFT processes. On highly adherent surfaces, flagella exhibit high-frequency Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. Flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane, suggesting that Ca2+ acts by directly disrupting an interaction between retrograde IFT trains and flagella membrane glycoproteins. By regulating the extent to which glycoproteins on the flagella surface interact with IFT motor proteins on the axoneme, this signalling mechanism allows precise control of traction force and gliding motility in adherent flagella.

Mesoscale oceanographic meanders influence protist community function and structure in the southern Indian Ocean.

The interface between the nutrient-rich Southern Ocean and oligotrophic Indian Ocean creates unique environmental conditions that can strongly influence biological processes. We investigated protist communities across a mesoscale meander of the Subtropical Front within the Southern Indian Ocean. 18S V9 rDNA metabarcoding suggests a diverse protist community in which the dinoflagellates and parasitic Syndiniales were abundant. Diversity was highest in frontal waters of the mesoscale meander, with differences in community structure inside and outside the meander. While the overall community was dominated by mixotrophic taxa, the frontal boundary of the meander had increased abundances of heterotrophic taxa, with potential implications for net atmospheric CO2 drawdown. Pulse amplitude modulated (PAM) fluorimetry revealed significant differences in the photophysiology of phytoplankton communities inside and outside the meander. By using single-cell PAM microscopy, we identified physiological differences between dinoflagellate and coccolithophore taxa, which may have contributed to changes in photophysiology observed at community level. Overall, our results demonstrate that frontal areas have a strong impact on the composition of protist communities in the Southern Ocean with important implications for understanding biological processes in this region.

Characterization of the molecular mechanisms of silicon uptake in coccolithophores.

Coccolithophores are an important group of calcifying marine phytoplankton. Although coccolithophores are not silicified, some species exhibit a requirement for Si in the calcification process. These species also possess a novel protein (SITL) that resembles the SIT family of Si transporters found in diatoms. However, the nature of Si transport in coccolithophores is not yet known, making it difficult to determine the wider role of Si in coccolithophore biology. Here, we show that coccolithophore SITLs act as Na+ -coupled Si transporters when expressed in heterologous systems and exhibit similar characteristics to diatom SITs. We find that CbSITL from Coccolithus braarudii is transcriptionally regulated by Si availability and is expressed in environmental coccolithophore populations. However, the Si requirement of C. braarudii and other coccolithophores is very low, with transport rates of exogenous Si below the level of detection in sensitive assays of Si transport. As coccoliths contain only low levels of Si, we propose that Si acts to support the calcification process, rather than forming a structural component of the coccolith itself. Si is therefore acting as a micronutrient in coccolithophores and natural populations are only likely to experience Si limitation in circumstances where dissolved silicon (DSi) is depleted to extreme levels.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Cold-induced [Ca2+]cyt elevations function to support osmoregulation in marine diatoms.

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As intertidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signaling interacts with osmotic signaling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signaling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii.

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.

Spatiotemporal patterns of intracellular Ca2+ signalling govern hypo-osmotic stress resilience in marine diatoms.

Diatoms are globally important phytoplankton that dominate coastal and polar-ice assemblages. These environments exhibit substantial changes in salinity over dynamic spatiotemporal regimes. Rapid sensory systems are vital to mitigate the harmful consequences of osmotic stress. Population-based analyses have suggested that Ca2+ signalling is involved in diatom osmotic sensing. However, mechanistic insight of the role of osmotic Ca2+ signalling is limited. Here, we show that Phaeodactylum Ca2+ elevations are essential for surviving hypo-osmotic shock. Moreover, employing novel single-cell imaging techniques we have characterised real-time Ca2+ signalling responses in single diatom cells to environmental osmotic perturbations. We observe that intracellular spatiotemporal patterns of osmotic-induced Ca2+ elevations encode vital information regarding the nature of the osmotic stimulus. Localised Ca2+ signals evoked by mild or gradual hypo-osmotic shocks are propagated globally from the apical cell tips, enabling fine-tuned cell volume regulation across the whole cell. Finally, we demonstrate that diatoms adopt Ca2+ -independent and dependent mechanisms for osmoregulation. We find that efflux of organic osmolytes occurs in a Ca2+ -independent manner, but this response is insufficient to mitigate cell damage during hypo-osmotic shock. By comparison, Ca2+ -dependent signalling is necessary to prevent cell bursting via precise coordination of K+ transport, and therefore is likely to underpin survival in dynamic osmotic environments.

Role of silicon in the development of complex crystal shapes in coccolithophores.

The development of calcification by the coccolithophores had a profound impact on ocean carbon cycling, but the evolutionary steps leading to the formation of these complex biomineralized structures are not clear. Heterococcoliths consisting of intricately shaped calcite crystals are formed intracellularly by the diploid life cycle phase. Holococcoliths consisting of simple rhombic crystals can be produced by the haploid life cycle stage but are thought to be formed extracellularly, representing an independent evolutionary origin of calcification. We use advanced microscopy techniques to determine the nature of coccolith formation and complex crystal formation in coccolithophore life cycle stages. We find that holococcoliths are formed in intracellular compartments in a similar manner to heterococcoliths. However, we show that silicon is not required for holococcolith formation and that the requirement for silicon in certain coccolithophore species relates specifically to the process of crystal morphogenesis in heterococcoliths. We therefore propose an evolutionary scheme in which the lower complexity holococcoliths represent an ancestral form of calcification in coccolithophores. The subsequent recruitment of a silicon-dependent mechanism for crystal morphogenesis in the diploid life cycle stage led to the emergence of the intricately shaped heterococcoliths, enabling the formation of the elaborate coccospheres that underpin the ecological success of coccolithophores.

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes.

The evolution of Na+-selective four-domain voltage-gated channels (4D-Navs) in animals allowed rapid Na+-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na+-selective voltage-gated channels (BacNav), they typically exhibit much slower kinetics than 4D-Navs, and are not thought to have crossed the prokaryote-eukaryote boundary. As such, the capacity for rapid Na+-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na+-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Nav blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na+-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Navs. The EukCatB channels therefore represent an independent evolution of fast Na+-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae.

The requirement for calcification differs between ecologically important coccolithophore species.

Coccolithophores are globally distributed unicellular marine algae that are characterized by their covering of calcite coccoliths. Calcification by coccolithophores contributes significantly to global biogeochemical cycles. However, the physiological requirement for calcification remains poorly understood as non-calcifying strains of some commonly used model species, such as Emiliania huxleyi, grow normally in laboratory culture. To determine whether the requirement for calcification differs between coccolithophore species, we utilized multiple independent methodologies to disrupt calcification in two important species of coccolithophore: E. huxleyi and Coccolithus braarudii. We investigated their physiological response and used time-lapse imaging to visualize the processes of calcification and cell division in individual cells. Disruption of calcification resulted in major growth defects in C. braarudii, but not in E. huxleyi. We found no evidence that calcification supports photosynthesis in C. braarudii, but showed that an inability to maintain an intact coccosphere results in cell cycle arrest. We found that C. braarudii is very different from E. huxleyi as it exhibits an obligate requirement for calcification. The identification of a growth defect in C. braarudii resulting from disruption of the coccosphere may be important in considering their response to future changes in ocean carbonate chemistry.

Coccolithophore biomineralization: New questions, new answers.

Coccolithophores are unicellular phytoplankton that are characterized by the presence intricately formed calcite scales (coccoliths) on their surfaces. In most cases coccolith formation is an entirely intracellular process - crystal growth is confined within a Golgi-derived vesicle. A wide range of coccolith morphologies can be found amongst the different coccolithophore groups. This review discusses the cellular factors that regulate coccolith production, from the roles of organic components, endomembrane organization and cytoskeleton to the mechanisms of delivery of substrates to the calcifying compartment. New findings are also providing important information on how the delivery of substrates to the calcification site is co-ordinated with the removal of H(+) that are a bi-product of the calcification reaction. While there appear to be a number of species-specific features of the structural and biochemical components underlying coccolith formation, the fluxes of Ca(2+) and a HCO3(-) required to support coccolith formation appear to involve spatially organized recruitment of conserved transport processes.

Spatial and temporal specificity of Ca2+ signalling in Chlamydomonas reinhardtii in response to osmotic stress.

Ca2+ -dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+ -responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Ocean acidification with (de)eutrophication will alter future phytoplankton growth and succession.

Human activity causes ocean acidification (OA) though the dissolution of anthropogenically generated CO2 into seawater, and eutrophication through the addition of inorganic nutrients. Eutrophication increases the phytoplankton biomass that can be supported during a bloom, and the resultant uptake of dissolved inorganic carbon during photosynthesis increases water-column pH (bloom-induced basification). This increased pH can adversely affect plankton growth. With OA, basification commences at a lower pH. Using experimental analyses of the growth of three contrasting phytoplankton under different pH scenarios, coupled with mathematical models describing growth and death as functions of pH and nutrient status, we show how different conditions of pH modify the scope for competitive interactions between phytoplankton species. We then use the models previously configured against experimental data to explore how the commencement of bloom-induced basification at lower pH with OA, and operating against a background of changing patterns in nutrient loads, may modify phytoplankton growth and competition. We conclude that OA and changed nutrient supply into shelf seas with eutrophication or de-eutrophication (the latter owing to pollution control) has clear scope to alter phytoplankton succession, thus affecting future trophic dynamics and impacting both biogeochemical cycling and fisheries.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

Plant cell growth and stress signaling require Ca²âº influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²âº-permeable conductance that permits Ca²âº influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²âº-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²âº- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²âº in response to OH•. An OH•-activated Ca²âº conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²âº-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²âº in plants.

A voltage-gated H+ channel underlying pH homeostasis in calcifying coccolithophores.

Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths) in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO3⁻ as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan H(v)1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the H(v)1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate use of H+ derived from calcification for intracellular CO2 generation. Furthermore, the presence of H(v)1 class ion channels in a wide range of extant eukaryote groups indicates they evolved in an early common ancestor.