Production and Characterization of the Intra‐ and Extracellular Carbohydrates and Polymeric Substances (EPS) of Three Sea‐Ice Diatom Species,and Evidence for a Cryoprotective Role for EPS

Diatoms and their associated extracellular polymeric substances (EPS) are major constituents of the microalgal assemblages present within sea ice. Yields and chemical composition of soluble and cell‐associated polysaccharides produced by three sea‐ice diatoms, Synedropsis sp., Fragilariopsis curta, and F. cylindrus, were compared. Colloidal carbohydrates (CC) contained heteropolysaccharides rich in mannose, xylose, galactose, and glucose. Synedropsis sp. CC consisted mainly of carbohydrates <8 kDa size, with relatively soluble EPS, compared to high proportions of less‐soluble EPS produced by both Fragilariopsis spp. F. curta colloidal EPS contained high concentrations of amino sugars (AS). Both Fragilariopsis species had high yields of hot bicarbonate (HB) soluble EPS, rich in xylose, mannose, galactose, and fucose (and AS in F. cylindrus). All species had frustule‐associated EPS rich in glucose–mannose. Nutrient limitation resulted in declines in EPS yields and in glucose content of all EPS fractions. Significant similarities between EPS fractions from cultures and different components of natural EPS from Antarctic sea ice were found. Increased salinity (52) reduced growth, but increased yields of EPS in Fragilariopsis cylindrus. Ice formation was inhibited byF. cylindrus, EPS, and by enhanced EPS content (additional xanthan gum) down to ?12°C, with growth rate reduced in the presence of xanthan. Differences in the production and composition of EPS between Synedropsis sp. and Fragilariopsis spp., and the association between EPS, freezing and cell survival, supports the hypothesis that EPS production is a strategy to assist polar ice diatoms to survive the cold and saline conditions present in sea ice.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Protists in the marine ice of the Amery Ice Shelf, East Antarctica

Samples of marine ice were collected from the Amery Ice Shelf, a large embayed ice shelf in East Antarctica, during the Austral summer of 2001–2002. The samples came from a site ∼90 km from the iceberg calving front of the shelf, where the ice is 479 m thick and the lower 203 m is composed of accreted marine ice. Protists identified within the marine ice layer of the Amery Ice Shelf include diatoms, chrysophytes, silicoflagellates and dinoflagellates. The numerical dominance of sea ice indicator diatoms such as Fragilariopsis curta, Fragilariopsis cylindrus, Fragilariopsis rhombica and Chaetoceros resting spores, and the presence of cold open water diatoms such as Fragilariopsis kerguelensis and species of Thalassiosira suggest the protist composition of the Amery marine ice is attributable to seeding from melting pack and/or fast ice protist communities in the highly productive waters of Prydz Bay to the north.     

The diatom Fragilariopsis cylindrus: A model alga to understand cold-adapted life

Diatoms are significant primary producers especially in cold, turbulent, and nutrient-rich surface oceans. Hence, they are abundant in polar oceans, but also underpin most of the polar food webs and related biogeochemical cycles. The cold-adapted pennate diatom Fragilariopsis cylindrus is considered a keystone species in polar oceans and sea ice because it can thrive under different environmental conditions if temperatures are low. In this perspective paper, we provide insights into the latest molecular work that has been done on F. cylindrus and discuss its role as a model alga to understand cold-adapted life.     

Heterologous expression,refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

DEVELOPMENT OF IMMUNOASSAYS FOR THE IRON‐REGULATED PROTEINS FERREDOXIN AND FLAVODOXIN IN POLAR MICROALGAE1

While the growth of Southern Ocean phytoplankton is often limited by iron availability, there are no comparable experiments on sea‐ice algae. Here we assess the use of ferredoxin and flavodoxin to investigate the iron nutritional status of sea‐ice algae and describe the development of a quantitative immunoassay for both proteins in marine diatoms. High‐affinity monoclonal antibodies toward both proteins were produced from Cylindrotheca closterium (Ehrenb.) J. M. Lewin et Reimann, and these were used to develop Western blots. Western blots run on whole protein extracts detected both proteins with little cross‐reactivity toward other proteins. The two proteins could be successfully quantitated when applied to gels at between 5 and 50 ng in a volume of 25 μL (0.2–2 μg · mL^?1). Flavodoxin and ferrodoxin expression was examined in the Antarctic diatoms Entomoneis kjellmannii (Cleve) Poulin et Cardinal, Navicula directa (W. Sm.) Ralfs, Fragilariopsis curta (Van Heurck) Hust., Pseudo‐nitzschia sp., Porosira glacialis (Grunow) E. G. Jørg., Fragilariopsis cylindrus (Grunow) Willi Krieg., Fragilariopsis sublinearis (Van Heurck) Heiden et Kolbe, C. closterium, Nitzschia lecointei Van Heurck, and the dinoflagellate Polarella glacialis Montresor, Procaccini et Stoecker. Two Arctic isolates were also examined, Nitzschia frigida (Grunow) and Fragilariopsis oceanica (Cleve) Hasle. Significant heterogeneity of protein expression was observed despite all cultures being grown in iron‐replete f/2 medium. Only one species, F. cylindrus, displayed the expected expression of ferredoxin only in iron‐replete medium. Four were observed to produce both proteins under iron‐replete conditions. Ferredoxin was not detected at all in F. curta and Pseudo‐nitzschia sp., but distinct flavodoxin bands were observed in both of these organisms. All species examined were observed to express either flavodoxin or ferredoxin or both of the proteins as determined by Western immunoblotting.     

Diversity of the diatom genus Fragilariopsis in the Argentine Sea and Antarctic waters: morphology, distribution and abundance

Fragilariopsis species composition and abundance from the Argentine Sea and Antarctic waters were analyzed using light and electron microscopy. Twelve species (F. curta, F. cylindrus, F. kerguelensis, F. nana, F. obliquecostata, F. peragallii, F. pseudonana, F. rhombica, F. ritscheri, F. separanda, F. sublinearis and F. vanheurckii) are described and compared with samples from the Frenguelli Collection, Museo de La Plata, Argentina. F. peragallii was examined for the first time using electron microscopy, and F. pseudonana was recorded for the first time in Argentinean shelf waters. New information on the girdle view is included, except for the species F. curta, F. cylindrus and F. nana, for which information already existed. In the Argentine Sea, F. pseudonana was the most abundant Fragilariopsis species, and in Antarctic waters, F. curta was most abundant. Of the twelve species of Fragilariopsis documented, four occurred in the Argentine Sea, nine in the Drake Passage and twelve in the Weddell Sea. F. curta, F. kerguelensis, F. pseudonana and F. rhombica were present everywhere.     

EFFECT OF HYPEROXIA ON THE GROWTH AND PHOTOSYNTHESIS OF POLAR SEA ICE MICROALGAE1

Sea ice algal communities are naturally exposed to very high concentrations of dissolved oxygen, which are likely to lead to increasing stress levels and declines in productivity. To test this hypothesis, cultures of Fragilariopsis cylindrus (Grun?) Hasle, Pseudo‐nitzschia sp., Fragilariopsis curta (Van Heurch), Porosira glacialis (Grunow), and Entomoneis kjellmannii (Cleve) from Antarctic sea ice and Nitzschia frigida from Arctic sea ice were exposed to elevated dissolved oxygen levels, and their growth, maximum quantum yield, relative maximum electron transport rate, and photosynthetic efficiency were measured. At oxygen concentrations equivalent to approximately four times air saturation (89% oxygen), the growth rate and maximum quantum yield were significantly reduced in all taxa. When the oxygen concentration was regularly allowed to drop, the effect on growth and quantum yield was reduced. At lower dissolved oxygen concentrations (52%), the declines in growth and quantum yield were reduced but were still mostly significantly different from the controls (21% oxygen). It is likely that the generation of excess active oxygen radicals in the presence of free oxygen is responsible for most of the decline in growth, maximum quantum yield, relative maximum electron transport rate, and photosynthetic efficiency in all species.     

ICE‐BINDING PROTEINS FROM SEA ICE DIATOMS (BACILLARIOPHYCEAE)1

Sea ice diatoms thrive under conditions of low temperature and high salinity, and as a result are responsible for a significant fraction of polar photosynthesis. Their success may be owing in part to secretion of macromolecules that have previously been shown to interfere with the growth of ice and to have the ability to act as cryoprotectants. Here we show that one of these molecules, produced by the sea ice diatom Navicula glaciei Vanheurk, is a ～25 kDa ice‐binding protein (IBP). A cDNA obtained from another sea ice diatom, Fragilariopsis cylindrus Grunow, was found to encode a protein that closely matched the partially sequenced N. glaciei IBP, and enabled the amplification and sequencing of an N. glaciei IBP cDNA. Similar proteins are not present in the genome of the mesophilic diatom Thalassiosira pseudonana. Both proteins closely resemble antifreeze proteins from psychrophilic snow molds, and as a group represent a new class of IBPs that is distinct from other IBPs found in fish, insects and plants, and bacteria. The diatom IBPs also have striking similarities to three prokaryotic hypothetical proteins. Relatives of both snow molds and two of the prokaryotes have been found in sea ice, raising the possibility of a fungal or bacterial origin of diatom IBPs.     

The Contributions of Sea Ice Algae to Antarctic Marine Primary Production

The seasonally ice-covered regions of the Southern Ocean havedistinctive ecological systems due to the growth of microalgaein sea ice. Although sea ice microalgal production is exceededby phytoplankton production on an annual basis in most offshoreregions of the Southern Ocean, blooms of sea ice algae differconsiderably from the phytoplankton in terms of timing and distribution.Thus sea ice algae provide food resources for higher trophiclevel organisms in seasons and regions where water column biologicalproduction is low or negligible. A flux of biogenic materialfrom sea ice to the water column and benthos follows ice melt,and some of the algal species are known to occur in ensuingphytoplankton blooms. A review of algal species in pack iceand offshore plankton showed that dominance is common for threespecies: Phaeocystis antarctica, Fragilariopsis cylindrus andFragilariopsis curta. The degree to which dominance by thesespecies is a product of successional processes in sea ice communitiescould be an important in determining their biogeochemical contributionto the Southern Ocean and their ability to seed blooms in marginalice zones.     

Physical parameters influencing diatom community structure in eastern Antarctic sea ice

Diatom assemblages obtained from fast ice around Prydz Bay, Antarctica, are distinctly different from those obtained from pack ice in the same area. The dominant species in all ice cores were Fragilariopsis curta, F. cylindrus, Nitzschia stellata and Pseudonitzschia turgiduloides. Entomoneis kjellmanii and Cocconeis spp. were more characteristic of fast ice samples and F. curta of pack ice samples. Ice crystal type (i.e. whether frazil or congelation crystal) is an important factor determining the algal composition of the ice. Other significant influences include the time of year the ice forms and the salinity of the ice.     

Phenotypic Plasticity of Southern Ocean Diatoms: Key to Success in the Sea Ice Habitat?

Diatoms are the primary source of nutrition and energy for the Southern Ocean ecosystem. Microalgae, including diatoms, synthesise biological macromolecules such as lipids, proteins and carbohydrates for growth, reproduction and acclimation to prevailing environmental conditions. Here we show that three key species of Southern Ocean diatom (Fragilariopsis cylindrus, Chaetoceros simplex and Pseudo-nitzschia subcurvata) exhibited phenotypic plasticity in response to salinity and temperature regimes experienced during the seasonal formation and decay of sea ice. The degree of phenotypic plasticity, in terms of changes in macromolecular composition, was highly species-specific and consistent with each species’ known distribution and abundance throughout sea ice, meltwater and pelagic habitats, suggesting that phenotypic plasticity may have been selected for by the extreme variability of the polar marine environment. We argue that changes in diatom macromolecular composition and shifts in species dominance in response to a changing climate have the potential to alter nutrient and energy fluxes throughout the Southern Ocean ecosystem.     

Sea ice seasonality during the Holocene, Adélie Land, East Antarctica

Thin sections of laminated cores from different Antarctic coastal areas have demonstrated the potential of diatom species to document climate change at the seasonal scale. Here we present the relative abundances of four diatom species and species groups (Fragilariopsis curta group as a proxy for yearly sea ice cover, F. kerguelensis as a proxy for summer sea-surface temperature, Chaetoceros Hyalochaete resting spores as a proxy for spring sea ice melting and the Thalassiosira antarctica group as a proxy for autumn sea ice formation) in core MD03-2601 retrieved off Adélie Land on the Antarctic continental shelf. These abundances were compared to surface temperatures and sea ice cover modelled over the last 9000 years. Both the marine records and the simulated climate demonstrated a cooler Early Holocene (9000–7700 years BP), a warmer Mid-Holocene (7700–4000 years BP) and a colder Late Holocene (4000–1000 years BP). Yearly sea ice cover followed an inverse pattern to temperatures with less sea ice during the Mid-Holocene Hypsithermal than during the Late Holocene Neoglacial. However, diatom census counts and model output indicate that sea ice spring melting happened earlier in the season, as expected, but that autumn sea ice formation also occurred earlier in the season during the Hypsithermal than during the colder Neoglacial, thereby following seasonal changes in local insolation.     

STRATEGIES AND RATES OF PHOTOACCLIMATION IN TWO MAJOR SOUTHERN OCEAN PHYTOPLANKTON TAXA: PHAEOCYSTIS ANTARCTICA (HAPTOPHYTA) AND FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE)1

We investigated rates and mechanisms of photoacclimation in cultures of Phaeocystis antarctica G. Karst. and Fragilariopsis cylindrus (Grunow) Willi Krieg, phytoplankton taxa that each dominate distinct areas of the Ross Sea, Antarctica. Both P. antarctica and F. cylindrus acclimated to increases in irradiance by reducing the effective size of the pigment antenna (σPSII) via xanthophyll‐cycle activity and reductions in chl. While enhanced photoprotection facilitated increases in specific growth rate and eventually led to higher light‐saturated photosynthetic rates (P^cell_m) in P. antarctica, increases in those variables were much smaller in F. cylindrus. In response to a lower irradiance, relaxation of xanthophyll‐cycle activity led to an increase in σPSII in both taxa, which occurred much more slowly in F. cylindrus. A surprising increase in specific growth rate over the first 36 h of acclimation in P. antarctica may have facilitated the significant reductions in P^cell_m observed in that taxon. In general, P. antarctica acclimated more quickly to changes in irradiance than F. cylindrus, exhibited a wider range in photosynthetic rates, but was more susceptible to photoinhibition. This acclimation strategy is consistent with growth in deeply mixed water columns with variations in irradiance that allow time for repair. In contrast, the slower acclimation rates, extensive photoprotection, and low photoinhibition exhibited by F. cylindrus suggest that it does not require the same period for repair as P. antarctica and is best suited for growth in habitats with relatively uniform irradiance, such as shallow mixed layers or sea ice.     

Psycrophilic organisms in snow and ice]

Psychrophilic and psycrotrophic organisms are important in global ecology as a large proportion of our planet is cold. Two-third of sea-water covering more than 70% of Earth is cold deep sea water with temperature around 2 degrees C, and more than 90% of freshwater is in polar ice-sheets and mountain glaciers. Though biological activity in snow and ice had been believed to be extremely limited, various specialized biotic communities were recently discovered at glaciers of various part of the world. The glacier is relatively simple and closed ecosystem with special biotic community containing various psychrophilic and psycrotrophic organisms. Since psychrophilic organisms was discovered in the deep ice-core recovered from the antarctic ice-sheet and a lake beneath it, snow and ice environments in Mars and Europa are attracting a great deal of scientific attention as possible extraterrestrial habitats of life. This paper briefly reviews the results of the studies on ecology of psychrophilic organisms living in snow and ice environments and their physiological and biochemical adaptation to low temperature.     

Fragilariopsis cylindrus (Grunow) Krieger: The most abundant diatom in water column assemblages of Antarctic marginal ice-edge zones

Summary Planktonic diatoms were sampled in the ice-edge zone of the Bellingshausen Sea during the early austral spring of 1990 and of the Weddell Sea during the late spring of 1983, the autumn of 1986, and the winter of 1988. The four cruises in the Antarctic marginal ice edge zones, combined with the summer cruise in Prydz Bay during a brief ice-free period (1988) provided us with opportunities for spatial and seasonal studies of diatom abundance and distribution in the water column. Cells from discrete water samples from 73 stations near the marginal ice-edge zones during all seasons were counted to gain quantitative information on the composition, abundance, and distribution of diatoms. Diatom abundance was dominated by the pennate diatom, usually nanoplanktonic, Fragilariopsis cylindrus (Grunow) Krieger, during all five cruises. The highest integrated numbers of F. cylindrus were found during the summer cruise with 7.9 × 10¹⁰ cells m^–2 and the lowest numbers were found during the winter cruise with 1.1 × 10⁸ cells m^–2. The average integrated abundance of F. cylindrus from the five cruises was about 35% of the total diatom abundance. The overall spatial pattern of F. cylindrus near the marginal ice-edge zones during the five seasonal cruises were similar with the highest number of cells in open waters compared to ice-covered waters. When all 73 stations during the five cruises were included in the correlation analysis, the abundance of total diatoms was positively correlated with the abundance of F. cylindrus, suggesting that the ice-edge pulses of diatom assemblages in the water column largely reflected its abundance. Cluster analysis revealed that the stations in marginal ice-edge zones were not only separated by seasons and locations, but they also separated based on location of stations in relation to the ice edge (open water stations vs. ice-covered stations).     

An inter-order horizontal gene transfer event enables the catabolism of compatible solutes by Colwellia psychrerythraea 34H

Colwellia is a genus of mostly psychrophilic halophilic Gammaproteobacteria frequently isolated from polar marine sediments and sea ice. In exploring the capacity of Colwellia psychrerythraea 34H to survive and grow in the liquid brines of sea ice, we detected a duplicated 37 kbp genomic island in its genome based on the abnormally high G + C content. This island contains an operon encoding for heterotetrameric sarcosine oxidase and is located adjacent to several genes used in the serial demethylation of glycine betaine, a compatible solute commonly used for osmoregulation, to dimethylglycine, sarcosine, and glycine. Molecular clock inferences of important events in the adaptation of C. psychrerythraea 34H to compatible solute utilization reflect the geological evolution of the polar regions. Validating genomic predictions, C. psychrerythraea 34H was shown to grow on defined media containing either choline or glycine betaine, and on a medium with sarcosine as the sole organic source of carbon and nitrogen. Growth by 8 of 9 tested Colwellia species on a newly developed sarcosine-based defined medium suggested that the ability to catabolize glycine betaine (the catabolic precursor of sarcosine) is likely widespread in the genus Colwellia. This capacity likely provides a selective advantage to Colwellia species in cold, salty environments like sea ice, and may have contributed to the ability of Colwellia to invade these extreme niches.