Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis

Fragilariopsis is a dominating psychrophilic diatom genus in polar sea ice. The two species Fragilariopsis cylindrus and Fragilariopsis curta are able to grow and divide below freezing temperature of sea water and above average sea water salinity. Here we show that antifreeze proteins (AFPs), involved in cold adaptation in several psychrophilic organisms, are widespread in the two polar species. The presence of AFP genes (afps) as a multigene family indicated the importance of this group of genes for the genus Fragilariopsis, possibly contributing to its success in sea ice. Protein phylogeny showed the potential mobility of afps, which appear to have crossed kingdom and domain borders, occurring in Bacteria, diatoms, crustaceans and fungi. Our results revealed a broad distribution of AFPs not only in polar organisms but also in taxa apparently not related to cold environments, suggesting that these proteins may be multifunctional. The relevance of AFPs to Fragilariopsis was also shown by gene expression analysis. Under stress conditions typical for sea ice, with subzero temperatures and high salinities, F. cylindrus and F. curta strongly expressed selected afps. An E/G point mutation in the Fragilariopsis AFPs may play a role in gene expression activity and protein function.     

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.     

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.     

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.     

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.     

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.     

Use of exogenous glycine betaine and its precursor choline as osmoprotectants in Antarctic sea‐ice diatoms1

Wide salinity ranges experienced during the seasonal freeze and melt of sea ice likely constrain many biological processes. Microorganisms generally protect against fluctuating salinities through the uptake, production, and release of compatible solutes. Little is known, however, about the use or fate of glycine betaine (GBT hereafter), one of the most common compatible solutes, in sea‐ice diatoms confronted with shifts in salinity. We quantified intracellular concentrations and used [¹⁴C]‐labeled compounds to track the uptake and fate of the nitrogen‐containing osmolyte GBT and its precursor choline in three Antarctic sea‐ice diatoms Nitzschia lecointei, Navicula cf. perminuta, and Fragilariopsis cylindrus at ?1°C. Experiments show that these diatoms have effective transporters for GBT, but take up lesser amounts of choline. Neither compound was respired. Uptake of GBT protected cells against hyperosmotic shock and corresponded with reduced production of extracellular polysaccharides in N. lecointei cells, which released 85% of the retained GBT following hypoosmotic shock. The ability of sea‐ice diatoms to rapidly scavenge and release compatible solutes is likely an important strategy for survival during steep fluctuations in salinity. The release and recycling of compatible solutes may play an important role in algal–bacterial interactions and nitrogen cycling within the semi‐enclosed brines of sea ice.     

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.     

Growth limitation of three Arctic sea ice algal species: effects of salinity, pH, and inorganic carbon availability

The effect of salinity, pH, and dissolved inorganic carbon (TCO₂) on growth and survival of three Arctic sea ice algal species, two diatoms (Fragilariopsis nana and Fragilariopsis sp.), and one species of chlorophyte (Chlamydomonas sp.) was assessed in controlled laboratory experiments. Our results suggest that the chlorophyte and the two diatoms have different tolerance to fluctuations in salinity and pH. The two species of diatoms exhibited maximum growth rates at a salinity of 33, and growth rates at a salinity of 100 were reduced by 50% compared to at a salinity of 33. Growth ceased at a salinity of 150. The chlorophyte species was more sensitive to high salinities than the two diatom species. Growth rate of the chlorophyte was greatly reduced already at a salinity of 50 and it could not grow at salinities above 100. At salinity 33 and constant TCO₂ concentration, all species exhibited maximal growth rate at pH 8.0 and/or 8.5. The two diatom species stopped growing at pH > 9.5, while the chlorophyte species still was able to grow at a rate which was 1/3 of its maximum growth rate at pH 10. Thus, Chlamydomonas sp. was able to grow at high pH levels in the succession experiment and therefore outcompeted the two diatom species. Complementary experiments indicated that growth was mainly limited by pH, while inorganic carbon limitation only played an important role at very high pH levels and low TCO₂ concentrations.     

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.     

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.     

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.     

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.     

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.     

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES1

The photosynthetic efficiency and photoprotective capacity of the sea‐ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (?1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen‐replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (F_V/F_M). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (F_M) could only be obtained by adding 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). reoxidation curves showed the presence of Q_B nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (Q_A) to the secondary (Q_B) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of Q_B nonreducing PSII centers within the photosystem. On a physiological level, the formation of Q_B nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient‐limiting conditions.     

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.     

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.     

PHYTOPLANKTON SELENIUM REQUIREMENTS: THE CASE FOR SPECIES ISOLATED FROM TEMPERATE AND POLAR REGIONS OF THE SOUTHERN HEMISPHERE1

A series of laboratory culture experiments was used to investigate the effect of selenium (Se, 0–10 nM) on the growth, cellular volume, photophysiology, and pigments of two temperate and four polar oceanic phytoplankton species [coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, cyanobacterium Synechococcus sp., prymnesiophyte Phaeocystis sp., and three diatoms—Fragilariopsis cylindrus (Grunow) Kriegar, Chaetoceros sp., and Thalassiosira antarctica G. Karst.]. Only Synechoccocus sp. and Phaeocystis sp. did not show any requirement for Se. Under Se‐deficient conditions, the growth rate of E. huxleyi was decreased by 1.6‐fold, whereas cellular volume was increased by 1.9‐fold. Se limitation also decreased chl a (2.5‐fold), maximum relative electron transport rate (1.9‐fold), and saturating light intensity (2.8‐fold), suggesting that Se plays a role in photosynthesis or high‐light acclimation. Pigment analysis for Antarctic taxa provided an interesting counterpoint to the physiology of E. huxleyi. For all Se‐dependent Antarctic diatoms, Se limitation decreased growth rate and chl a content, whereas cellular volume was not affected. Pigment analysis revealed that other pigments were affected under Se deficiency. Photoprotective pigments increased by 1.4‐fold, while diadinoxanthin:diatoxanthin ratios decreased by 1.5‐ to 4.9‐fold under Se limitation, supporting a role for Se in photoprotection. Our results demonstrate an Se growth requirement for polar diatoms and indicate that Se could play a role in the biogeochemical cycles of other nutrients, such as silicic acid in the Southern Ocean. Se measurements made during the austral summer in the Southern Ocean and Se biological requirement were used to discuss possible Se limitation in phytoplankton from contrasting oceanographic regions.