Multiple ice‐binding proteins of probable prokaryotic origin in an Antarctic lake alga,Chlamydomonas sp. ICE‐MDV (Chlorophyceae)

Ice‐associated algae produce ice‐binding proteins (IBPs) to prevent freezing damage. The IBPs of the three chlorophytes that have been examined so far share little similarity across species, making it likely that they were acquired by horizontal gene transfer (HGT). To clarify the importance and source of IBPs in chlorophytes, we sequenced the IBP genes of another Antarctic chlorophyte, Chlamydomonas sp. ICE‐MDV (Chlamy‐ICE). Genomic DNA and total RNA were sequenced and screened for known ice‐associated genes. Chlamy‐ICE has as many as 50 IBP isoforms, indicating that they have an important role in survival. The IBPs are of the DUF3494 type and have similar exon structures. The DUF3494 sequences are much more closely related to prokaryotic sequences than they are to sequences in other chlorophytes, and the chlorophyte IBP and ribosomal 18S phylogenies are dissimilar. The multiple IBP isoforms found in Chlamy‐ICE and other algae may allow the algae to adapt to a greater variety of ice conditions than prokaryotes, which typically have a single IBP gene. The predicted structure of the DUF3494 domain has an ice‐binding face with an orderly array of hydrophilic side chains. The results indicate that Chlamy‐ICE acquired its IBP genes by HGT in a single event. The acquisitions of IBP genes by this and other species of Antarctic algae by HGT appear to be key evolutionary events that allowed algae to extend their ranges into polar environments.     

Possible role of horizontal gene transfer in the colonization of sea ice by algae

Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Proteins with similar sequences and ice-binding activities are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 47% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes.     

NOVEL ICE‐BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE,CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.     

Annealing condition influences thermal hysteresis of fungal type ice-binding proteins

The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption–inhibition mechanism model at the ice–water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.     

An extracellular ice-binding glycoprotein from an Arctic psychrophilic yeast

A psychrophilic yeast was isolated from an Arctic pond and its culture supernatant showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of ∼26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yeast IBP was determined and was found to encode a 261 amino acid protein with molecular weight of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria, clustering with a class of ice-active proteins.     

Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation

To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.     

GROWTH RESPONSES TO SALINITY VARIATION IN FOUR ARCTIC ICE DIATOMS1,2

During spring, extensive blooms of microalgae grow on the underside of arctic sea ice. The brownish, algal layer penetrates ca. 2 cm into the bottom surface of the ice and the algae are potentially exposed to very high salinities. Four diatom species, Melosira juergensii Ag., Porosira glacialis (Grun.) Jørg., Navicula transitans var. derasa (Grun.) Cleve, and Coscinodiscus lacustris Grun., isolated from, sea ice samples taken from the Beaufort and Chukchi seas near Barrow, Alaska, were grown at 11 salinities ranging from 5 to 70‰ at 5 C under constant illumination. All of the species grew at 5‰ except N. transitans whose lower growth limit was 15‰. Growth was high over a broad range of salinities, but none of the species grew at salinities above 60‰. These diatom species appear to be well suited to tolerate the salinities in the brine pockets near the bottom of annual arctic sea ice where they are found. High brine-cell salinity, however, may limit the upward, penetration of ice algae into the bottom of 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.     

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish¹, plants^{2, 3}, arthropods^{4, 5}, fungi⁶, and bacteria⁷. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested^8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears¹¹, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments.We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously^{9, 10}, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)¹². This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies'' group (Queens University)¹⁰. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs^{5, 13}. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations^{14, 15}. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber¹³. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere¹⁶.

The Cold Stage System

The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 μm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).     

SEA ICE MICROBIAL COMMUNITIES. V. THE VERTICAL ZONATION OF DIATOMS IN AN ANTARCTIC FAST ICE COMMUNITY1

A distinct vertical zonation was observed among diatoms in a bottom congelation ice community at McMurdo Sound, Antarctica during the 1981 spring bloom. The bottom 20 cm of ice collected in December from four stations with variable snow cover was subdivided into 5 cm sections for analysis of algal distribution. Algal abundance was inversely related to the depth of snow cover, and generally decreased with increasing distance above the ice-water interface. Most diatoms, including the dominant species Nitzschia stellata Manguin, Amphiprora kufferathii Manguin and Fragilaria islandica var. adeliae Manguin showed peak abundance in the bottom 10 cm of the ice, where the proportion of living to empty cells was also highest. Two species, however, an Auricula Castracane sp. and Navicula glaciei van Heurck, reached highest concentrations at depths 10–20 cm above the ice-water interface. We considered two factors as contributing to the observed vertical zonation: (1) successive blooms at the ice-water interface become spatially stratified within the ice by further accretion below; (2) a differential growth of species occurs along physicochemical gradients within the ice column. A comparison of early versus late season profiles suggests the latter mechanism may prevail once ice accretion has ceased.     

Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium

Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the ‘ice-binding domain’ [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes’. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using ‘anchored clathrate mechanism’ have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.     

Changes in Phytoplankton Biomass and Nutrient Quantities in Sea Ice as Responses to Light/Dark Manipulations during different Phases of the Baltic Winter 2003

The response of Baltic Sea ice communities to changing light climate was studied in three subsequent 3 week in situ experiments on the SW coast of Finland. The investigation covered three different winter periods, short day with low solar angles leading to limited light in the ice, late winter with deep snow cover and early spring with melting snow and increasing light availability. The experimental setup consisted of transparent (no snow) and completely darkened (heavy snow cover) plexiglass tubes in which the ice cores were incubated in situ from 1 to 2 weeks. Changes in the concentrations of inorganic nutrients (NO₃⁻-–N, PO₄³⁻-–P, SiO₄⁻-–Si) and chlorophyll-a concentration in the phytoplankton community composition were recorded as responses to different light manipulations. Changes in inner ice light intensity in untreated ice as well as the temperature both in air and ice were recorded over the entire study period. Increased irradiance in late winter/early spring and during meltdown affected the chlorophyll-a amount in the sea ice. During these periods the phytoplankton community in the top layers decreased possibly as a consequence of photo-acclimation. Closer to the bottom of the ice, however, the increased inner ice light intensity induced algal growth. Complete exclusion of light stopped the algal growth in the whole ice column. Darkening the ice cores also slowed down the ice melting opposite to accelerated melting caused by increased light. The significant differences found in nutrient concentrations between the light and dark treatments were mostly explicable by changes in algal biomass. No obvious changes were observed in the phytoplankton community composition due to light manipulation, diatoms and heterotrophic flagellates dominating throughout the study period.     

Cumulative solar irradiance and potential large-scale sea ice algae distribution off East Antarctica (30°E–150°E)

We present a computational model of the large-scale cumulative light exposure of sea ice in the Southern Ocean off East Antarctica (30°E–150°E). The model uses remotely sensed or modelled sea ice concentration, snow depth over sea ice, and solar irradiance data, and tracks sea ice motion over the season of interest in order to calculate the cumulative exposure of the ice field to photosynthetically active radiation (PAR). Light is the limiting factor to sea ice algal growth over winter and early spring, and so the results have implications for the estimation of algal biomass in East Antarctica. The model results indicate that highly light-exposed ice is restricted to within a few degrees of the coast in the eastern part of the study region, but extends much further north in the 30°E–100°E sector. The relative influences of sea ice motion, solar flux, and snow depth variations on interannual variations in model predictions were evaluated. The model estimates of cumulative PAR were found to correlate with satellite estimates of subsequent open-water chlorophyll-a concentration, consistent with the notion that sea ice algae can provide inocula for phytoplankton blooms.     

Inflorescence bud proteins of Pistacia vera

 The Pistacia vera L. (common name pistachio) is a unique dioecious and deciduous tree species, which is productive under harsh desert climates. We have identified and purified an Inflorescence Bud Protein of 32 kDa (IBP32) from male pistachio trees. There is a close correlation between its accumulation and inflorescence bud development and its disappearance and flowering. Using antibodies raised against this protein, we have identified in female trees the IBP32 and in addition a 27 kDa protein (IBP27), which appears to be specific to female inflorescence buds. The accumulation and disappearance of IBP27 follows the same pattern as that of IBP32. These proteins are glycoproteins rich in glycine and alanine and are highly hydrophilic. Based on the analytical results and immunological cross-reactivity between dehydrin antibodies and the IBPs, it is assumed that the latter are dehydrin-like and may protect inflorescence bud meristems against cold injury during dormancy. The IBPs are the major proteins of the pistachio bud, therefore they may also serve as nitrogen storage during winter for inflorescence bud growth in spring. Received: 17 October 1997 / Accepted: 6 March 1998     

Protists in Arctic drift and land‐fast sea ice

Global climate change is having profound impacts on polar ice with changes in the duration and extent of both land‐fast ice and drift ice, which is part of the polar ice pack. Sea ice is a distinct habitat and the morphologically identifiable sympagic community living within sea ice can be readily distinguished from pelagic species. Sympagic metazoa and diatoms have been studied extensively since they can be identified using microscopy techniques. However, non‐diatom eukaryotic cells living in ice have received much less attention despite taxa such as the dinoflagellate Polarella and the cercozoan Cryothecomonas being isolated from sea ice. Other small flagellates have also been reported, suggesting complex microbial food webs. Since smaller flagellates are fragile, often poorly preserved, and are difficult for non‐experts to identify, we applied high throughput tag sequencing of the V4 region of the 18S rRNA gene to investigate the eukaryotic microbiome within the ice. The sea ice communities were diverse (190 taxa) and included many heterotrophic and mixotrophic species. Dinoflagellates (43 taxa), diatoms (29 taxa) and cercozoans (12 taxa) accounted for ~80% of the sequences. The sympagic communities living within drift ice and land‐fast ice harbored taxonomically distinct communities and we highlight specific taxa of dinoflagellates and diatoms that may be indicators of land‐fast and drift ice.     

Effects of snow removal and algal photoacclimation on growth and export of ice algae

Net growth of ice algae in response to changes in overlying snow cover was studied after manipulating snow thickness on land-fast, Arctic sea ice. Parallel laboratory experiments measured the effect of changing irradiance on growth rate of the ice diatom, Nitzschia frigida. After complete removal of thick snow (≥9 cm), in situ ice algae biomass declined (over 7–12 days), while removal of thin snow layers (4–5 cm), or partial snow removal, increased net algal growth. Ice bottom ablation sometimes followed snow removal, but did not always result in net loss of algae. Similarly, in laboratory experiments, small increases in irradiance increased algal growth rate, while greater light shifts suppressed growth for 3–6 days. However, N. frigida could acclimate to relatively high irradiance (110 μmol photons m² s⁻¹). The results suggest that algal loss following removal of a thick snow layer was due to the combination of photoinhibition and bottom ablation. The smaller relative increase in irradiance after removal of thin or partial snow layers allowed algae to maintain high specific-growth rates that compensated for loss from physical mechanisms. Thus, the response of ice algae to snow loss depends both on the amount of change in snow depth and algal photophysiology. The complex response of ice algae growth and export loss to frequently changing snow fields may contribute to horizontal and temporal patchiness of ecologically and biogeochemically important variables in sea ice and should be considered in predictions of how climate change will affect Arctic marine ecosystems.