Living inside Sea Ice : Distribution and Functional Characterisation of Antifreeze Proteins in Polar Diatoms

Antifreeze proteins (AFPs) are an important adaptation mechanism for organisms subjected to subzero temperatures. The motivation of this thesis was to elucidate the distribution of AFPs in sea ice diatoms and to study their function. The findings were used to deduce the mechanisms of action and relevance in vivo. Diatom isolates were tested in culture experiments for the presence of recrystallisation inhibition activity, as a measure for AFP activity. Seven Arctic and four Antarctic diatom isolates were subjected to a temperature decrease and salinity shift resembling the inclusion of the diatoms into sea ice. All tested polar diatom species showed AFP activity, ten thereof even without being stressed. In three species, AFP activity was furthermore up-regulated by the temperature and salinity shift. Cell numbers and photosynthetic quantum yield indicated that cellular damage caused by the stress was more severe for water column isolates than for isolates from the ice column. The correlation of recrystallisation to the protein concentration for a Fragilariopsis nana Ant cell extract and a recombinant AFP allowed the calculation of AFP concentrations as AFP equivalents. Intracellular concentrations of 0.3 µM to 68.5 µM AFP equivalents confirmed the function of AFP as recrystallisation inhibitor and even come close to the concentrations required for thermal hysteresis. As AFP equivalents made up 0.1% to 5% of the total cell protein, diatoms invest a large amount of energy to produce AFPs. This indicates that AFPs are an important factor for diatom success in sea ice. Further, two isoforms of AFPs from Fragilariopsis nana were heterologously expressed in Escherichia coli, to study their molecular function. Recombinant proteins were deposited in inclusion bodies, but successfully refolded to functionally active proteins with respect to crystal deformation, recrystallisation inhibition and thermal hysteresis. The two isoforms showed different characteristics. One isoform produced a thermal hysteresis of up to 1.53 °C ± 0.53 °C and modified ice crystal growth to formation of hexagonal bi-pyramidal shapes, whereas the other isoform produced a thermal hysteresis of up to 2.34 °C ± 0.25 °C and ice crystals in the form of hexagonal columns. Thermal hysteresis activity of both proteins was positively correlated with protein concentration. The AFP activity increased with increasing buffer salinity in a linear correlation. High AFP concentrations or buffer salinities caused radial dendritic burst patterns of ice crystals in AFP solution. Thermal hysteresis potentials, crystal deformation habits and burst patterns led to the classification of both AFPs as hyperactive AFPs. In addition, the two isoforms differed with respect to the signal for subcellular localisation. Preceding the antifreeze domain, the gene of the first isoform carries a signal peptide indicating the secretion into the extracellular space, whereas the second isoform has a long N-terminal sequence of unknown function. We thus propose that AFPs have different functions in vivo, with distinct localisations of the proteins inside or outside the cell. To investigate AFP activity in situ, five cDNA libraries of eukaryotic communities from Arctic and Antarctic sea ice were analysed for abundance and phylogenetic relationship of AFP sequences. AFP transcripts were found in all sea ice cDNA libraries from the Arctic (Kongsfjord) and Antarctic (Weddell Sea and Dumont d´Urville Sea). Abundances of AFPs ranged from 115 to 1824 AFP transcripts per 100.000 reads. The different abundances did not correlate with taxonomic distribution or environmental parameters of the respective samples. Phylogenetic placement assigned 90% of the sequences to a clade of AFPs from the diatoms Navicula glacei, Chaetoceros neogracile and the crustacean Stephos longipes. The remaining 10% were placed into a separate clade of Fragilariopsis AFP sequences. In the Arctic sample all AFP sequences were assigned to the Fragilariopsis clade, whereas in only one Antarctic sample were some sequences found in this clade. These findings indicate that AFPs play an important role for eukaryotic sea ice organisms in their natural habitat and that the Navicula/Chaetoceros clade is more prominent than the Fragilariopsis clade under the conditions investigated.