Salt stress tolerance in the psychrophilic diatom Fragilariopsis cylindrus

Abstract

This thesis was conducted to find mechanisms responsible for the adaptation success of Fragilariopsis cylindrus to the extreme polar environment, especially sea ice, manifested in the genetic repertoire. The generated molecular information was afterwards utilized in expression studies focused on the regulation of the proline metabolism during acclimatisation to elevated external salt concentrations. An expressed sequence tag (EST) approach was used to establish two complementaryDNA (cDNA) datasets, based on cultures subjected to temperature and salt stress conditions, the major abiotic constraints in sea ice. The genetic information (ca. 2600 tentative unique sequences) gathered with these two cDNA libraries covered about 20 % of all genes present in F. cylindrus, taken the genome of the centric diatom Thalassiosira pseudonana as a reference. A comparison of the salt stress cDNA library to the genomes of the mesophilic species T. pseudonana and Phaeodactylum tricornutum revealed about one third of the sequences to be unique to F. cylindrus, indicating substantial genomic variation between the mesophilic and psychrophilic lifestyle. This indicates the potential of yet unknown adaptation mechanisms. A putative function could be assigned to 44 % of the sequences and a large number of genes involved in transport processes, oxidative stress defence, osmolyte synthesis and protein turnover as well as chaperones could be identified, stressing the importance of these mechanisms in salt stress acclimatisation. Furthermore, four different full length sequences encoding a new class of ice-binding proteins yet unknown in animals and plants were found and further studies proved its occurrence in a number of polar diatom species, but not in mesophilic ones. These proteins are most probably exuded into the extracellular space and hence might be of fundamental importance in enabling survival in the brine channel system. The finding of all relevant proteins involved in the ana- and catabolic pathways of proline metabolism enabled a detailed expression study of these genes in a physiological salt shock (elevation from 34 to 70 PSU) experiment. Expression levels of Delta1-pyrroline-5-carboxylate synthase (P5CS), the rate limiting enzyme in synthesis from glutamate, strongly decreased, whereas copy numbers of ornithine delta-aminotransferase (delta-OAT) increased, indicating a shift from the glutamate to the ornithine route under elevated external salinities. This contrasts with findings in higher plants, where the opposite regulation of P5CS and delta-OAT was observed. A shortage in reduction equivalents caused by a severe inhibition of linear electron transport revealed by the measurement of the photosynthetic quantum yield might force F. cylindrus to use energy saving pathways of proline synthesis. Interestingly, a further temperature decrease in addition to the elevated salt concentration exhibited no more negative effects in a number of physiological parameters, thus leading to the conclusion that salt is the dominating abiotic stressor in sea ice.