Functional genomic insights into cellular processes related to harmful bloom formation in ichthyotoxic prymnesiophytes

Not much information is available about the genetic background of growth and toxicity- related processes in toxic Haptophyta species. The aim of my thesis was to contribute to better understanding of these issues using functional and comparative genomic approaches with the ichthyotoxic prymnesiophytes Chrysochromulina polylepis and Prymnesium parvum. In particular, I explored different gene-expression profiling methods in order to monitor the transcriptomic responses in these species to different environmental conditions. Through the sequencing of a cDNA library, a transcriptomic database (Expressed Sequence Tag library) was established for both prymnesiophyte species. Approximately 2900 and 6300 contigs were found in the Chrysochromulina polylepis and Prymnesium parvum datasets, respectively. The sequences were annotated and compared to similar data sets available from other Haptophyta species (Pavlova lutherii, Isochrysis galbana and Emiliania huxleyi). This analysis revealed a `core set` of approx. 1500 genes which were found in all Haptophyta species investigated in this study. Moreover, 67 and 362 genes were present only in C. polylepis and P. parvum, respectively. The physiological background and cellular regulation of synthesis and liberation of Chrysochromulina and Prymnesium toxin(s) is still poorly understood, but the involvement of PKS genes in the biosynthesis of certain compounds is likely. The presence of the conserved ketosynthase (KS) domains - an obligatory part of PKS genes were shown in both species, represented by fourteen and four copies in C. polylepis and P. parvum, respectively. In order to indirectly test the hypothesis invoking a role of PKS genes in toxin biosynthesis, the correlation between toxicity and PKS gene expression was monitored in both species. The observed positive correlation strengthens the hypothesis on the involvement of PKS genes in toxin production C. polylepis as well as in P. parvum. A gene expression microarray was generated based on the EST data originating from P. parvum, and this tool was used to monitor gene-expression changes during growth in nutrient replete and phosphorus (P)- or nitrogen (N)-deprived P. parvum cells. In accord with previously published data, elevated intracellular toxicity was observed in P-deprived cells, whereas it did not change in N-depleted or nutrient replete cells. As a response to P limitation, the upregulation of different genes related to transport and acquisition of phosphate could be observed. On the other hand, N limitation did not lead to such a clear effect on the gene expression level, since most genes likely involved in the uptake, storage and transport of N sources were not upregulated. Utilizing the tools of ecophysiology and functional genomics we identified gene-expression patterns indicative of physiological (nutrient, toxicity) and growth status of C. polylepis and P. parvum. With reference to this data set, knowledge about cellular processes in toxic Prymnesiophyceae species was expanded considerably, and pointed the way forward for incorporation of functional genomic approaches to determining regulatory factors involved in prymnesiophyte bloom dynamics through gene expression studies.