|A first look at the genetic control mechanisms shaping metabolic changes during nitrogen starvation in Phaeodactylum tricornutum|
Matthijs, M. (2014). A first look at the genetic control mechanisms shaping metabolic changes during nitrogen starvation in Phaeodactylum tricornutum. PhD Thesis. Ghent University, Faculty of Sciences, Department of Plant Biotechnology and Bioinformatics/VIB, Department of Plant Systems Biology: Gent. 135 + appx. pp.
Genetic control; Metabolism; Phaeodactylum tricornutum Bohlin, 1897 [WoRMS]; Marine
Diatoms are unicellular algae that are successful fresh and marine environments. Their photosynthesis supplies a fifth of all the oxygen we breathe and forms a large part of the primary oceanic productivity that feeds our seas and ultimately us. Diatoms have exquisitely symmetric cell walls that have fascinated microscopists since the 19th century. Besides their ecological importance, they also have a commercial application since, among other things, they produce lipids rich in polyunsaturated omega-3 fatty acids. This accumulation mainly occurs during conditions adverse for growth like when a nutrient such as nitrogen is no longer present in sufficient quantities to support growth. In chapter three, the known reactions to macronutrient starvation are summarized and they contrast to those of green algae. The arrival of next generation sequencing has accelerated the molecular study of diatoms by making the sequencing of genomes and transcriptomes fast and affordable. The molecular toolbox for P. tricornutum is limited but it can be transformed and the genome is publically available. In this study, the model diatom Phaeodactylum tricornutum is used to further the understanding of metabolic and transcriptomic changes during nitrogen starvation. These findings were contrasted with three other stress conditions. The aforementioned changes are steered by a set of controlling genes that was virtually unknown. On the RNA level, these changes are mainly steered by transcription factors (TF’s). In chapter four, the search is described for transcription factors that steer adaptations during N starvation. The problem was approached by identifying overrepresented cis-regulatory motifs in genes responsive to N starvation. Using a yeast one hybrid screening, a transcription factor termed NMB1 was identified which binds one of these motifs and is induced during N scarcity. This gene is part of a previously unknown transcription factor family conserved in heterokonts. In chapter five, the most striking find of our RNA-seq dataset is analyzed: a coordinated upregulation of the genes involved in the Krebs or tricarboxylic acid (TCA) cycle. This cycle plays a central role in the primary metabolism as it is able to turn sugars, lipids and amino acid into reducing equivalents that can be turned into energy. The TCA cycle can also move in the reverse reaction providing the carbon skeletons for synthesizing these molecules. In this work it was shown that the intermediates of the TCA cycle mainly consist out of carbon that was already present in the cell. Since the pattern of transcription for these enzymes was so similar, it was hypothesized that there was a common transcription factor steering the expression all these genes. Chapter five also details the identification of the conserved transcription factor bZIP14 by co-expression analysis. This transcription factor had a similar expression pattern as several TCA enzymes and was induced during N starvation. Using a protein binding microarray, the motif bound by bZIP14 was identified, which was present in several promoters of TCA genes. Upon overexpression of this TF several genes involved in the TCA cycle were also upregulated, a clear indication that this factor is involved in the regulation of the Krebs cycle.