Nitrate metabolism in the dinoflagellate Lingulodinium polyedrum

Dinoflagellates are unicellular eukaryotes found in most aquatic ecosystems of the world. They are major contributors to carbon fixation in the oceans, either as free-living phytoplankton or as symbionts to corals. Dinoflagellates are also infamous because some species can form spectacular blooms called red tides, which can cause serious damage to ecosystems, human health, fisheries and tourism. One of the factors often correlated with algal blooms are increases in nutrients, particularly nitrogen and phosphorus. Nitrate is one of the main components of agricultural runoffs, but also the most abundant bioavailable form of nitrogen in marine environments. Thus, agricultural activities have globally contributed to the magnification of the problems associated with red tides. However, bloom formation and persistence cannot be ascribed to human pollution alone, because other biotic and abiotic factors are at play. Particularly, it is difficult to assess the relative importance of nitrate addition over these other factors, because nitrate metabolism in dinoflagellate is mostly unknown. Filling part of this gap was the main goal of this thesis. I selected Lingulodinium polyedrum as a model for studying nitrate metabolism, because this dinoflagellate can easily be cultured in the lab and a recent transcriptomic survey has provided an almost complete gene catalogue for this species. It is also interesting that some molecular components of the nitrate pathway in this organism have been reported to be under circadian control. Thus, in this project, I used physiological, biochemical, transcriptomic and bioinformatic approaches to enrich our understanding of dinoflagellate nitrate metabolism and to increase our appreciation of the role of the circadian clock in regulating this important primary metabolic pathway. I first studied the particular case of dinoflagellate blooms that occur and persist in conditions of nitrogen depletion. This idea may seems counterintuitive, because nitrogen addition rather than depletion, is generally associated with algal blooms. However, I discovered that when nitrate was added to nitrogen-deficient or nitrogen-sufficient cultures, those that had been acclimated to nitrogen stress were able to survive for about two months at high cell densities, while non-acclimated cells died after two weeks. In conditions of severe nitrogen limitation, cells could survive a little bit more than two weeks by arresting cell division and reducing photosynthetic rates. The incapacity to synthesize new amino acids for these deprived cells in a context of on-going photosynthesis led to the accumulation of reduced carbon in the form of starch granules and lipid bodies. Interestingly, both of these carbon storage compounds were polarized in Lingulodinium cells, suggesting a functional role. The second contribution of my thesis was to identify and characterize the first nitrate transporters in dinoflagellates. I found that in contrast to plants, Lingulodinium had a reduced suite of nitrate transporters and only members of the high-affinity nitrate transporter 2 (NRT2) family were predicted to be functionally relevant in the transport of nitrate. The main transporter was constitutively expressed, which suggested that nitrate uptake in Lingulodinium was mostly a constitutive process rather than an inducible one. I also discovered that nitrate uptake in this organism was light-dependent and not a circadian-regulated process, as previously suggested. Finally, I used RNA-seq to verify if any transcripts involved in the nitrate metabolism of Lingulodinium were under circadian control. Not only did I discovered that there were no daily variations in the level of transcripts involved in nitrate metabolism, but also that there were no changes for any transcripts present in the whole transcriptome of Lingulodinium. This discovery showed that the circadian timer in this species did not require rhythmic transcription to generate biological rhythms, as observed in other eukaryotes.

Keyphrases: Acclimatation, Nitrate, Transcriptome, Acclimation, Carbon, Circadian clock, Daily rhythms, Dinoflagellates, Harmful algal blooms, High-affinity nitrate transporters, Lipid bodies, Nitrate uptake, Nitrogen stress, Photosynthesis, Starch, Transcription-translation feedback loops