Marine eukaryotic microalgae are important primary producers in the ocean. As phosphorus (P) limitation has been widely observed in different marine regions, investigating the response of marine eukaryotic microalgae to P-deficiency can help us to gain a better understanding of the biogeochemical cycles for phosphorus and carbon. A special bloom-forming marine alga with high P-limitation tolerance, Aureoumbra lagunensis, was chosen to examine the whole cell response to nutrient depletion on protein level. By using two-dimensional electrophoresis and tandem mass spectrometry analysis combined with de novo cross-species protein identification method, differentially expressed proteins were identified under P- and N-limitation. The proteomic and physiological response to P-limitation sugges...[ Read more ]

Marine eukaryotic microalgae are important primary producers in the ocean. As phosphorus (P) limitation has been widely observed in different marine regions, investigating the response of marine eukaryotic microalgae to P-deficiency can help us to gain a better understanding of the biogeochemical cycles for phosphorus and carbon. A special bloom-forming marine alga with high P-limitation tolerance, Aureoumbra lagunensis, was chosen to examine the whole cell response to nutrient depletion on protein level. By using two-dimensional electrophoresis and tandem mass spectrometry analysis combined with de novo cross-species protein identification method, differentially expressed proteins were identified under P- and N-limitation. The proteomic and physiological response to P-limitation suggest that A. lagunensis may adopt intracellular nutrient compensation and oxidative damage protection to thrive under P-depleted environment. Additionally, a highly abundant P-limitation specific protein that was identified as a putative alkaline phosphatase (APase) was further characterized by enzyme activity assay on non-denaturing gel and confocal microscopy, which confirmed that this protein has APase activity, and is a cytoplasmic protein located adjacent to cell membrane. As utilization of organic P by using APase is one of the most important processes of P metabolism, and the lack of characterized APase is the main obstacle of applying quantitative gene expression of alkaline phosphatase on determining the cellular P physiology in marine eukaryotic microalgae, in this study a reverse genetic route was used to identify more APase in marine eukaryotic microalgae. Two APases (PhtrAP1 and PhtrAP2) were identified and characterized in Phaeodactylum tricornutum with this method. Further analysis of algal physiology, in situ enzymatic labeling, and protein sequence topology showed that PhtrAP1 was controlled by intracellular P physiology when P-limitation is severe, and it was an integral membrane protein located inside the vacuole-like organelles; PhtrAP2 is controlled by environment phosphate concentration, and it located in periplasm. Additionally, the BLAST result in various marine eukaryotic microalgae genomes showed that the PhoA and PhoD like APases are dominant in marine eukaryotic microalgae, and PhtrAP2 maybe a new type APase found in diatoms. All together, this thesis provides new insight into the adaptations in response to P-limitation and the utilization of organic P in marine eukaryotic microalgae.