Collaborative Research: An Integrated Approach Towards Understanding Iron Uptake in Marine Eukaryotic Phytoplankton

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Marine algae are responsible for about half of the annual removal of carbon dioxide from the atmosphere through photosynthesis, but their ability to do so is often hampered by low iron availability. Exactly how different species compete with each other for this iron is poorly understood. This project uses state-of-the-art methods to evaluate whether one well established mechanism in brewer's yeast may also be operating in marine diatoms or whether these organisms use novel, previously undescribed, mechanisms for iron uptake. The collection of massive data sets on thousands of proteins will reveal proteins that are turned on in response to low iron conditions, and newly emerging genetic tools will allow us to robustly test which proteins could be directly responsible for iron uptake. This will dramatically improve the understanding of these ecologically important organisms. More broadly speaking, this may lead to a better understanding of iron uptake in other related organisms, which include human and agricultural pathogens. The investigators will work closely with high school students from under-represented groups and engage them in STEM activities tightly linked to the project. Hands on experiments will be conducted to communicate the concepts of nutrient uptake, using lakes across an urban-rural gradient as the classroom. Additional exercises will introduce students to the cutting-edge approaches that identify thousands of proteins. Select HS students will work in the investigator's laboratories. The attitudes and perceptions of these students will be evaluated to gauge the effect of the experience on their understanding of, and desire to engage in, STEM disciplines. The chromalveolates, which include diatoms and haptophytes, are the most successful and biogeochemically significant eukaryotic phytoplankton in the contemporary ocean. The evidence for iron (Fe) limitation in the oceans has led to an emphasis to understand how phytoplankton compete for and acquire Fe, but this is in its adolescence (diatoms) or infancy (Phaeocystis). This work will resolve long-standing controversies regarding the mechanisms of Fe uptake in a model diatom, T. pseudonana, and the haptophyte Phaeocystis globosa. Objectives include to quantify the proteomes of these species grown under low and high Fe (with recent advances in proteomic methodology), further utilize robust emergent reverse genetics tools to evaluate the localization of key proteins and couple these approaches with kinetics to determine which proteins and redox states are important for Fe uptake. MS^E-based proteomics with ion mobility spectrometry, ideally suited for quantifying cell surface proteins, will be used. Then, for T. pseudonana, knockdown and over-expression clones will be used to confirm localization and test phenotypic responses under varied conditions. Fe uptake and Fe(II) production rates by these clones will help determine which proteins and Fe redox states are important for Fe uptake. Manipulations of gene expression for putative diatom Fe acquisition proteins will be emphasized to address specific hypotheses, but these approaches may also be adapted to take advantage of any novel proteins derived from proteomics. Full participation of under-represented minorities in STEM disciplines will be realized at various educational levels, and the success of these efforts with HS students will be evaluated.