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

每年通过光合作用从大气中去除的二氧化碳中，约有一半是由海藻负责的，但它们的这一能力往往受到铁利用率低的阻碍。不同物种究竟是如何相互竞争这种铁的，目前还知之甚少。该项目使用最先进的方法来评估啤酒酵母中一种公认的机制是否也适用于海洋硅藻，或者这些生物是否使用以前未描述的新的铁吸收机制。对数千种蛋白质的海量数据集的收集将揭示出对低铁条件做出反应的蛋白质，新出现的遗传工具将使我们能够有力地测试哪些蛋白质可能直接导致铁的摄取。这将极大地提高人们对这些具有生态重要性的生物的了解。更广泛地说，这可能有助于更好地了解其他相关生物体的铁吸收情况，包括人类和农业病原体。调查人员将与代表不足群体的高中生密切合作，让他们参与与该项目密切相关的STEM活动。将进行动手实验，以跨越城乡坡度的湖泊作为课堂，交流养分吸收的概念。额外的练习将向学生介绍识别数千种蛋白质的尖端方法。选定的HS学生将在研究人员的实验室工作。这些学生的态度和认知将被评估，以衡量经验对他们对STEM学科的理解和意愿的影响。嗜铬藻类是当代海洋中最成功、最具生物地球化学意义的真核浮游植物，包括硅藻和浮游植物。海洋中铁(Fe)限制的证据导致了对浮游植物如何竞争和获取铁的重视，但这是在其青春期(硅藻)或婴儿期(棕囊藻)。这项工作将解决长期存在的关于模式硅藻、假单胞藻和球状棕囊藻吸收铁的机制的争议。目标包括量化这些物种在低铁和高铁条件下生长的蛋白质组(随着蛋白质组学方法学的最新进展)，进一步利用强大的新兴反向遗传学工具来评估关键蛋白质的定位，并将这些方法与动力学相结合，以确定哪些蛋白质和氧化还原状态对铁的吸收是重要的。将使用基于MS^E的蛋白质组学和离子迁移率光谱，非常适合于定量细胞表面蛋白质。然后，对于假单胞菌，将使用敲除和过表达克隆来确认定位并测试在不同条件下的表型反应。这些克隆的铁吸收和铁(II)产生速率将有助于确定哪些蛋白质和铁氧化还原状态对铁吸收是重要的。对可能的硅藻铁获取蛋白的基因表达的操纵将被强调以解决特定的假设，但这些方法也可能被调整以利用来自蛋白质组学的任何新的蛋白质。将在各级教育中实现未被充分代表的少数群体充分参与STEM学科，并将评估这些努力在HS学生中的成功。

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.