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

合作研究：了解海洋真核浮游植物铁吸收的综合方法

• 批准号: 1558030
• 负责人: Lewis Brown
• 金额: $ 15.36万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1558030&HistoricalAwards=false
• 关键词: Collaborative; Research; Integrated; Approach; Towards

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.

海洋藻类每年通过光合作用从大气中去除大约一半的二氧化碳，但它们这样做的能力经常受到铁含量低的阻碍。不同的物种究竟是如何为铁而竞争的，人们知之甚少。该项目使用最先进的方法来评估啤酒酵母中一个已建立的机制是否也可能在海洋硅藻中起作用，或者这些生物是否使用新的，以前未描述的铁摄取机制。对数千种蛋白质的大量数据集的收集将揭示在低铁条件下被激活的蛋白质，而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负责铁的摄取。这将极大地提高对这些重要生态有机体的认识。更广泛地说，这可能会导致更好地了解其他相关生物的铁摄取，包括人类和农业病原体。调查人员将与来自代表性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。将以跨城乡梯度的湖泊作为课堂，进行实践实验来传达营养吸收的概念。额外的练习将向学生介绍识别数千种蛋白质的前沿方法。选定的HS学生将在研究者的实验室工作。这些学生的态度和看法将被评估，以衡量这段经历对他们对STEM学科的理解和参与意愿的影响。包括硅藻和附生植物在内的染色藻是当代海洋中最成功的、具有生物地球化学意义的真核浮游植物。海洋中铁（Fe）有限的证据导致人们强调了解浮游植物如何竞争和获取铁，但这是在其青春期（硅藻）或婴儿期（Phaeocystis）。这项工作将解决长期以来关于模型硅藻、T. pseudonana和球状褐囊藻（Phaeocystis globosa）铁吸收机制的争议。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学（利用蛋白质组学方法的最新进展），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摄取很重要。将使用基于质谱的蛋白质组学和离子迁移谱法，非常适合于定量细胞表面蛋白质。然后，利用敲低和过表达克隆来确定假单胞菌的定位，并测试不同条件下的表型反应。这些克隆的铁摄取和铁（II）生成速率将有助于确定哪些蛋白质和铁的氧化还原状态对铁摄取很重要。将强调对假定的硅藻铁获取蛋白的基因表达的操作，以解决特定的假设，但这些方法也可以适应于利用来自蛋白质组学的任何新蛋白质。将在各个教育层次实现未被充分代表的少数民族充分参与STEM学科，并对高中学生的这些努力的成功进行评估。