Defining the Competitive Edge: Cellular Systems that Enable Nitrate Assimilation in Marine Diatoms

海洋覆盖了地球表面的70%，包含了极其多样化的微生物生命，构成了地球上最大的生态系统。地球上每年约有一半的有机物质光合作用生产发生在海洋环境中水柱的光区，使海洋成为全球碳循环的主要组成部分。硅藻是最主要的浮游植物类群之一，对维持海洋系统的生态和生物地球化学平衡至关重要。据估计，硅藻光合作用占每年固定在海洋中的450亿至500亿吨有机碳的25%至40%。硅藻在上升流诱导的周期性营养丰富的条件下茁壮成长的能力，使它们成为世界上最短、最节能的食物网的基础。世界上一些最大的渔业主要是由硅藻为基础的新生产驱动和维持的：由上升的硝酸盐推动的二氧化碳固定。在海洋浮游植物中，硅藻是硝酸盐水平升高的最好竞争者之一，但在沿海上升流事件中提供硅藻竞争优势的细胞系统仍未得到充分描述。拓宽我们对海洋硅藻氮素传感和细胞内代谢的生理和分子理解是微生物科学和全球海洋氮循环的重要研究热点。我们的项目旨在提高对硅藻细胞系统的了解，这些系统能够有效和快速地对细胞外环境中硝酸盐可用性的增加和储备耗尽时细胞内硝酸盐浓度的降低做出反应。具体地说，我们将表征系统的主要组件的活性，包括传感器、膜运输蛋白和膜脂。该项目还将开展以小学生为对象的微生物学、生物地球化学循环和生物技术领域的教育推广活动。这些科学推广活动得益于与总部设在圣地亚哥的组织非凡科学家和工程师联盟(LXS)的合作。这个项目试图了解在沿海上升流事件中为硅藻提供竞争优势的鲜为人知的细胞系统。归根结底，对硅藻硝酸盐吸收途径的更详细、更机械的了解将有助于大大提高预测海洋营养物质输送和相关硅藻吸收的预期变化的影响的能力。这一关键的细胞和生物地球化学问题集中在确定硅藻中硝酸盐感知的分子组成，识别调节细胞对氮素充足和有限条件的整体反应的代谢信号，表征液泡硝酸盐跨膜通道和泵，以及研究液泡膜脂质的组成、生物合成和循环。具体地说，该项目将结合生理学、接合附体克隆、基因敲除、蛋白质定位、标记-ATP生物化学和尖端分子生物学实验方法来表征系统的主要组成部分的活性：a)传感器，传递关于硝酸盐可用性的信息，以及在叶绿体中产生的代谢物信号，触发激活转录所需的一系列分子事件；B)膜转运蛋白I)在充足条件下将硝酸盐泵过外膜并进入细胞的MFS蛋白；ii)液泡膜中的硝酸盐通道和H+泵，将多余的硝酸盐输送到液泡中进行储存；iii)嵌入叶绿体膜中的亚硝酸盐转运蛋白，通过亚硝酸还原酶和GS-GOGAT酶输送亚硝酸盐同化；以及c)新合成和回收的膜脂，在储存过量的硝酸盐时，这是液泡膜扩张所必需的。研究活动的总体目标包括开发和验证关键海洋浮游植物中硝酸盐传感、储存和吸收的精细化概念模型。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The oceans cover 70% of the Earth's surface, contain an extraordinary diversity of microbial life, and constitute the largest ecosystem on our planet. Approximately half of the annual photosynthetic production of organic matter on Earth takes place in the photic zone of the water column in marine environments, making the ocean a major component of the global carbon cycle. Diatoms are one of the most prominent phytoplankton groups and are crucial to maintaining the ecological and biogeochemical equilibrium of marine systems. Diatom photosynthesis is estimated to account for between 25% and 40% of the 45-50 billion tons of organic carbon fixed annually in the sea. The ability of diatoms to thrive in upwelling-induced, periodically nutrient-rich conditions makes them the basis for the world's shortest and most energy-efficient food webs. Some of the world's largest fisheries are driven and maintained primarily by diatom-based new production: CO2 fixation fueled by upwelled nitrate. Among marine phytoplankton, diatoms are among the best competitors for elevated levels of nitrate, yet the cellular systems providing the competitive advantage of diatoms during coastal upwelling events remain to be fully described. Broadening our physiological and molecular understanding of nitrogen sensing and intracellular metabolism in marine diatoms is an important research focus for microbial sciences and the global marine nitrogen cycle. Our project aims to improve understanding of the cellular systems in diatoms that efficiently and rapidly respond to both increased availability of nitrate in the immediate extracellular environment and reduced concentrations of intracellular nitrate as stores are depleted. Specifically, we will characterize the activity of the major components of the system including sensors, membrane transport proteins, and membrane lipids. The project will also develop educational outreach activities which target primary school students in the areas of microbiology, biogeochemical cycles and biotechnology. These science outreach activities benefit from collaborations with the San Diego-based organization: The League of Extraordinary Scientists and Engineers (LXS). This project seeks to understand the poorly understood cellular systems which provide the competitive advantage of diatoms during coastal upwelling events. Ultimately a more thoroughly detailed, mechanistic understanding of diatom nitrate uptake pathways will facilitate a much-improved ability to forecast the impact of anticipated changes in ocean nutrient delivery and associated uptake by diatoms. This critical cellular and biogeochemical issue is centered around efforts to define the molecular components of nitrate sensing in diatoms, identify the metabolic signals that regulate the overall cellular response to nitrogen-replete and nitrogen-limited conditions, characterize vacuolar nitrate transmembrane channels and pumps, and investigate the composition, biosynthesis and recycling of vacuolar membrane lipids. Specifically the project will employ combination of physiological, conjugative episomal cloning, gene knockouts, protein localization, tagged-ATP biochemistry and cutting edge molecular biological experimental approaches to characterize the activity of the major components of the system: a) sensors that relay information on the availability of nitrate, and metabolite signals, generated in the chloroplast, that trigger the cascade of molecular events necessary to activate transcription; b) membrane transport proteins i) MFS proteins that pump nitrate across the outer membrane and into the cell under replete conditions, ii) nitrate channels and H+ pumps in vacuolar membranes that transport excess nitrate into vacuoles for storage, iii) the nitrite transporter, embedded in the chloroplast membrane, that delivers nitrite for assimilation via nitrite reductase and GS-GOGAT enzymes; and c) membrane lipids, both newly synthesized and recycled, that are necessary for expansion of vacuolar membranes as excess nitrate is stored. Overall aims of the research activities include, development and validation of a refined conceptual model of nitrate sensing, storage, and uptake in key marine phytoplankton.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.