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

海洋覆盖了地球表面的70%，包含了微生物生命的非凡多样性，并构成了我们星球上最大的生态系统。地球上每年光合作用产生的有机物约有一半发生在海洋环境中水柱的透光区，使海洋成为全球碳循环的主要组成部分。硅藻是最重要的浮游植物类群之一，对维持海洋系统的生态和地球化学平衡至关重要。据估计，硅藻的光合作用占海洋中每年固定的450 - 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和最节能的食物网的基础。世界上一些最大的渔业主要是由以生物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佳竞争者之一，但在沿海上升流事件中提供硅藻竞争优势的细胞系统仍有待充分描述。扩大我们的生理和分子理解的氮传感和细胞内代谢的海洋硅藻是微生物科学和全球海洋氮循环的一个重要研究热点。我们的项目旨在提高对硅藻细胞系统的理解，这些硅藻细胞系统有效且快速地对直接细胞外环境中硝酸盐的可用性增加和细胞内硝酸盐浓度降低做出反应。具体来说，我们将表征系统的主要组成部分，包括传感器，膜转运蛋白和膜脂质的活动。该项目还将在微生物学、地球化学循环和生物技术领域开展针对小学生的教育推广活动。这些科学推广活动得益于与圣地亚哥组织的合作：杰出科学家和工程师联盟（LXS）。 该项目旨在了解知之甚少的细胞系统，提供沿海涌升事件中硅藻的竞争优势。最终，对硅藻硝酸盐吸收途径的更彻底详细的机械理解将有助于大大提高预测海洋营养物质输送和硅藻相关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是围绕努力定义硅藻硝酸盐传感的分子组成，确定调节氮充足和氮限制条件下的整体细胞反应的代谢信号，表征液泡硝酸盐跨膜通道和泵，并调查液泡膜脂质的组成，生物合成和再循环。具体而言，该项目将采用生理学，接合附加型克隆，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的组合来表征系统主要组分的活性：a）传递关于叶绿体中产生的硝酸盐的可用性和代谢物信号的信息的传感器，触发激活转录所必需的分子事件级联; B）膜转运蛋白i）MFS蛋白，其在充满条件下将硝酸盐泵送穿过外膜并进入细胞，ii）液泡膜中的硝酸盐通道和H+泵，其将过量的硝酸盐转运到液泡中以进行储存，其通过亚硝酸盐还原酶和GS-GOGAT酶递送用于同化的亚硝酸盐;和c）膜脂质，其是新合成的和再循环的，其是当过量的硝酸盐被储存时液泡膜扩张所必需的。研究活动的总体目标包括，开发和验证硝酸盐敏感，存储和吸收的关键海洋phytoplankton.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.