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

J. Craig Venter Institute, Inc., La Jolla CA

Investigators

Abstract

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.