Award: OCE-1638838

Intellectual Merit Coccolithophores are unicellular marine phytoplankton that possess the remarkable ability to biomineralize minute calcium carbonate plates (coccoliths) within their cells that are subsequently secreted onto the cell surface to form an outer cell layer known as the coccosphere. Coccolithophores are widespread phytoplankton and are one of the most significant producers of biogenic calcite in the surface oceans, responsible for precipitating ~1.6 Pg CaCO3/year. Much of this calcite is exported to the deep ocean through sinking of individual coccoliths and as aggregates with organic matter. Over geological timescales this results in the formation of chalk deposits such as the White Cliffs of Dover. Therefore, through both photosynthesis and calcification, coccolithophores play a major role in the biogeochemical carbon cycle. Despite their biological importance in surface oceans, there is much to be learned about the mechanisms of intracellular calcification and the sensitivity of coccolithophores to changing climate and surface ocean pH. For example, unlike diatoms, coccolithophores are not silicified, yet some species exhibit a requirement for Si in the calcification process. These Si-requiring species also possess silicon transporters (SITLs) that share homology to diatom SITs. The project therefore integrated molecular characterization of coccolithophore SITs with physiological experiments to understand the role of Si in coccolithophore calcification and to address how the differing requirements for Si in calcifying coccolithophores may have shaped competitive interactions with other phytoplankton over both contemporary and evolutionary timescales. We discovered that coccolithophore SITLs act much the same as diatom Na+-coupled Si transporters when expressed in heterologous systems, and that SITLs are transcriptionally regulated by Si availability. However, the Si requirement of coccolithophores is very low, with uptake of Si into cells below the level of detection of sensitive assays of Si transport. We concluded that Si acts to support the calcification process but does not form part of the coccolith structure itself. Si is thus acting as a micronutrient in coccolithophores, and natural populations are unlikely experience Si limitation in surface oceans unless dissolved silicon is depleted to extreme levels. The project also allowed for a comparison of the Si requirements for calcification in the two major life phases of coccolithophores (diploid heterococcolithophores, and haploid holococcolithophores). We use advanced microscopy techniques to discover that the holococcoliths, comprising minute simple rhombohedral calcite crystals, are also formed within a specialized vesicle inside the cell in a similar manner to the more complex crystal heterococcoliths. However, silicon is not required for holococcolith formation and the requirement for silicon therefore relates specifically to the process of crystal morphogenesis in heterococcoliths. This suggests the lower complexity holococcoliths are an ancestral form of calcification in coccolithophores. The subsequent recruitment of a silicon-dependent mechanism for crystal morphogenesis in the diploid life cycle stage likely led to the emergence of the intricately shaped heterococcoliths, enabling the formation of the elaborate coccospheres that underpin a variety of functional roles and the ecological success of coccolithophores. During the course of this research additional collaborations and discoveries were made that yielded important insights into the physiology, ecology and evolution of coccolithophores. For example, we discovered that the ability of coccolithophores to regulate internal pH levels (pH homeostasis) relies on novel ion transport mechanisms that remove H+ ions for the cytoplasm. Interestingly, the H+ mechanisms employed likely depend on the type of coccolith produced (holococcolith vs heterococcolith) and species (heavily calcified heterococcoliths vs lightly calcified hetercoccoliths). This has important implications for understanding how different species may be affected by ocean acidification. We also developed a new tool to monitor coccolithophore calcification in single cells and populations which allowed visualization of coccolith production, release, and reabsorption by cells. Overall, the research provided important information on the physiology, ecology, and evolution of coccolithophores, which is vital to understand how coccolithophores have been influenced by past changes in the Earth's climate, and their potential responses to future oceans. Broader Impacts The project provided numerous research experiences for a range of participants including 9 undergraduates, 4 graduates, and two postdoctoral staff. Each of the 4 female graduates continued their STEM careers in the USA as research associates or post-doctoral staff. The two postdocs involved in the project have secured permanent research specialist positions in applied marine sciences. The project therefore provided multiple advanced training and professional development opportunities that supported advancement and retention of female and underrepresented minorities in STEM. The research team provided numerous outreach activities over the course of the project that were designed to attract high school students to STEM college programs. This included working with local schools and counties to provide exploratory workshops in biological imaging and marine microbes for students interested in marine science at the college level. Last Modified: 01/04/2023 Submitted by: Alison R Taylor