The Mechanisms of Calcification in Coccolithophores - The molecular basis of calcium and inorganic carbon transport in Emiliania huxleyi

Coccolithophores are calcifying marine phytoplankton that through the fixation of inorganic carbon into calcite and particulate organic carbon play a fundamental role in global carbon cycles. As the CO2 concentration of the surface ocean increases through the anthropogenic release of CO2 by burning fossil fuels both a decrease in pH (ocean acidification) and a increase in dissolved inorganic carbon (ocean carbonation) are taking place. To understand the impact of these ocean changes on coccolithophores it is essential that we rapidly increase our knowledge of the cellular processes underlying coccolithophore physiology. This doctoral thesis focuses on the cellular and molecular processes involved in the transport of Ca2+, inorganic carbon and H+ in relation to calcification and photosynthesis in the coccolithophore species Emiliania huxleyi. The thesis comprises 7 chapters: Chapter 1 is a general introduction to coccolithophore cellular biology and global carbon cycling; Chapters 2-6 are a combination of publications and data chapters; Chapter 7 provides a synthesis of the results placing the presented data into context of coccolithophores in a changing ocean, highlighting future research areas. Chapter 2 is a published review of the current literature on the molecular aspects of calcification in coccolithophores. It identifies key gaps in our knowledge of coccolithophore cellular biology and presents new hypotheses for the transport of substrates to the site of calcification. Chapter 3 investigates some of the proposed hypotheses by examining the role of several candidate Ca2+, H+ and inorganic carbon transport genes in calcifying and non-calcifying cells of E. huxleyi, using quantitative reverse transcriptase PCR. The data provides strong evidence that a putative HCO3- transporter (AEL1), a Ca2+/H+ exchanger (CAX3), a vacuolar H+-ATPase pump (ATPVc/c’) and a gene encoding for a coccolith-associated protein, GPA, play key roles in E. huxleyi biomineralization. CAX3 and AEL1 were chosen for further analysis and were successfully cloned and expressed in Saccharomyces cerevisiae and Human Embryonic Kidney cells (HEK293) respectively (Chapter 6). However complete characterization of CAX3 and AEL1 was unsuccessful. CAX3 failed to complement the Ca2+ sensitive phenotype of a S. cerevisiae mutant, with further expression in a Ca2+ sensitive Escherichia coli mutant resulting in a lethal phenotype. The investigation of HCO3- transport in HEK293 cells expressing AEL1 gave negative results, potentially due to the poor localization of AEL1 to the plasma membrane. The data highlights the importance of developing genetic transformation techniques in coccolithophores to reduce the dependency of using foreign expression systems for the characterization of genes. The influence of the individual carbonate system components (CO2, HCO3-, CO32- and H+) on coccolithophore physiology and genetic response is relatively unknown. Chapter 4 disentangles the individual carbonate system components investigating their influence on calcification, particulate organic carbon fixation and gene expression in E. huxleyi. It identifies, for the first time, the genetic basis of a carbon concentrating mechanism (CCM) in coccolithophores, with the transcription of multiple CCM associated genes up-regulated at low concentrations of HCO3- and CO2. Physiological data combined with expression data indicates that calcification does not function as a CCM under carbon limitation and is instead reduced to allow the redistribution of inorganic carbon from calcification to photosynthesis. Furthermore, the data confirms previous studies that the substrate for calcification is HCO3- and that growth and organic carbon fixation rates are primarily influenced by CO2. The recent sequencing of the E. huxleyi genome has provided vast quantities of genetic data that requires detailed analysis to realise its full potential. Chapter 5 analyses the genome for calcification and photosynthesis related transport genes discovering that E. huxleyi has a diverse range of inorganic carbon, Ca2+ and H+ transporters, from classical plant, animal and bacterial families. The presence of multiple Na+/Ca2+ exchangers, a family of almost exclusively animal transporters indicates that coccolithophores have the potential to use both H+ and Na+ electrochemical gradients to drive secondary transport. Furthermore the identification of green algal CCM genes may provide a strong basis for investigating the evolution of CCMs in eukaryotic algae. The data presented in this thesis provides a significant step in our understanding of coccolithophore physiology at a cellular and molecular level. It offers a solid platform for future research in coccolithophore cell biology an area of research that is essential to comprehend the role of coccolithophores in gobal carbon cycling and how they will respond and adapt to future ocean changes.


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