Why do coccolithophores calcify ? - investigating the metabolic role of calcification.

Overview: We propose a comprehensive analysis of metabolic processes in coccolithophores in order to gain insight into the mechanisms and biological function of calcification and into the environmental factors which regulate this process. Through a detailed assessment of primary metabolism in different coccolithophore strains, we will assess diversity in the response of calcifying and non-calcifying strains to changes in their environment and the interactions between calcification and the processes of photosynthesis, osmoregulation and phosphate acquisition. Rationale: Calcification by coccolithophores plays a major role in the global carbon cycle and climate change may have a significant impact on this process. However, the biological function of calcification in coccolithophores remains unclear. Various physical roles have been proposed, including protection from grazers, increasing sinking rates and protection from high light as well as metabolic roles such as enhanced acquisition of nutrients and as a source of carbon for photosynthesis, but definitive evidence for these roles is lacking. In order to further our understanding of the biological role of calcification, we will examine metabolic processes in calcifying and non-calcifying coccolithophores. Haptophyte algae accumulate two principal compatible solutes, the tertiary sulfonium compound dimethylsulfonioproprionate (DMSP) and the cyclitol, D-1,4/2,5-cyclohexanetetrol (CHT). Our preliminary evidence suggests that CHT is absent from calcifying E. huxleyi strains, but is a major metabolite in a non-calcifying E. huxleyi strain. This unexpected finding suggests that calcification has a major impact on metabolic processes in coccolithophores. We propose that detailed examination of coccolithophore metabolism will provide important information on the biological role of calcification. Experimental Approach: Hypothesis 1: Metabolite profiling of diverse coccolithophore strains can be used to identify distinct metabolic traits associated with calcification. Non-targeted metabolite profiling techniques (including GC-MS, liquid-chromatography-mass spectrometry [LC-MS] and 1H, 13C and 31P NMR) will be used to examine the low molecular weight metabolome of a range of calcifying and non-calcifying strains of E. huxleyi and other coccolithophore species present in the MBA's culture collection. In collaboration with Exeter's bioinformatics group, we will identify which areas of metabolism are intimately linked to calcification. In addition, these studies will be used to determine whether specific metabolites (such as CHT) may be used in environmental samples as a proxy for calcification rate. Hypothesis 2: Calcification is linked to osmoregulation in coccolithophores. The osmoregulatory mechanisms in E. huxleyi isolates from coastal, shelf and ocean environments will be quantified in calcifying and non-calcifying strains grown at a range of salinities, using a range of biochemical and physiological techniques. We aim to determine the role of calcification in the physiological and metabolic responses to osmotic changes. Hypothesis 3: Calcifying cells demonstrate a remodelling of phosphate utilisation and storage Calcification in E. huxleyi is closely linked to the availability of phosphate. We will use 31P-NMR profiles to determine the accumulation of inorganic phosphate and polyphosphates in calcifying/non-calcifying E. huxleyi strains in P-replete and P-limited cultures. Further studies will examine how calcification affects the response of primary metabolism (e.g. carbon metabolism / compatible solute accumulation) to phosphate limitation.