Award: EF-1041143

This research effort identified, for the first time, the proteins responsible for the formation of the carbonate minerals in corals. Specifically, by sequencing a coral genome and analyzing the proteins in the coral skeleton, we discovered a novel set of very acidic proteins, which we called coral acid rich proteins (CARPs). We subsequently constructed genes for four of these proteins and expressed them in a bacterial system. The proteins were purified and precipitated aragonite (the mineral form of calcium carbonate in corals) in pure, unammended seawater. We made antibodies to these and several other proteins and showed their distribution in coral skeletons and the living animal. Further, we demonstrated that aragonite is precipitated by CARPs at ph 8.2 and 7.6, strongly suggesting that in principle ocean acidification should not substantially influence the formation of carbonate in the coming century, in spite of the acidification of the upper ocean. Our research has substantially increased our mechanistic understanding of the biomineralization process in stony corals and has strong parallels to biomineralization of bones and teeth in animals. Among the specific outcomes of this grant is the development of the methodology for calcium carbonate precipitation using coral tissue cultures that aggregate to form "proto-polyps" of the hermatypic, zooxanthellate coral, Stylophora pistillata. This novel experimental system in which calcification is facilitated at the cellular level, has been used to assess the effects of increasing pCO2 on the calcification process. To that end we developed a new method for quantifying the calcification rates in the tissue culture based on the incorporation of strontium into the coral skeleton. Cell cultures of S. pistillata incubated in four CO2 treatments (400, 700, 1000 and 2000 ppm pCO2) were evaluated for photosynthetic efficiency, calcification rates, skeletal organic matter (protein) expression rates, and mineralogical, elemental and isotopic composition. The primary cell cultures assembled into organic "proto-polyps" precipitating aragonite crystals, which formed on the external face of the proto-polyps and were identified by their distinctive elongated crystallography and X-ray diffraction pattern. The data suggest an apparent link between the protein expression and CaCO3 mass accumulation. CaCO3 mass decreased substantially at pCO2 levels above 700 ppm, which could be related to the apparently inhibited protein expression at high pCO2 although re-dissolution of mineral following initial formation in more acidic media cannot be excluded. To better understand the geochemical controls on coralline proxies we also measured the elemental composition of the precipitates (e.g., Li/Ca, B/Ca, Na/Ca) and boron isotopes. We found that boron isotope ratios, a proxy for pH, of culture-precipitated aragonite follow the inorganic precipitation line with a relatively constant positive offset, consistent with observations from nubbin cultures, providing evidence for pH modification (increase) at the calcification site occurring at the molecular or cellular level. Preliminary boron isotope measurements of CaCO3 crystals precipitated directly by the CARP proteins suggest that the pH modification is likely occurring at the cellular level and not exerted by the protein itself. Combined, the biological and geochemical evidence supports in vivo observations that the pH of the calcifying fluid in corals is elevated relative to ambient seawater, thus suggesting that ocean acidification should not affect coral calcification to the degree that suggested previously. This project trained 3 post docs. Two of them now hold academic position while the third is continuing his post-doctoral studies elsewhere. In addition, we trained one PhD student and several undergraduates. Last Modified: 11/02/2014 Submitted by: Yair Rosenthal