Jeana L. Drake

Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata

It has long been recognized that a suite of proteins exists in coral skeletons that is critical for the oriented precipitation of calcium carbonate crystals, yet these proteins remain poorly characterized. Using liquid chromatography-tandem mass spectrometry analysis of proteins extracted from the cell-free skeleton of the hermatypic coral, Stylophora pistillata, combined with a draft genome assembly from the cnidarian host cells of the same species, we identified 36 coral skeletal organic matrix proteins. The proteome of the coral skeleton contains an assemblage of adhesion and structural proteins as well as two highly acidic proteins that may constitute a unique coral skeletal organic matrix protein subfamily. We compared the 36 skeletal organic matrix protein sequences to genome and transcriptome data from three other corals, three additional invertebrates, one vertebrate, and three single-celled organisms. This work represents a unique extensive proteomic analysis of biomineralization-related proteins in corals from which we identify a biomineralization “toolkit,” an organic scaffold upon which aragonite crystals can be deposited in specific orientations to form a phenotypically identifiable structure.

Comparative genomics explains the evolutionary success of reef-forming corals

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years. DOI: http://dx.doi.org/10.7554/eLife.13288.001

Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata

Significance Although various aspects of biomineralization in corals have been studied for decades, the basic mechanism responsible for the precipitation of the aragonite skeleton remains enigmatic. To address this issue, we used antibodies against key biomineralization proteins derived from the common zooxanthellate coral Stylophora pistillata to elucidate the spatial arrangement of specific skeletal matrix proteins in the skeleton and in the animal tissue. To our knowledge, our results reveal for the first time that the biomineral is produced in discrete nanoscale packages in which the secreted organic matrices remain entrapped within the crystalline units whose growth they control, leading to the formation of highly ordered, microscopic, heterologous structures, which are aggregated to form a macroscopic skeleton. The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral’s metabolic pathways.

Aragonite Precipitation by “Proto-Polyps” in Coral Cell Cultures

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form “proto-polyps”. Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ωarag∼4), the primary cell cultures assemble into “proto-polyps” which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis.

Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization ‘toolkit’ in the early life history of a stony coral.

Reply to Ramos-Silva et al.: Regarding coral skeletal proteome

We thank Ramos-Silva et al. (1) for their thoughtful comments on our work recently published in PNAS (2). We agree that careful cleaning of biomineral samples is indeed necessary for appropriate proteomic analysis. However, we respectfully disagree with their interpretation of our cleaning methods and our decision to include certain proteins in our final list of 36 potential biomineralization proteins.

Evolution of biomineralization proteins in Cnidaria

Recently, the effects of ocean acidification on coral calcification have received significant interest. However, similar information is lacking for soft corals, which produce internal calcite sclerites. This poor understanding of the calcifying mechanisms makes it difficult to predict the response of soft corals to increased ocean acidification. In order to better understand the potential effects of increased atmospheric CO2, it is crucial that we expand and enhance our knowledge of soft coral calcification biomolecules. Toward this end, we use molecular biology techniques to understand the evolutionary history of a Cnidarian highly acidic protein, previously thought to be limited to stony corals. Coral acid rich protein (CARP) 4, is a coralspecific protein exhibiting a high proportion (>20%) of the acidic amino acids (aspartic and glutamic acids). Previously thought to be stony coral-specific, the partial sequence of a soft coral sclerite protein appears similar to CARP4. An alignment of homologues of four stony corals and CARP4 proteins with an N-terminal peptide from Lobophytum crassum sclerites suggests that soft corals possess a member of the CARP4 subfamily. Through a series of molecular biology techniques, the presence of CARP4 in soft corals can be verified. If L. crassum does indeed possess the CARP4 gene, phylogenetic comparison of soft versus stony coral CARP4 will be performed to trace the evolutionary track of this Cnidarian-specific biomineralization protein.