Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification

Abstract

The bicarbonate ion (HCO3(-)) is involved in two major physiological processes in corals, biomineralization and photosynthesis, yet no molecular data on bicarbonate transporters are available. Here, we characterized plasma membrane-type HCO3(-) transporters in the scleractinian coral Stylophora pistillata. Eight solute carrier (SLC) genes were found in the genome: five homologs of mammalian-type SLC4 family members, and three of mammalian-type SLC26 family members. Using relative expression analysis and immunostaining, we analyzed the cellular distribution of these transporters and conducted phylogenetic analyses to determine the extent of conservation among cnidarian model organisms. Our data suggest that the SLC4γ isoform is specific to scleractinian corals and responsible for supplying HCO3(-) to the site of calcification. Taken together, SLC4γ appears to be one of the key genes for skeleton building in corals, which bears profound implications for our understanding of coral biomineralization and the evolution of scleractinian corals within cnidarians.

Figure 1

Structural histology and model of DIC transport through the different coral tissue layers (modified from Bertucci et al60). Dotted arrows represent CO2 diffusion, the projected involvement of BATs is indicated by red circles. Zoox.: zooxanthellae; M.: mitochondrion. CA: Carbonic Anhydrase; PMCA: plasma membrane Calcium ATPase. Unknown mechanisms of DIC transport are indicated with a question mark

Figure 2

Comparison of Stylophora pistillata sequences of (A) SLC4 family proteins, and (B) SLC26 family proteins using ClustalW alignment using Genious. Legend for colored boxes is 100% similar in black, >80% in dark grey, >60% in medium grey, less than 60% in light grey.

Figure 3

Phylogenetic relationships of human and anthozoan Bicarbonate Anion Transporter protein sequences inferred from Maximum Likelihood (ML) and Bayesian analyses. Bootstrap network of BAT sequences based on ML distances are estimated with a LG+G model (α = 0.749) using PHYML. Bayesian posterior probabilities are indicated in black whereas ML bootstrap values are in red.

Figure 4

Exon/intron organization of the different BATs in the genome of Stylophora pistillata. Exons are represented as boxes whereas introns are depicted as lines.

Figure 5

Relative expression of the different BAT proteins in coral tissues based on qPCR. (A) cDNA are prepared from total tissues (whole coral fragment), or from oral disc. (B) Gene expression normalized to 36B4 expression in total tissues (Total), or in oral disc (Oral). Grey bars represent different colonies. Error bars represent the SD of three technical replicates of each colony.

Figure 6

Immunolocalization of SpiSLC26β and SpiSLC4γ. Embedded cross-section of Stylophora pistillata tissues labeled by (A) preimmune serum for SpiSLC26β, (B) and (C) anti-SpiSLC26β, (D) preimmune serum for SpiSLC4γ, and (E) and (F) anti- SpiSLC4γ antibody. Rows (A), (B), (D), and (E) are views of the four tissues composing the coral. (C) are magnifications of oral tissues and (F) are magnifications of aboral tissues. Nuclei are labeled in blue in first column (DAPI), streptavidin AlexaFluor 568 fluorescence appears in orange in second column, merged is in the third column. The background red color in cross-section (A) and (D) with preimmune serum corresponds to autofluorescence of coral tissues. AEnd = Aboral Endoderm; AT = Aboral Tissue; CEct = Calicoblastic Ectoderm; Co = Coelenteron; m = Mesoglea; OEct = Oral Ectoderm; OEnd = Oral Endoderm; OT = Oral Tissue; Sw = Seawater; Sk = Skeleton