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SpiAMT1d: molecular characterization, localization, and potential role in coral calcification of an ammonium transporter in Stylophora pistillata

Abstract

Members of the ammonium transporter family occur in all domains of life. However, they have been characterized, at the molecular level, mainly in bacteria and plants, whereas in animals their molecular characterization is limited to a few species. In marine invertebrates, recent studies have reported a multitude of physiological functions in which ammonium could take part. Among them, calcification is one for which very few data are available. In scleractinian corals, it has been reported that external sources of ammonium and/or ammonium containing compounds enhance calcification. However, despite these physiological insights, the underlying transport mechanisms are still unknown at the molecular level. In this study, we performed a first molecular characterization of ammonium transporters in the scleractinian coral Stylophora pistillata. By performing differential gene expression analysis, through quantitative real-time PCR, we show that S. pistillata ammonium transporters possess different tissue specificities. Most notably, one ammonium transporter, spiAMT1d, is specifically expressed in the coral tissue containing the calcifying cells. Furthermore, we determined spiAMT1d subcellular localization by immunostaining S. pistillata histological cross-sections and show that it localizes on the apical side of the calcifying cells and in their intracellular compartments. Taken together our results strongly suggest a role of ammonium transporters in coral calcification and lay the groundwork for many future studies aiming to better elucidate the potential role of ammonium in calcification.

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Data availability

The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files. Genomic and transcriptomic data were obtained from the publicly available database of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/).

Abbreviations

AMT:

Ammonium transporter

ECM:

Extracellular calcifying medium

H+ :

Protons

MEP:

Methylammonium permease

NH3 :

Ammonia

NH4 + :

Ammonium

PMCA:

Ca+2-ATPase

qPCR:

Real-time PCR

Rh:

Rhesus proteins

SLC4γ:

Solute carrier 4γ-bicarbonate transporter

SOMPs:

Skeletal organic matrix proteins

TMs:

Transmembrane segments

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Acknowledgements

We would like to thank Dominique Desgré for coral maintenance, Alexander Venn for comments, Christine Ferrier-Pagès for kindly revising the text of the manuscript, and Renaud Grover for fruitful discussions.

Funding

This work was supported by the Government of the Principality of Monaco and baseline funding from King Abdullah University of Science and Technology (KAUST) to MA. The funding body played no role in the design, collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.

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ST and DZ were involved in conceptualization, formal analysis, and writing–review and editing; LC was involved in formal analysis, investigation, and writing–original draft; PG and MA were involved in validation and writing–review and editing. All authors gave final approval for publication and agree to be held accountable for the work performed therein.

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Correspondence to Sylvie Tambutté.

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Ethical approval

Coral was collected from the aquaria of the Centre Scientifique de Monaco (CSM), and select coral fragments (less than 3 cm in length) were killed for further laboratory analysis.

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338_2022_2256_MOESM1_ESM.tiff

Figure S1: Immunofluorescence staining of CHO cells transfected with a pIRES2-DsRed-Express (Clonotech) co-expressing spiAMT1d and Red Fluorescent Protein (RFP) with (A, C) pre-immune serum and (B, D) anti-spiAMT1d. Rows (C) and (D) are magnifications of the cells identified by a square in rows (A) and (B), respectively. Immunofluorescence staining with pre-immune serum showed no labelling (Figure S1 A and C), whereas when using anti-spiAMT1d, spiAMT1d accumulated at the cell membrane and co-localized with RFP (Figure S1 B and D). Controls using pre-immune and anti-spiAMT1d on CHO cells not transfected showed no labelling (not shown). Nuclei are labelled in blue in the first column (DAPI), RFP is shown in red in the second column, Alexa Fluor 488 fluorescence appears in green in the third column and merge is shown in the fourth column. (TIFF 25399 kb)

338_2022_2256_MOESM2_ESM.tiff

Figure S2 : Flow cytometry assay performed on CHO-transfected cells transiently expressing spiAMT1d. These cells were treated with Preimmune serum (PPI) (a), anti-spiAMT1d (b), and anti-spiAMT1d pre-incubated with peptides 1 and 2 (P1 and P2) (c). Short and long horizontal bars delimitate, respectively, negative and positive fluorescent intensity signals associated with anti-spiAMT1d. (TIFF 4000 kb)

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Capasso, L., Zoccola, D., Ganot, P. et al. SpiAMT1d: molecular characterization, localization, and potential role in coral calcification of an ammonium transporter in Stylophora pistillata. Coral Reefs 41, 1187–1198 (2022). https://doi.org/10.1007/s00338-022-02256-5

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Keywords

  • Ammonium transporters
  • Scleractinian corals
  • Differential gene expression
  • Immunolocalization
  • Calcification
  • Biomineralization