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

Coral Reefs volume 41, pages 1187–1198 (2022)Cite this article

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

References

  • Allemand D, Ferrier-Pagès C, Furla P, Houlbrèque F, Puverel S, Reynaud S, Tambutté É, Tambutté S, Zoccola D (2004) Biomineralisation in reef-building corals: From molecular mechanisms to environmental control. Comptes Rendus - Palevol 3:453–467

  • Aranda M, Li Y, Liew YJ, Baumgarten S, Simakov O, Wilson MC, Piel J, Ashoor H, Bougouffa S, Bajic VB, Ryu T, Ravasi T, Bayer T, Micklem G, Kim H, Bhak J, LaJeunesse TC, Voolstra CR (2016) Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle. Sci Rep 6:1–15

  • Baday S, Orabi EA, Wang S, Lamoureux G, Bernèche S (2015) Mechanism of NH4+ Recruitment and NH3 Transport in Rh Proteins. Structure 23:1550–1557

  • Béraud E, Gevaert F, Rottier C, Ferrier-Pagès C (2013) The response of the scleractinian coral Turbinaria reniformis to thermal stress depends on the nitrogen status of the coral holobiont. J Exp Biol 216:2665–2674

  • Bertucci A, Tambutté S, Supuran CT, Allemand D, Zoccola D (2011) A New Coral Carbonic Anhydrase in Stylophora pistillata. Mar Biotechnol 13:992–1002

  • Biscéré T, Ferrier-Pagès C, Grover R, Gilbert A, Rottier C, Wright A, Payri C, Houlbrèque F (2018) Enhancement of coral calcification via the interplay of nickel and urease. Aquat Toxicol 200:247–256

  • Biver S, Belge H, Bourgeois S, Van Vooren P, Nowik M, Scohy S, Houillier P, Szpirer J, Szpirer C, Wagner CA, Devuyst O, Marini AM (2008) A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility. Nature 456:339–343

  • Burland TG (2000) DNASTAR’s Lasergene Sequence Analysis Software. Humana Press Totowa, NJ 132:71–91

  • Burris RH (1983) Uptake and assimilation of 15NH4+ by a variety of corals. Mar Biol 75:151–155

  • Cameron JN, Batterton CV (1978) Antennal gland function in the freshwater crab Callinectes sapidus: Water, electrolyte, acid-base, and ammonia excretion. J Comp Physiol 123:143–148

  • Campbell, J. W. and BDB (1976) On the acid-base balance of gastropod molluscs. University of South Carolina Press, Columbia,

  • Campbell, James W. and KVS (1969) Carbonate, Ammonia and biological deposition of calcium. Nature 224.5220:725–726

  • Campbell JW (1997) Mitochondrial Ammonia Metabolism and the pdf. 308–321

  • Capasso L, Ganot P, Planas-Bielsa V, Tambutté S, Zoccola D (2021) Intracellular pH Regulation: Characterization and Functional Investigation of H+ Transporters in Stylophora Pistillata. BMC Mol Cell Biol 1–19

  • Chen JC, Lin CY (1992) Lethal effects of ammonia on Penaeus chinensis Osbeck juveniles at different salinity levels. J Exp Mar Biol Ecol 156:139–148

  • Cleves PA, Strader ME, Bay LK, Pringle JR, Matz MV (2018) CRISPR/Cas9-mediated genome editing in a reef-building coral. Proc Natl Acad Sci USA 115(20):5235–5240. https://doi.org/10.1073/pnas.1722151115

  • Conant GC, Wolfe KH (2008) Turning a hobby into a job: How duplicated genes find new functions. Nat Rev Genet 9:938–950

  • Conroy MJ, Jamieson SJ, Blakey D, Kaufmann T, Engel A, Fotiadis D, Merrick M, Bullough PA (2004) Electron and atomic force microscopy of the trimeric ammonium transporter AmtB. EMBO Rep 5:1153–1158

  • Conroy MJ, Bullough PA, Merrick M, Avent ND (2005) Modelling the human rhesus proteins: Implications for structure and function. Br J Haematol 131:543–551

  • Cooper AJ, Plum F (1987) Biochemistry and physiology of brain ammonia. Physiol Rev 67:440–519

  • Crossland CJ, Barnes DJ (1974) The role of metabolic nitrogen in coral calcification. Mar Biol 28:325–332

  • Fernandes de Barros Marangoni L, Ferrier-Pagès C, Rottier C, Bianchini A, Grover R (2020) Unravelling the different causes of nitrate and ammonium effects on coral bleaching. Sci Rep 10:1–14

  • Furla P, Galgani I, Durand I, Allemand D (2000) Sources and mechanisms of inorganic carbon transport for coral calcification and photosynthesis. J Exp Biol 203:3445–3457

  • Furla P, Allemand D, Shick JM, Ferrier-Pagès C, Richier S, Plantivaux A, Merle PL, Tambutté S (2005) The symbiotic anthozoan: A physiological chimera between alga and animal. Integr Comp Biol 45:595–604

  • Ganot P, Moya A, Magnone V, Allemand D, Furla P, Sabourault C (2011) Adaptations to endosymbiosis in a Cnidarian-Dinoflagellate association: Differential gene expression and specific gene duplications. PLoS Genet 7:

  • Ganot P, Zoccola D, Tambutté E, Voolstra CR, Aranda M, Allemand D, Tambutté S (2015) Structural Molecular Components of Septate Junctions in Cnidarians Point to the Origin of Epithelial Junctions in Eukaryotes. Mol Biol Evol 32:44–62

  • Graham TE, MacLean DA (1998) Ammonia and amino acid metabolism in skeletal muscle: Human, rodent and canine models. Med Sci Sports Exerc 30:34–46

  • Grover R, Maguer JF, Reynaud-Vaganay S, Ferrier-Pagès C (2002) Uptake of ammonium by the scleractinian coral Stylophora pistillata: Effect of feeding, light, and ammonium concentrations. Limnol Oceanogr 47:782–790

  • Hans S, Quijada-Rodriguez AR, Allen GJP, Onken H, Treberg JR, Weihrauch D (2018) Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus. J Exp Biol 221:

  • Hansuebsai A, Rungsupa S, Kiyoki Y, Sasaki S, Chawakitchareon P (2018) Study the effect of ammonia by image analysis on healthiness detection for coral quality of lifetitle. Front Artif Intell Appl 301:343–353

  • Hazel RH, Burkhead CE, Huggins DG (1982) Development of water quality criteria for ammonia and total residual chlorine for the protection of aquatic life in two johnson county, kansas streams. In: Pearson JG, Foster RB, Bishop WE (eds) Proceedings 5th annual symposium aquatic toxicology. Philadelphia, PA, pp 381–388

  • Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2008) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:

  • Henry RP, Lucu Č, Onken H, Weihrauch D (2012) Multiple functions of the crustacean gill: Osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals. Front Physiol 3 NOV:1–33

  • Hu MY, Sung PH, Guh YJ, Lee JR, Hwang PP, Weihrauch D, Tseng YC (2017) Perfused gills reveal fundamental principles of pH regulation and ammonia homeostasis in the cephalopod Octopus vulgaris. Front Physiol 8:

  • Huang CH, Peng J (2005) Evolutionary conservation and diversification of Rh family genes and proteins. Proc Natl Acad Sci U S A 102:15512–15517

  • Karako-Lampert S, Zoccola D, Salmon-Divon M, Katzenellenbogen M, Tambutté S, Bertucci A, Hoegh-Guldberg O, Deleury E, Allemand D, Levy O (2014) Transcriptome analysis of the scleractinian coral stylophora pistillata. PLoS One 9:

  • Katju V (2012) In with the Old, in with the New: The Promiscuity of the Duplication Process Engenders Diverse Pathways for Novel Gene Creation. Int J Evol Biol 2012:1–24

  • Khademi S, O’Connell J, Remis J, Robles-Colmenares Y, Miercke LJW, Stroud RM (2004) Mechanism of ammonia transport by Amt/MEP/Rh: Structure of AmtB at 135 Å. Science (80- ) 305:1587–1594

  • Kustu S, Inwood W (2006) Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels. Transfus Clin Biol 13:103–110

  • LaJeunesse TC, Parkinson JE, Gabrielson PW, Jeong HJ, Reimer JD, Voolstra CR, Santos SR (2018) Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts. Curr Biol 28:2570-2580.e6

  • Lamoureux G, Javelle A, Baday S, Wang S, Bernèche S (2010) Mécanismes de transport dans le pore des transporteurs d’ammonium. Transfus Clin Biol 17:168–175

  • Le Moullac G, Haffner P (2000) Environmental factors affecting immune responses in Crustacea. Aquaculture 191(1–3):121–131

  • Levy S, Elek A, Tanay A, Grau-bove X (2021) A stony coral cell atlas illuminates the molecular and cellular basis of coral symbiosis, calcification, and immunity. Cell 1–15

  • Li XD, Lupo D, Zheng L, Winkler F (2006) Structural and functional insights into the AmtB/Mep/Rh protein family. Transfus Clin Biol 13:65–69

  • Loest RA (1979) Ammonia-Forming Enzymes and Calcium-Carbonate Deposition in Terrestrial Pulmonates. Physiol Zool 52:470–483

  • Ludewig U, Neuhäuser B, Dynowski M (2007) Molecular mechanisms of ammonium transport and accumulation in plants. FEBS Lett 581:2301–2308

  • Lupo D, Li XD, Durand A, Tomizaki T, Cherif-Zahar B, Matassi G, Merrick M, Winkler FK (2007) The 1.3-Å resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins. Proc Natl Acad Sci U S A 104:19303–19308

  • Martinelle K, Häggström L (1993) Mechanisms of ammonia and ammonium ion toxicity in animal cells: Transport across cell membranes. J Biotechnol 30:339–350

  • McDonald TR, Ward JM (2016) Evolution of electrogenic ammonium transporters (AMTs). Front Plant Sci 7:1–9

  • Merrick M, Javelle A, Durand A, Severi E, Thornton J, Avent ND, Conroy MJ, Bullough PA (2006) The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins. Transfus Clin Biol 13:97–102

  • Moya A, Tambutté S, Bertucci A, Tambutté E, Lotto S, Vullo D, Supuran CT, Allemand D, Zoccola D (2008) Carbonic anhydrase in the scleractinian coral Stylophora pistillata: Characterization, localization, and role in biomineralization. J Biol Chem 283:25475–25484

  • Muscatine L (1978) L. Muscatine. 23:725–734

  • O’Donnell MJ, Wright JC (1995) Nitrogen excretion in terrestrial crustaceans. In: Walsh PJ, Wright P (ed) Nitrogen metabolism and excretion. CRC Press, Boca Raton, FL, pp 105–118.

  • Ostrensky A, Marchiori MA, Poersch LH (1992) Aquatic toxicity of ammonia in the metamorphosis of post-larvae Penaeus paulensis Perez-Farfante. An Acad Bras Cienc 64:383–389

  • Pantoja O (2012) High affinity ammonium transporters: Molecular mechanism of action. Front Plant Sci 3:1–10

  • Pernice M, Meibom A, Van Den Heuvel A, Kopp C, Domart-Coulon I, Hoegh-Guldberg O, Dove S (2012) A single-cell view of ammonium assimilation in coral-dinoflagellate symbiosis. ISME J 6:1314–1324

  • Puverel S, Tambutté E, Zoccola D, Domart-Coulon I, Bouchot A, Lotto S, Allemand D, Tambutté S (2005) Antibodies against the organic matrix in scleractinians: A new tool to study coral biomineralization. Coral Reefs 24:149–156

  • Rädecker N, Pogoreutz C, Voolstra CR, Wiedenmann J, Wild C (2015) Nitrogen cycling in corals: The key to understanding holobiont functioning? Trends Microbiol 23:490–497

  • Rahav O, Dubinsky Z, Achituv Y, Falkowski PG (1989) Ammonium metabolism in the zooxanthellate coral, stylophora pistillata . Proc R Soc London B Biol Sci 236:325–337

  • Roberty S, Béraud E, Grover R, Ferrier-Pagès C (2020) Coral productivity is co-limited by bicarbonate and ammonium availability. Microorganisms 8:

  • Sproles AE, Kirk NL, Kitchen SA, Oakley CA, Grossman AR, Weis VM, Davy SK (2018) Phylogenetic characterization of transporter proteins in the cnidarian-dinoflagellate symbiosis. Mol Phylogenet Evol 120:307–320

  • Sun CY, Stifler CA, Chopdekar R V., Schmidt CA, Parida G, Schoeppler V, Fordyce BI, Brau JH, Mass T, Tambutté S, Gilbert PUPA (2020) From particle attachment to space-filling coral skeletons. Proc Natl Acad Sci U S A 117:30159–30170

  • Tambutté É, Allemand D, Mueller E, Jaubert J (1996) A compartmental approach to the mechanism of calcification in hermatypic corals. J Exp Biol 199:1029–1041

  • Tambutté S, Holcomb M, Ferrier-Pagès C, Reynaud S, Tambutté É, Zoccola D, Allemand D (2011) Coral biomineralization: From the gene to the environment. J Exp Mar Bio Ecol 408:58–78

  • Tang J, Ni X, Wen J, Wang L, Luo J, Zhou Z (2020) Increased Ammonium Assimilation Activity in the Scleractinian Coral Pocillopora damicornis but Not Its Symbiont After Acute Heat Stress. Front Mar Sci 7:1–10

  • Thies A, Quijada-Rodriguez AR, Zhouyao H, Weihrauch D, Tresguerres M (2021) A novel nitrogen concentrating mechanism in the coral-algae symbiosome. bioRxiv 1–22

  • Udomsap B, Chawakitchareon P, Rungsupa S (2018) Effects of temperature and ammonia on coral health status: a case study of disc coral (Turbinaria peltata). Int Trans J Eng Manag Appl Sci Technol 9:31–41

  • Venn A, Tambutté E, Holcomb M, Allemand D, Tambutté S (2011) Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater. PLoS One 6:

  • Voolstra CR, Li Y, Liew YJ, Baumgarten S, Zoccola D, Flot JF, Tambutté S, Allemand D, Aranda M (2017) Comparative analysis of the genomes of Stylophora pistillata and Acropora digitifera provides evidence for extensive differences between species of corals. Sci Rep 7:

  • Weihrauch D, Allen GJP (2018) Ammonia excretion in aquatic invertebrates: New insights and questions. J Exp Biol 221:0–2

  • Weihrauch D, Becker W, Postel U, Luck-Kopp S, Siebers D (1999) Potential of active excretion of ammonia in three different haline species of crabs. J Comp Physiol B 169:25–37

  • Weihrauch D, Morris S, Towle DW (2004) Ammonia excretion in aquatic and terrestrial crabs. J Exp Biol 207(26):4491–4504

  • Wood CM, Matsuo AY, Gonzalez RJ, Wilson RW, Patrick ML, Val AL (2002) Mechanisms of ion transport in Potamotrygon, a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro. J Exp Biol 205:3039–3054

  • Yellowlees D, Rees TA V., Leggat W (2008) Metabolic interactions between algal symbionts and invertebrate hosts. Plant, Cell Environ 31:679–694

  • Zheng L, Kostrewa D, Bernèche S, Winkler FK, Li XD (2004) The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli. Proc Natl Acad Sci U S A 101:17090–17095

  • Zoccola D, Tambutté E, Kulhanek E, Puverel S, Scimeca JC, Allemand D, Tambutté S (2004) Molecular cloning and localization of a PMCA P-type calcium ATPase from the coral Stylophora pistillata. Biochim Biophys Acta - Biomembr 1663:117–126

  • Zoccola D, Ganot P, Bertucci A, Caminiti-Segonds N, Techer N, Voolstra CR, Aranda M, Tambutté E, Allemand D, Casey JR, Tambutté S (2015) Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification. Sci Rep 5:

  • Zoccola D, Innocenti A, Bertucci A, Tambutté E, Supuran CT, Tambutté S (2016) Coral carbonic anhydrases: Regulation by ocean acidification. Mar Drugs 14:

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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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Authors and Affiliations

  1. Marine Biology Department, Centre Scientifique de Monaco (CSM), 8 Quai Antoine 1er, 98000, Monaco City, Monaco

    Laura Capasso, Didier Zoccola, Philippe Ganot & Sylvie Tambutté

  2. Sorbonne Université, Collège Doctoral, 75005, Paris, France

    Laura Capasso

  3. Division of Biological and Environmental Science and Engineering (BESE), Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia

    Manuel Aranda

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  1. Laura Capasso
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Contributions

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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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