Letter | Published:

Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

Nature volume 555, pages 534537 (22 March 2018) | Download Citation

Abstract

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton1,2. Although most dissolved iron in the marine environment is complexed with organic molecules3, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone4 and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms5. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts6,7, including the ferric iron-concentrating protein ISIP2A8, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution8 and are abundant in marine environmental genomic datasets9,10, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

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References

  1. 1.

    et al. Processes and patterns of oceanic nutrient limitation. Nat. Geosci. 6, 701–710 (2013)

  2. 2.

    et al. Mesoscale iron enrichment experiments 1993–2005: synthesis and future directions. Science 315, 612–617 (2007)

  3. 3.

    & The organic complexation of iron in the marine environment: a review. Front. Microbiol. 3, 69 (2012)

  4. 4.

    , , & Photochemical cycling of iron in the surface ocean mediated by microbial iron(iii)-binding ligands. Nature 413, 409–413 (2001)

  5. 5.

    , & The role of unchelated Fe in the iron nutrition of phytoplankton. Limnol. Oceanogr. 53, 400–404 (2008)

  6. 6.

    et al. Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc. Natl Acad. Sci. USA 105, 10438–10443 (2008)

  7. 7.

    et al. Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation. Genome Biol. 13, R66 (2012)

  8. 8.

    et al. A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake. Curr. Biol. 25, 364–371 (2015)

  9. 9.

    et al. Comparative metatranscriptomics identifies molecular bases for the physiological responses of phytoplankton to varying iron availability. Proc. Natl Acad. Sci. USA 109, E317–E325 (2012)

  10. 10.

    et al. Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge. Proc. Natl Acad. Sci. USA 112, 9938–9943 (2015)

  11. 11.

    , , & Evolution of the transferrin family: conservation of residues associated with iron and anion binding. Comp. Biochem. Physiol. 142, 129–141 (2005)

  12. 12.

    , & Stoichiometric and site characteristics of the binding of iron to human transferrin. J. Biol. Chem. 253, 1930–1937 (1978)

  13. 13.

    , & Dealing with iron: common structural principles in proteins that transport iron and heme. Proc. Natl Acad. Sci. USA 100, 3579–3583 (2003)

  14. 14.

    , , & A structurally novel transferrin-like protein accumulates in the plasma membrane of the unicellular green alga Dunaliella salina grown in high salinities. J. Biol. Chem. 272, 1565–1570 (1997)

  15. 15.

    & The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thalassiosira weissflogii. Limnol. Oceanogr. 27, 789–813 (1982)

  16. 16.

    et al. Structure of Haemophilus influenzae Fe+3-binding protein reveals convergent evolution within a superfamily. Nat. Struct. Biol. 4, 919–924 (1997)

  17. 17.

    & The Neoproterozoic oxygenation event: environmental perturbations and biogeochemical cycling. Earth Sci. Rev. 110, 26–57 (2012)

  18. 18.

    et al. Transcriptional orchestration of the global cellular response of a model pennate diatom to diel light cycling under iron limitation. PLoS Genet. 12, e1006490 (2016)

  19. 19.

    et al. Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6, 6925 (2015)

  20. 20.

    , & Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol. 97, 329–339 (1983)

  21. 21.

    , & Iron uptake by the halotolerant alga Dunaliella is mediated by a plasma membrane transferrin. J. Biol. Chem. 273, 17553–17558 (1998)

  22. 22.

    & The synergistic binding of anions and Fe3+ by transferrin. Implications for the interlocking sites hypothesis. J. Biol. Chem. 250, 2182–2188 (1975)

  23. 23.

    , , , & The oxalate effect on release of iron from human serum transferrin explained. J. Mol. Biol. 339, 217–226 (2004)

  24. 24.

    ., ., & Ocean acidification: the other CO2 problem. Annu. Rev. Mar. Sci. 1, 169–192 (2009)

  25. 25.

    & Effect of pH, light, and temperature on Fe–EDTA chelation and Fe hydrolysis in seawater. Mar. Chem. 84, 35–47 (2003)

  26. 26.

    , , & Effect of ocean acidification on iron availability to marine phytoplankton. Science 327, 676–679 (2010)

  27. 27.

    , , & FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii. Eukaryot. Cell 6, 1841–1852 (2007)

  28. 28.

    , & Cloning and characterization of high-CO2-specific cDNAs from a marine microalga, Chlorococcum littorale, and effect of CO2 concentration and iron deficiency on the gene expression. Plant Cell Physiol. 39, 131–138 (1998)

  29. 29.

    , & Some thoughts on the concept of colimitation: three definitions and the importance of bioavailability. Limnol. Oceanogr. 53, 276–290 (2008)

  30. 30.

    , , , & Iron bioavailability to phytoplankton: an empirical approach. ISME J. 9, 1003–1013 (2015)

  31. 31.

    et al. Probing the evolution, ecology and physiology of marine protists using transcriptomics. Nat. Rev. Microbiol. 15, 6–20 (2017)

  32. 32.

    & MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013)

  33. 33.

    , & SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27, 221–224 (2010)

  34. 34.

    RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014)

  35. 35.

    , & PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25, 2286–2288 (2009)

  36. 36.

    & A molecular time-scale for eukaryote evolution recalibrated with the continuous microfossil record. Proc. R. Soc. B 273, 1867–1872 (2006)

  37. 37.

    , , & Estimating the timing of early eukaryotic diversification with multigene molecular clocks. Proc. Natl Acad. Sci. USA 108, 13624–13629 (2011)

  38. 38.

    , , & Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973 (2012)

  39. 39.

    , , & SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011)

  40. 40.

    , , & Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300, 1005–1016 (2000)

  41. 41.

    , , & Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997)

  42. 42.

    , & Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17, 646–653 (2001)

  43. 43.

    , & PredGPI: a GPI-anchor predictor. BMC Bioinformatics 9, 392 (2008)

  44. 44.

    , & in Algal Culturing Techniques (ed. ) 35–63 (Elsevier, 2005)

  45. 45.

    , , , & Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J. Phycol. 36, 379–386 (2000)

  46. 46.

    et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009)

  47. 47.

    , , , & Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. 1, 239–251 (1999)

  48. 48.

    & A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter–terminator cassette. FEBS J. 272, 3413–3423 (2005)

  49. 49.

    et al. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. Plant Biotechnol. J. 13, 460–470 (2015)

  50. 50.

    & Distinguishing between extra- and intracellular iron in marine phytoplankton. Limnol. Oceanogr. 34, 1113–1120 (1989)

  51. 51.

    , , & (eds) Guide to Best Practices for Ocean CO2 Measurements (North Pacific Marine Science Organization, 2007)

  52. 52.

    , & Sequence analysis and transcriptional regulation of iron acquisition genes in two marine diatoms. J. Phycol. 43, 715–729 (2007)

  53. 53.

    , , & Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis. PLoS ONE 7, e45799 (2012)

  54. 54.

    , , & An automated system for spectrophotometric seawater pH measurements. Limnol. Oceanogr. Methods 11, 16–27 (2013)

  55. 55.

    ., & Program developed for CO2 System Calculations (Carbon Dioxide Information Analysis Center, 1998)

  56. 56.

    The statistics of synergism. J. Mol. Cell. Cardiol. 30, 723–731 (1998)

  57. 57.

    R Core Team. R: A Language and Environment for Statistical Computing. (R Foundation for Statistical Computing, Vienna, Austria, 2014)

Download references

Acknowledgements

We thank J. Badger for early contributions to phylogenetic analyses, A. Dickson for pH analysis, K. Forsch for CSV measurements and E. Bertrand for trace-metal clean techniques. This study was supported by the National Science Foundation (NSF-MCB-1024913, NSF-ANT-1043671 and NSF-OCE-0727997), United States Department of Energy Genomics Science program (DE-SC00006719 and DE-SC0008593), and the Gordon and Betty Moore Foundation grant GBMF3828 (A.E.A.); NSF-1557928 (A.B.K.); and the Czech Science Foundation, project 15-17643S (M.O. and A.H.).

Author information

Author notes

    • Bogumil J. Karas

    Present address: Designer Microbes Inc., London, Ontario N6G4X8, Canada.

Affiliations

  1. J. Craig Venter Institute, Microbial and Environmental Genomics, La Jolla, California 92037, USA

    • Jeffrey B. McQuaid
    • , John P. McCrow
    • , Bogumil J. Karas
    • , Hong Zheng
    •  & Andrew E. Allen
  2. Scripps Institution of Oceanography, University of California, La Jolla, California 92093, USA

    • Jeffrey B. McQuaid
    • , Theodor Kindeberg
    • , Andreas J. Andersson
    • , Katherine A. Barbeau
    •  & Andrew E. Allen
  3. Rutgers University–Newark, Earth and Environmental Sciences, Newark, New Jersey 07102, USA

    • Adam B. Kustka
  4. Biology Centre CAS, Institute of Parasitology, Branišovská 31, 370 05 České Budějovice, Czech Republic

    • Miroslav Oborník
    •  & Aleš Horák
  5. University of South Bohemia, Faculty of Science, Branišovská 31, 370 05 České Budějovice, Czech Republic

    • Miroslav Oborník
    •  & Aleš Horák

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Contributions

J.B.M., A.B.K., M.O. and A.E.A. designed the study and interpreted the results. J.B.M., M.O. and A.H. generated and analysed phylogenetic and molecular clock data. J.B.M. and B.J.K. generated mutant cell lines, J.B.M. and A.B.K. with assistance from K.A.B. performed physiology experiments. J.B.M. performed microscopy, and H.Z. performed western blots. T.K. and A.J.A. analysed inorganic carbon species. J.B.M. and J.P.M. conducted statistical analyses. J.B.M. wrote the paper with input from A.E.A., A.B.K., M.O., J.P.M, A.J.A. and K.A.B. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andrew E. Allen.

Reviewer Information Nature thanks S. Amin, E. DeLong and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature25982

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