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Evolution and metabolic significance of the urea cycle in photosynthetic diatoms

Abstract

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world’s most productive marine food webs1,2,3,4. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont5)1,6,7,8,9,10, yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline12 and the precursors for long-chain polyamines required for silica precipitation during cell wall formation11. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

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Figure 1: Carbamoyl phosphate synthase phylogeny and divergence timing.
Figure 2: Transcript levels of genes encoding components of the urea cycle and the nitrate assimilation pathway in wild-type P. tricornutum cells.
Figure 3: Growth characteristics and metabolite abundance in wild-type and unCPS RNAi P. tricornutum lines.
Figure 4: Conceptual overview of the roles of unCPS and the diatom urea cycle on the basis of metabolite data from wild-type and RNAi lines.

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References

  1. Falkowski, P. G. et al. The evolution of modern eukaryotic phytoplankton. Science 305, 354–360 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Falkowski, P. G. & Oliver, M. J. Mix and match: how climate selects phytoplankton. Nature Rev. Microbiol. 5, 813–819 (2007)

    Article  CAS  Google Scholar 

  3. Nelson, D. M., Treguer, P., Brzezinski, M. A., Leynaert, A. & Queguiner, B. Production and dissolution of biogenic silica in the ocean - revised global estimates, comparison with regional data and relationship to biogenic sedimentation. Glob. Biogeochem. Cycles 9, 359–372 (1995)

    Article  ADS  CAS  Google Scholar 

  4. Smetacek, V. Diatoms and the ocean carbon cycle. Protist 150, 25–32 (1999)

    Article  CAS  Google Scholar 

  5. Hamm, C. & Smetacek, V. in Evolution of Primary Producers in the Sea (eds Falkowski, P. G. & Knoll, A. H. ) (Academic Press, 2007)

    Google Scholar 

  6. Allen, A. E., Vardi, A. & Bowler, C. An ecological and evolutionary context for integrated nitrogen metabolism and related signaling pathways in marine diatoms. Curr. Opin. Plant Biol. 9, 264–273 (2006)

    Article  CAS  Google Scholar 

  7. Bowler, C. et al. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456, 239–244 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Moustafa, A. et al. Genomic footprints of a cryptic plastid endosymbiosis in diatoms. Science 324, 1724–1726 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Bowler, C., Vardi, A. & Allen, A. E. Oceanographic and biogeochemical insights from diatom genomes. Ann. Rev. Mar. Sci. 2, 333–365 (2010)

    Article  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  11. Kröger, N. & Poulsen, N. Diatoms—from cell wall biogenesis to nanotechnology. Annu. Rev. Genet. 42, 83–107 (2008)

    Article  Google Scholar 

  12. Krell, A., Funck, D., Plettner, I., John, U. & Dieckmann, G. Regulation of proline metabolism under salt stress in the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae). J. Phycol. 43, 753–762 (2007)

    Article  CAS  Google Scholar 

  13. Anderson, P. M. Glutamine- and N-acetylglutamate-dependent carbamoyl phosphate synthetase in elasmobranchs. Science 208, 291–293 (1980)

    Article  ADS  CAS  Google Scholar 

  14. Hong, J., Salo, W. L., Lusty, C. J. & Anderson, P. M. Carbamoyl-phosphate synthetase-III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamoyl-phosphate synthetases. J. Mol. Biol. 243, 131–140 (1994)

    Article  CAS  Google Scholar 

  15. Mommsen, T. P. & Walsh, P. J. Evolution of urea synthesis in vertebrates: the piscine connection. Science 243, 72–75 (1989)

    Article  ADS  CAS  Google Scholar 

  16. Lawson, F. S., Charlebois, R. L. & Dillon, J. A. R. Phylogenetic analysis of carbamoylphosphate synthetase genes: Complex evolutionary history includes an internal duplication within a gene which can root the tree of life. Mol. Biol. Evol. 13, 970–977 (1996)

    Article  CAS  Google Scholar 

  17. Guppy, M. The hibernating bear: why is it so hot, and why does it cycle urea through the gut. Trends Biochem. Sci. 11, 274–276 (1986)

    Article  CAS  Google Scholar 

  18. Holden, H. M., Thoden, J. B. & Raushel, F. M. Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product. Cell. Mol. Life Sci. 56, 507–522 (1999)

    Article  CAS  Google Scholar 

  19. Armbrust, E. V. et al. The genome of the diatom Thalassiosira pseudonana: Ecology, evolution, and metabolism. Science 306, 79–86 (2004)

    Article  ADS  CAS  Google Scholar 

  20. Ast, M. et al. Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl Acad. Sci. USA 106, 3621–3626 (2009)

    Article  ADS  CAS  Google Scholar 

  21. Maheswari, U. et al. Digital expression profiling of novel diatom transcripts provides insight into their biological functions. Genome Biol. 11, R85 (2010)

    Article  Google Scholar 

  22. Esteban-Pretel, G. et al. Vitamin A deficiency increases protein catabolism and induces urea cycle enzymes in rats. J. Nutr. 140, 792–798 (2010)

    Article  CAS  Google Scholar 

  23. Lee, B. et al. In vivo urea cycle flux distinguishes and correlates with phenotypic severity in disorders of the urea cycle. Proc. Natl Acad. Sci. USA 97, 8021–8026 (2000)

    Article  ADS  CAS  Google Scholar 

  24. De Riso, V. et al. Gene silencing in the marine diatom Phaeodactylum tricornutum . Nucleic Acids Res. 37, e96 (2009)

    Article  Google Scholar 

  25. Young, E. B. & Beardall, J. Photosynthetic function in Dunaliella tertiolecta (Chlorophyta) during a nitrogen starvation recovery cycle. J. Phycol. 39, 897–905 (2003)

    Article  CAS  Google Scholar 

  26. Morris, S. M. Regulation of enzymes of the urea cycle and arginine metabolism. Annu. Rev. Nutr. 22, 87–105 (2002)

    Article  CAS  Google Scholar 

  27. Nunes-Nesi, A., Fernie, A. R. & Stitt, M. Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol. Plant 3, 973–96 (2010)

    Article  CAS  Google Scholar 

  28. Parker, M. S., Mock, T. & Armbrust, E. V. Genomic insights into marine microalgae. Annu. Rev. Genet. 42, 619–645 (2008)

    Article  CAS  Google Scholar 

  29. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L. & Fernie, A. R. Gas chromatography mass spectrometry-based metabolite profiling in plants. Nature Protocols 1, 387–396 (2006)

    Article  CAS  Google Scholar 

  30. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and models. Bioinformatics 22, 2688–2690 (2006)

    Article  CAS  Google Scholar 

  31. Siaut, M. et al. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum . Gene 406, 23–35 (2007)

    Article  CAS  Google Scholar 

  32. Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C. & Bowler, C. Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. 1, 239–251 (1999)

    Article  CAS  Google Scholar 

  33. Schauer, N. et al. GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. 579, 1332–1337 (2005)

    Article  CAS  Google Scholar 

  34. R Development Core Team R: A language and environment for statistical computing. ISBN 3-900051-07-0 (R Foundation for Statistical Computing, 2008)

  35. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. TREE-PUZZLE: a maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–504 (2002)

    Article  CAS  Google Scholar 

  38. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003)

    Article  Google Scholar 

  39. Lartillot, N. & Philippe, H. A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Mol. Biol. Evol. 21, 1095–1109 (2004)

    Article  CAS  Google Scholar 

  40. Quang, L. S., Gascuel, O. & Lartillot, N. Empirical profile mixture models for phylogenetic reconstruction. Bioinformatics 24, 2317–2323 (2008)

    Article  CAS  Google Scholar 

  41. Anisimova, M. & Gascuel, O. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552 (2006)

    Article  Google Scholar 

  42. Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755 (2001)

    Article  CAS  Google Scholar 

  43. Ronquist, F. & Huelsenbeck, J. P. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003)

    Article  CAS  Google Scholar 

  44. Lartillot, N., Lepgae, T. & Blanquart, S. PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25, 2286–2288 (2009)

    Article  CAS  Google Scholar 

  45. Sanderson, M. J. A nonparametric approach to estimating divergence times in the absence of rate consistency. Mol. Biol. Evol. 19, 1218–1231 (1997)

    Article  Google Scholar 

  46. Sanderson, M. J. Estimating absolute rates of molecular evolution and divergence times: a penalized likelihood approach. Mol. Biol. Evol. 19, 101–109 (2002)

    Article  CAS  Google Scholar 

  47. Thorne, J. L., Kishino, H. & Painter, I. S. Estimating the rate of evolution of the rate of molecular evolution. Mol. Biol. Evol. 15, 1647–1657 (1998)

    Article  CAS  Google Scholar 

  48. Berney, C. & Pawlowski, J. A molecular timescale for eukaryote evolution recalibrated with the continuous microfossil record. Proc. R. Soc. A/B 273, 1867–1872 (2006)

    Article  CAS  Google Scholar 

  49. Benton, M. J. & Donoghue, P. C. J. Paleontological evidence to date the tree of life. Mol. Biol. Evol. 24, 26–53 (2007)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Meichenin and C. Lichtlé for assistance with electron microscropy, J. C. Thomas for CPS purification and activity experiments, A. Falciatore for advice on RNAi constructs and A. Main for screening and evaluation of RNAi lines. This study was supported by the National Science Foundation (NSF-OCE-0722374, NSF-OCE-0727997, NSF-MCB-1024913) and JCVI internal funding to A.E.A., the European Commission Diatomics project and Agence Nationale de la Recherche (France) (C.B.) and the Czech Science Foundation (206/08/1423) (M.O.).

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A.E.A. and C.B. designed the study. A.E.A. performed CPS localization, confocal microscopy, protein purification, activity, overexpression and other laboratory experiments. A.E.A. and C.L.D. designed nitrogen-recovery experiments and the physiological characterization of the RNAi as well as wild-type experiments, which were performed by H.Z and C.L.D.. H.Z. generated and screened RNAi lines. H.Z. and H.H. performed long-term and short-term nitrogen-recovery and related qPCR experiments. D.A.J. ran qPCR reactions and assisted with analyses of qPCR data. M.O., A.H. and A.E.A. generated and analysed phylogenetic and molecular clock data. A.N-N. and A.R.F. performed metabolite profiling of samples collected from RNAi and wild-type cultures. J.P.M., C.L.D. and A.E.A. analysed qPCR, metabolite and western blot data in detail. A.E.A. wrote the paper with assistance from C.L.D., A.R.F., C.B. and M.O. All the authors discussed the results and commented on the manuscript.

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Correspondence to Andrew E. Allen.

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Allen, A., Dupont, C., Oborník, M. et al. Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473, 203–207 (2011). https://doi.org/10.1038/nature10074

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