Abstract

Nitrogen (N) is a limiting nutrient in vast regions of the world’s oceans, yet the sources of N available to various phytoplankton groups remain poorly understood. In this study, we investigated inorganic carbon (C) fixation rates and nitrate (NO3), ammonium (NH4+) and urea uptake rates at the single cell level in photosynthetic pico-eukaryotes (PPE) and the cyanobacteria Prochlorococcus and Synechococcus. To that end, we used dual 15N and 13C-labeled incubation assays coupled to flow cytometry cell sorting and nanoSIMS analysis on samples collected in the North Pacific Subtropical Gyre (NPSG) and in the California Current System (CCS). Based on these analyses, we found that photosynthetic growth rates (based on C fixation) of PPE were higher in the CCS than in the NSPG, while the opposite was observed for Prochlorococcus. Reduced forms of N (NH4+ and urea) accounted for the majority of N acquisition for all the groups studied. NO3 represented a reduced fraction of total N uptake in all groups but was higher in PPE (17.4 ± 11.2% on average) than in Prochlorococcus and Synechococcus (4.5 ± 6.5 and 2.9 ± 2.1% on average, respectively). This may in part explain the contrasting biogeography of these picoplankton groups. Moreover, single cell analyses reveal that cell-to-cell heterogeneity within picoplankton groups was significantly greater for NO3 uptake than for C fixation and NH4+ uptake. We hypothesize that cellular heterogeneity in NO3 uptake within groups facilitates adaptation to the fluctuating availability of NO3 in the environment.

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References

  1. 1.

    Tyrrell T. The relative influences of nitrogen and phosphorus on oceanic primary production. Nature. 1999;400:525–31.

  2. 2.

    Moore JK, Geider RJ, Guieu C, Jaccard SL, Jickells TD, LaRoche J, et al. Processes and patterns of nutrient limitation. Nat Geosci. 2013;6:701–10.

  3. 3.

    Chisholm SW. Phytoplankton size. In: Falkowski PG, Woodhead AD, Vivirito K, eds. Primary productivity and biogeochemical cycles in the sea. Boston, MA: Springer US; 1992. p. 213–37.

  4. 4.

    de Vargas C, Audic S, Henry N, Decelle J, Mahe F, Logares R, et al. Eukaryotic plankton diversity in the sunlit ocean. Science. 2015;348:1261605.

  5. 5.

    Kirkham AR, Lepère C, Jardillier LE, Not F, Bouman H, Mead A, et al. A global perspective on marine photosynthetic picoeukaryote community structure. ISME J. 2013;7:922–36.

  6. 6.

    Sohm JA, Ahlgren NA, Thomson ZJ, Williams C, Moffett JW, Saito MA, et al. Co-occurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature, macronutrients and iron. ISME J. 2016;10:333–45.

  7. 7.

    Biller SJ, Berube PM, Lindell D, Chisholm SW. Prochlorococcus: the structure and function of collective diversity. Nat Rev Microbiol. 2014;13:13–27.

  8. 8.

    Flombaum P, Gallegos JL, Gordillo RA, Rincon J, Zabala LL, Jiao N, et al. Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus. Proc Natl Acad Sci. 2013;110:9824–9.

  9. 9.

    Rii YM, Duhamel S, Bidigare RR, Karl DM, Repeta DJ, Church MJ. Diversity and productivity of photosynthetic picoeukaryotes in biogeochemically distinct regions of the South East Pacific Ocean. Limnol Oceanogr. 2016;61:806–24.

  10. 10.

    Rii YM, Karl DM, Church MJ. Temporal and vertical variability in picophytoplankton primary productivity in the North Pacific Subtropical Gyre. Mar Ecol Prog Ser. 2016;562:1–18.

  11. 11.

    Hartmann M, Gomez-Pereira P, Grob C, Ostrowski M, Scanlan DJ, Zubkov MV. Efficient CO2fixation by surface Prochlorococcusin the Atlantic Ocean. ISME J. 2014;8:2280–9.

  12. 12.

    Buitenhuis ET, Li WKW, Vaulot D, Lomas MW, Landry MR, Partensky F, et al. Picophytoplankton biomass distribution in the global ocean. Earth Syst. Sci Data. 2012;4:37–46.

  13. 13.

    Jardillier L, Zubkov MV, Pearman J, Scanlan DJ. Significant CO2 fixation by small prymnesiophytes in the subtropical and tropical northeast Atlantic Ocean. ISME J. 2010;4:1180–92.

  14. 14.

    Mackey KRM, Paytan A, Caldeira K, Grossman AR, Moran D, McIlvin M, et al. Effect of temperature on photosynthesis and growth in marine Synechococcus spp. Plant Physiol. 2013;163:815–29.

  15. 15.

    Johnson ZI, Zinser ER, Coe A, McNulty NP, Woodward EMS, Chisholm SW. Niche partitioning among Prochlorococcus ecotypes along ocean-scale environmental gradients. Science. 2006;311:1737–40.

  16. 16.

    Zinser ER, Johnson ZI, Coe A, Karaca E, Veneziano D, Chisholm SW. Influence of light and temperature on Prochlorococcus ecotype distributions in the Atlantic Ocean. Limnol Oceanogr. 2007;52:2205–20.

  17. 17.

    Moore LR, Goericke R, Chisholm SW. Comparative physiology of Synechococcusand Prochlorococcus: Influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Mar Ecol Prog Ser. 1995;116:259–76.

  18. 18.

    Chen B, Liu H, Huang B, Wang J. Temperature effects on the growth rate of marine picoplankton. Mar Ecol Prog Ser. 2014;505:37–47.

  19. 19.

    Martiny AC, Pham CTA, Primeau FW, Vrugt JA, Moore JK, Levin SA, et al. Strong latitudinal patterns in the elemental ratios of marine plankton and organic matter. Nat Geosci. 2013 ;6:279–83.

  20. 20.

    Weber TS, Deutsch C. Ocean nutrient ratios governed by plankton biogeography. Nature . 2010;467:550–4.

  21. 21.

    Karl DM, Bidigare RR, Church MJ, Dore JE, Letelier RM, Mahaffey C et al. The nitrogen cycle in the North Pacific trades biome: an evolving paradigm. In: Nitrogen in the marine environment. 2008. New-York, USA: Academic press, pp. 705–69. 

  22. 22.

    Fawcett SE, Lomas MW, Casey JR, Ward BB, Sigman DM. Assimilation of upwelled nitrate by small eukaryotes in the Sargasso Sea. Nat Geosci. 2011;4:717–22.

  23. 23.

    Martiny AC, Kathuria S, Berube PM. Widespread metabolic potential for nitrite and nitrate assimilation among P rochlorococcus ecotypes. Proc Natl Acad Sci Usa. 2009;106:10787–92.

  24. 24.

    Berube PM, Biller SJ, Kent AG, Berta-Thompson JW, Roggensack SE, Roache-Johnson KH, et al. Physiology and evolution of nitrate acquisition in Prochlorococcus. ISME J. 2014;9:1195–207.

  25. 25.

    Casey JR, Lomas MW, Mandecki J, Walker DE. Prochlorococcus contributes to new production in the Sargasso Sea deep chlorophyll maximum. Geophys Res Lett. 2007;34:L10604.

  26. 26.

    Kettler GC, Martiny AC, Huang K, Zucker J, Coleman ML, Rodrigue S, et al. Patterns and implications of gene gain and loss in the evolution of Prochlorococcus. PLoS Genet. 2007;3:2515–28.

  27. 27.

    Biller SJ, Berube PM, Berta-Thompson JW, Kelly L, Roggensack SE, Awad L, et al. Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus. Sci Data. 2014;1:140034.

  28. 28.

    Kashtan N, Roggensack SE, Rodrigue S, Thompson JW, Biller SJ, Coe A, et al. Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus. Science. 2014;344:416–20.

  29. 29.

    Lomas MW, Bronk DA, van den Engh G. Use of flow cytometry to measure biogeochemical rates and processes in the ocean. Ann Rev Mar Sci. 2011;3:537–66.

  30. 30.

    Musat N, Foster R, Vagner T, Adam B, Kuypers MM. Detecting metabolic activities in single cells, with emphasis on nanoSIMS. FEMS Microbiol Rev. 2012;36:486–511.

  31. 31.

    Gao D, Huang X, Tao Y. A critical review of NanoSIMS in analysis of microbial metabolic activities at single-cell level. Crit Rev Biotechnol. 2016;36:884–90.

  32. 32.

    López-Lozano A, Diez J, El Alaoui S, Moreno-Vivián C, García-Fernández JM. Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures. FEMS Microbiol Ecol. 2002;41:151–60.

  33. 33.

    Duhamel S, Van Wambeke F, Lefevre D, Benavides M, Bonnet S. Mixotrophic metabolism by natural communities of unicellular cyanobacteria in the western tropical South Pacific Ocean. Environ Microbiol. 2018;20:2743-56.

  34. 34.

    Worden AZ, Nolan JK, Palenik B. Assessing the dynamics and ecology of marine picophytoplankton: The importance of the eukaryotic component. Limnol Oceanogr. 2004;49:168–79.

  35. 35.

    Baer SE, Lomas MW, Terpis KX, Mouginot C, Martiny AC. Stoichiometry of Prochlorococcus, Synechococcus, and small eukaryotic populations in the western North Atlantic Ocean. Environ Microbiol. 2017;19:1568–83.

  36. 36.

    Moutin T, Raimbault P, Poggiale J-C. Primary production in surface waters of the western mediterranean sea. Calculation of daily production. Comptes Rendus l’Académie Des Sciences. 1999;322:651–9.

  37. 37.

    Harrison WG, Harris LR, Irwin BD. The kinetics of nitrogen utilization in the oceanic mixed layer: Nitrate and ammonium interactions at nanomolar concentrations. Limnol Oceanogr. 1996;41:16–32.

  38. 38.

    Rees AP, Joint I, Donald KM. Early spring bloom phytoplankton-nutrient dynamics at the Celtic Sea shelf edge. Deep Res Part I Oceanogr Res Pap. 1999;46:483–510.

  39. 39.

    Ribalet F, Swalwell J, Clayton S, Jiménez V, Sudek S, Lin Y, et al. Light-driven synchrony of Prochlorococcus growth and mortality in the subtropical Pacific gyre. Proc Natl Acad Sci. 2015;112:8008–112.

  40. 40.

    L’Helguen S, Slawyk G, Le Corre P. Seasonal patterns of urea regeneration by size-fractionated microheterotrophs in well-mixed temperate coastal waters. J Plankton Res. 2005;27:263–70.

  41. 41.

    Clark DR, Rees AP, Joint I. Ammonium regeneration and nitrification rates in the oligotrophic Atlantic Ocean: implications for new production estimates. Limnol Oceanogr. 2008;53:52–62.

  42. 42.

    Price NM, Harrison PJ. Urea uptake by Sargasso Sea phytoplankton: saturated and in situ uptake rates. Deep Sea Res Part A Oceanogr Res Pap. 1988;35:1579–93.

  43. 43.

    Dugdale RC, Goering JJ. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol Oceanogr. 1967;12:196–206.

  44. 44.

    Raimbault P, Garcia N. Evidence for efficient regenerated production and dinitrogen fixation in nitrogen-deficient waters of the South Pacific Ocean: impact on new and export production. Biogeosciences. 2008;323–38.

  45. 45.

    Solomon C, Collier J, Berg G, Glibert P. Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review. Aquat Microb Ecol. 2010;59:67–88.

  46. 46.

    Fawcett SE, Lomas MW, Ward BB, Sigman DM. Global Biogeochemical Cycles production in the Sargasso Sea. Glob Biogeochem Cycles. 2014;28:86–102.

  47. 47.

    Berube PM, Coe A, Roggensack SE, Chisholm SW. Temporal dynamics of Prochlorococcus cells with the potential for nitrate assimilation in the subtropical Atlantic and Pacific oceans. Limnol Oceanogr. 2016;61:482–95.

  48. 48.

    Massana R. Eukaryotic Picoplankton in Surface Oceans. Annu Rev Microbiol. 2011;65:91–110.

  49. 49.

    Vaulot D, Eikrem W, Viprey M, Moreau H. The diversity of small eukaryotic phytoplankton (≤3 μm) in marine ecosystems. Vol. 32, FEMS Microbiology Reviews. 2008. p. 795–820.

  50. 50.

    Ahlgren NA, Rocap G. Diversity and distribution of marine Synechococcus: Multiple gene phylogenies for consensus classification and development of qPCR assays for sensitive measurement of clades in the ocean. Front Microbiol. 2012;3:article 213.

  51. 51.

    Larkin AA, Blinebry SK, Howes C, Lin Y, Loftus SE, Schmaus CA, et al. Niche partitioning and biogeography of high light adapted Prochlorococcus across taxonomic ranks in the North Pacific. ISME J. 2016;10:1555–67.

  52. 52.

    Glibert PM, Wilkerson FP, Dugdale RC, Raven JA, Dupont CL, Leavitt PR, et al. Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions. Limnol Oceanogr. 2016;61:165–97.

  53. 53.

    Kent AG, Dupont CL, Yooseph S, Martiny AC. Global biogeography of Prochlorococcus genome diversity in the surface ocean. ISME J. 2016;10:1856-65.

  54. 54.

    Ohashi Y, Shi W, Takatani N, Aichi M, Maeda SI, Watanabe S, et al. Regulation of nitrate assimilation in cyanobacteria. J Exp Bot. 2011;62:1411–24.

  55. 55.

    Kang LK, Gong GC, Wu YH, Chang J. The expression of nitrate transporter genes reveals different nitrogen statuses of dominant diatom groups in the southern East China Sea. Mol Ecol. 2015;24:1374–86.

  56. 56.

    Song B, Ward BB. Molecular cloning and characterization of high-affinity nitrate transporters in marine phytoplankton. J Phycol. 2007;43:542–52.

  57. 57.

    Raven JA, Wollenweber B, Handley LL. A comparison of ammonium and nitrate as nitrogen sources for photolithotrophs. New Phytol. 1992;121:19–32.

  58. 58.

    Quesada A, Hidalgo J, Fernández E. Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii. Mol Gen Genet. 1998;258:373–7.

  59. 59.

    L’Helguen S, Maguer JF, Caradec J. Inhibition kinetics of nitrate uptake by ammonium in size-fractionated oceanic phytoplankton communities: Implications for new production and f-ratio estimates. J Plankton Res. 2008;30:1179–88.

  60. 60.

    Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, et al. Structure and function of the global ocean microbiome. Science. 2015;348:1261359.

  61. 61.

    Fuhrman JA. Microbial community structure and its functional implications. Nature . 2009;459:193–9.

  62. 62.

    Ackermann M. A functional perspective on phenotypic heterogeneity in microorganisms. Nat Rev Microbiol. 2015;13:497–508.

  63. 63.

    Schreiber F, Littmann S, Lavik G, Escrig S, Meibom A, Kuypers MMM, et al. Phenotypic heterogeneity driven by nutrient limitation promotes growth in fluctuating environments. Nat Microbiol. 2016;1:16055.

  64. 64.

    Bódi Z, Farkas Z, Nevozhay D, Kalapis D, Lázár V, Csörgő B, et al. Phenotypic heterogeneity promotes adaptive evolution. PLOS Biol. 2017;1:e2000644.

  65. 65.

    Magdanova LA, Golyasnaya NV. Heterogeneity as an adaptive trait of microbial populations. Microbiology . 2013;82:1–10.

  66. 66.

    Hashimoto M, Nozoe T, Nakaoka H, Okura R, Akiyoshi S, Kaneko K, et al. Noise-driven growth rate gain in clonal cellular populations. Proc Natl Acad Sci. 2016;113:3251–6.

  67. 67.

    Mohr W, Vagner T, Kuypers MMM, Ackermann M, LaRoche J. Resolution of Conflicting signals at the single-cell level in the regulation of cyanobacterial photosynthesis and nitrogen fixation. PLOS ONE. 2013;8:e66060.

  68. 68.

    Kopf SH, McGlynn SE, Green-Saxena A, Guan Y, Newman DK, Orphan VJ. Heavy water and 15N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats. Environ Microbiol. 2015;17:2542–56.

  69. 69.

    Finzi-Hart JA, Pett-Ridge J, Weber PK, Popa R, Fallon SJ, Gunderson T, et al. Fixation and fate of C and N in the cyanobacterium Tr ichodesmium using nanometer-scale secondary ion mass spectrometry. PNAS . 2009;106:6345–50.

  70. 70.

    Ploug H, Musat N, Adam B, Moraru CL, Lavik G, Vagner T, et al. Carbon and nitrogen fluxes associated with the cyanobacterium A phanizomenon sp. in the Baltic Sea. ISME J. 2010;4:1215–23.

  71. 71.

    Foster RA, Sztejrenszus S, Kuypers MM. Measuring carbon and N2 fixation in field populations of colonial and free-living unicellular cyanobacteria using nanometer-scale secondary ion mass spectrometry. Raven J, editor. J Phycol. 2013;49:502–16.

  72. 72.

    Flores E, Herrero A. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem Soc Trans. 2005;33:164–7.

  73. 73.

    Chisholm SW. Prochlorococcus. Curr Biol. 2017;27:R447–8.

  74. 74.

    Bec B, Husseini-Ratrema J, Collos Y, Souchu P, Vaquer A. Phytoplankton seasonal dynamics in a Mediterranean coastal lagoon: Emphasis on the picoeukaryote community. J Plankton Res. 2005;27:881–94.

  75. 75.

    Shilova IN, Mills MM, Robidart JC, Turk-Kubo KA, Björkman KM, Kolber Z et al. Differential effects of nitrate, ammonium, and urea as N sources for microbial communities in the North Pacific Ocean. Limnology and Oceanography. 2017;62:2550-74.

  76. 76.

    Van Mooy BAS, Devol AH. Assessing nutrient limitation of Prochlorococcus in the North Pacific subtropical gyre by using an RNA capture method. Limnol Oceanogr. 2008;53:78–88.

  77. 77.

    Mahaffey C, Björkman KM, Karl DM. Phytoplankton response to deep seawater nutrient addition in the North Pacific Subtropical Gyre. Mar Ecol Prog Ser. 2012;460:13–34.

  78. 78.

    Rii YM, Bidigare RR, Church MJ. Differential responses of eukaryotic phytoplankton to nitrogenous nutrients in the North Pacific Subtropical Gyre. Front Mar Sci. 2018;5:article 92.

  79. 79.

    Berube PM, Biller SJ, Kent AG, Berta-Thompson JW, Roggensack SE, Roache-Johnson KH, et al. Physiology and evolution of nitrate acquisition in Prochlorococcus. ISME J. 2015;9:1195–207.

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Acknowledgements

We would like to thank the Schmidt Ocean Institute for providing the vessel to conduct this research and the captain and crew of the R/V Falkor for their help during the cruise. We are grateful to Aimee Neeley (NASA) for providing us with the Chl a data. We also thank Smail Mostefaoui for his assistance with nanoSIMS analyses at the French National Ion MicroProbe Facility hosted by the Muséum National d’Histoire Naturelle (Paris). N. C. and H. B. were supported by the “Laboratoire d’Excellence” LabexMER (ANR-10-LABX-19) and co-funded by a grant from the French government under the program “Investissements d’Avenir”. SD was funded by the National Science Foundation (OCE-1434916 and OCE-1458070). IC was funded through Schmidt Ocean Institute and NASA's PACE mission.

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  1. Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/IFREMER, Institut Universitaire Européen de la Mer (IUEM), Brest, France

    • Hugo Berthelot
    • , Stéphane L’Helguen
    • , Jean-Francois Maguer
    •  & Nicolas Cassar
  2. Division of Biology and Paleo Environment, Lamont-Doherty Earth Observatory, PO Box 1000, 61 Route 9W, Palisades, NY, 10964, USA

    • Solange Duhamel
  3. Division of Earth and Ocean Sciences, Nicholas School of the Environment, Duke University, Durham, NC, 27708, USA

    • Seaver Wang
    •  & Nicolas Cassar
  4. NASA Goddard Space Flight Center, Ocean Ecology Laboratory, Code 616, Greenbelt, MD, USA

    • Ivona Cetinić
  5. GESTAR/Universities Space Research Association, Columbia, MD, USA

    • Ivona Cetinić

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The authors declare that they have no conflict of interest.

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Correspondence to Hugo Berthelot or Nicolas Cassar.

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https://doi.org/10.1038/s41396-018-0285-8