NanoSIMS single cell analyses reveal the contrasting nitrogen sources for small phytoplankton

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.

Fig. 1

Locations of the three stations sampled in the Northeast Pacific Ocean superimposed on surface chlorophyll a concentration (AQUA MODIS composite image of January and February 2017)

Fig. 2

Examples of nanoSIMS images showing the sum of ¹²C¹⁴N ions detected (a), the A¹⁵N enrichment (b), and the A¹³C enrichment (c) for Synechococcus. The white outlines define the identified cells. Scale bars are 5 µm

Fig. 3

Whisker plot of C-based specific division rate (d⁻¹) for each group (Prochlorococcus, Synechococcus, and PPE) in each daylight assay. Each dot represents an analyzed cell. Grey dots denote cells with rates not significantly different from zero. Colored lines denote mean division rates and standard deviations (plain horizontal and dashed vertical, respectively) in each assay. The percentages indicate proportions of detected active cells. Grey lines denote mean division rates and standard deviations (horizontal plain and vertical dashed, respectively) in the North Pacific Subtropical Gyre (NPSG) and in the California Current System (CCS) regions

Fig. 4

NH₄⁺, urea, and NO₃⁻ specific uptake rates (h⁻¹) of photosynthetic picoeukaryotes (PPE), Prochlorococcus, and Synechococcus in each assay. Each dot represents an analyzed cell. Colored and grey dots denote cells with detected and undetected activities, respectively. The percentages are the proportions of detected active cells in each assay

Fig. 5

a Group N specific uptake (sum of NO₃⁻, NH₄⁺, and urea specific uptake, h⁻¹) of photosynthetic picoeukaryotes (PPE), Prochlorococcus (pro), and Synechococcus (syn) in each assay. b Contribution of the different N sources to group N uptake (%) in each assay. c Contribution of the different N sources averaged over all assays for each population investigated (%)

Fig. 6

Box-and-whisker plot of the metabolic heterogeneity for C fixation and NH₄⁺, NO₃⁻, and N-urea uptake for photosynthetic pico-eukaryote (PPE), Prochlorococcus, and Synechococcus groups. Each dot represents an assay. Only daylight assays were considered for the C metabolic heterogeneity. For each group, metabolic heterogeneity medians that are not statistically different are indicated by the same letters (unpaired Kruskal-Wallis test, p > 0.05)

Fig. 7

Contribution to total community C fixation as a function of the contribution of NO₃⁻ to the group N specific uptake for each group in each daylight assay. The size of the dots represents the group N specific uptake (sum of NO₃⁻, NH₄⁺, and urea specific uptakes)