Introduction

Seagrass meadows are extremely productive aquatic habitats that provide important ecological functions, such as refuge and nutrient sources for a great variety of marine organisms [1, 2]. Nitrogen (N) is an essential element for maintaining seagrass growth and productivity [3, 4], with foliar uptake of N often contributing more of the plant total N requirement compared with roots (up to 74% [5]). Although seagrass leaves preferentially uptake inorganic nitrogen (DIN [6]), paradoxically, seagrasses flourish in coastal environments characterised by scarce inorganic nutrients, but high concentrations of dissolved organic nitrogen (DON [7, 8]).

Within the marine environment, microorganisms on the surface of plants and animals can be highly abundant [9], forming functional partnerships that can play a critical role in nutrient acquisition from oligotrophic waters [10]. Seagrass leaves provide a physical substratum for a rich epiphytic community of autotrophic and heterotrophic microorganisms [11, 12]. Surprisingly, while the contribution of the autotrophic microbiota (i.e. nitrogen-fixing bacteria) to enhance seagrass N availability has been extensively reported [13], the significance of heterotrophic microorganisms on seagrass leaves in facilitating the plant’s ability to uptake N from DON has been largely overlooked.

Materials and methods

Here, we tested whether seagrass microbiota, through the conversion of amino acids (aa) into bioavailable DIN, mediate the assimilation of N by seagrass leaves. We exposed seagrass (Posidonia sinuosa) leaves, with and without microorganisms, to 15N-enriched aa (50 µM of 98% 15N-aa; NLM-2161 Cambridge Isotope Laboratories) and 14N-aa (controls; 50 µM of 98% 14N-aa; ULM-2314 Cambridge Isotope Laboratories) over 12 h. Epiphytes and microorganisms were removed through scraping visible leaf epiphytes using a razor blade and incubation with antibiotics, followed by examination of potential damage by transmission electron microscopy and pulse amplitude modulated (PAM) fluorometry (see Supplementary Methods including Figs. S1, S2 & S3). 15N accumulation into seagrass leaves (n = 3 per treatment/control) was first measured by isotope ratio mass spectrometry (IRMS), after which a subset of samples was randomly selected and analysed by high-resolution mass spectrometry (NanoSIMS) to show 15N accumulation in microorganisms on the leaf surface and discrete sub-cellular components (cell wall, cytosol, vacuole and chloroplast) of seagrass leaves (see Supplementary Methods for details of techniques).

Results and discussion

Evidence that microorganisms associated with P. sinuosa leaves facilitate seagrass uptake of 15N derived from aa was provided by our results of bulk tissue analysis (IRMS) of seagrass leaves with and without intact microorganisms. At all times following incubation, 15N accumulation was greater in leaves with an associated microbiota compared to those where microorganisms had been removed, with levels 4.5 times higher in leaves with microorganisms by 12 h (p < 0.001; Supplementary Fig. S3). However, bulk isotope measures are unable to discern specific isotope tracer accumulation points or the source of slight 15N enrichment we detected in leaves removed of microorganisms. Consequently, high-resolution secondary ion mass spectrometry (NanoSIMS) was done on representative samples to trace the uptake of 15N derived from aa at the P. sinuosa leaf–microbial interface.

NanoSIMS analysis of seagrass leaves with intact microorganisms clearly distinguished the associated microorganisms from seagrass cells, with resolution sufficient to differentiate the sub-cellular components of the seagrass leaf (cell wall, vacuole, cytosol and chloroplast; Supplementary Fig. S5). After 0.5 h incubation in 15N-aa, analysis showed microorganisms on the surface of the seagrass leaf were highly enriched compared to the adjacent seagrass cells (Figs. 1 and 2). The microorganisms remained more 15N-enriched than adjacent seagrass cells at each time, and their enrichment was characterised by an exponential accumulation of 15N over time (Fig. 2). By 12 h, microorganisms were ~200 times more 15N-enriched than unlabelled reference samples, indicating the efficiency of microorganisms to utilise aa through the activity of intracellular and extracellular enzymes that break down aa to produce DIN [14].

Fig. 1
figure 1

15N enrichment images of seagrass (P. sinuosa) leaf cells after incubation in enriched 15N aa mixture with (a–d) or without (e–h) microorganisms on their leaves. Seagrass leaves were incubated for 0.5 h (a,e), 2 h (b,f), 6 h (c,g) and 12 h (d,h). Seagrass sub-cellular compartments are reported (CW cell wall, Ch chloroplast, Va vacuole, Cy cytosol), as well as microorganisms (M). 15N enrichment images generated from nanoSIMS collected 12C14N− and 12C15N− ion data and expressed as hue saturation intensity, where blue represents low isotopic abundance of nitrogen (δ15N‰ = 0; close to natural abundance) and enrichment is shown as a shift towards magenta (colour scale label in δ15N‰). The images show a 15N enrichment of seagrass cell wall at 0.5 h (a) and the appearance of enriched substrate within the cytosol and vacuole (b,c) and chloroplasts (d) over time. Where microorganisms are absent (e–h) a clear enrichment is absent. Scale bar represents 5 µm

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Fig. 2
figure 2

Box–Whisker plots (median, interquartile range = box, extremes = whiskers) of 15N enrichment of identifiable microorganisms and sub-cellular seagrass components (cell wall, cytosol, vacuole and chloroplast) from nanoSIMS analysis of seagrass leaves incubated with (left) or without (right) associated microorganisms for each incubation time, up to 12 h. Reference values (reference), representing δ15N natural abundance for microorganisms and seagrass sub-cellular compartments obtained from incubation with non-enriched 14N aa, are displayed for each graph. Note the difference in y-axis scales between microorganisms and plant sub-cellular components

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The presence of microorganisms, and their ability to mineralise aa, clearly influenced the accumulation of 15N into different components of the seagrass tissue over the experiment timescale (Figs. 1 and 2). Within 0.5 h of incubation, 15N was 26 times higher in the outer cell wall of epidermal cells of seagrass leaves with microorganisms present compared to those with microorganisms removed. While the mechanisms of absorption of solutes through the cuticle are not fully characterised [15], the enhanced and subsequent decreased enrichment of 15N in the cell wall (Figs. 1a–c and 2) suggests that: (i) the movement of N across the seagrass cuticle is a rapid process [16]; (ii) the added aa source was quickly exhausted; or less likely; and (iii) the plant is able to limit N uptake once it has reached substrate saturation [6]. Slight 15N accumulation was first observed in the cytosol and vacuole after 0.5 h, followed by an exponential accumulation over time (Fig. 1d). The chloroplasts that proliferate at the outer edge of the seagrass epidermal cells accumulated 15N more slowly than other seagrass sub-components, and still showed increasing enrichment at the final 12 h sampling time. Since microbial catabolism of aa results in exuded ammonium (NH4+ [14]) and NH4+ is the preferred DIN source for foliar seagrass acquisition, the microorganisms associated with seagrass above-ground tissue appear to provide a mechanism for the seagrass leaves to access N derived from aa. While NH4+is harmful to plant tissue (at concentrations as low as 25 µM, ambient concentration around 15 µM [17]), our data show that it quickly moves to chloroplasts where it is assimilated into organic compounds, some of which are released back into the cytosol [6].

The role of microorganisms in mediating the uptake of N by seagrass leaves is further supported by isolated uptake of 15N by seagrass cells that were adjacent to small ‘patches’ of microorganisms missed in the removal process. NanoSIMS analysis, and corresponding live/dead cell counts, revealed a few examples where microorganisms were not entirely removed (<1% leaf surface; Supplementary Figs. S1 & S6). In these specific areas, the few remaining microorganisms were 20 times more 15N-enriched than those of unlabelled reference samples, and only seagrass cells directly adjacent to those microorganisms exhibited 15N enrichment (Supplementary Fig. S4). Overall, our results cast doubt over the ability of seagrasses to utilise DON directly as a source of essential N. Previous studies [18, 19] that postulated seagrass ability to directly use DON did not completely remove microorganisms from seagrass tissues prior to incubation with 15N, and our findings suggest that even small numbers of microorganisms on seagrass leaves influence the uptake of N derived from aa.

Conclusions

In this study, we show that the association between microorganisms and the leaves of P. sinuosa can provide an alternative source of N for uptake by seagrass from the abundant organic nitrogen pool. By mineralising amino acids, epiphytic microorganisms on P. sinuosa leaves link the nitrogen elemental cycle in seagrass meadows via heterotrophic metabolism and are likely to contribute significantly to the high productivity of seagrass meadows. Thus, the maintenance of a ‘stable’ partnership between seagrass leaves and its microbiota, indirectly enables the provision of the pivotal ecosystem services provided by these important, globally distributed, benthic habitats [20].