Recently photoassimilated carbon and fungus-delivered nitrogen are spatially correlated in the ectomycorrhizal tissue of Fagus sylvatica

Abstract

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided ¹⁵ N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a ¹³ CO₂ atmosphere. We analysed the short-term distribution of ¹³ C and ¹⁵ N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more ¹³ C to root parts that received more ¹⁵ N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of ¹³ C and ¹⁵ N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Keywords: Fagus sylvatica (beech); NanoSIMS; carbon; ectomycorrhiza; nitrogen (N); recent photosynthates; reciprocal rewards; resource exchange.

Fig. 1

Experimental design of split‐root boxes. Shown is an illustration of the split‐root box setup (a) and a photograph of one of the experimental trees growing in such a box shortly before harvest (b). Plants were grown for c. 1 yr in split‐root boxes (155 × 135 × 125 mm) with their root systems divided between two separated compartments, each filled with soil (‘soil compartments’, each 121 × 60 × 125 mm inside). The plant stems were stabilized using a cylinder filled with quartz sand. Each soil compartment was connected to a litter compartment (17 × 60 × 125 mm inside) by a double‐layered 35 µm nylon mesh, which allowed fungal hyphae, but not roots, to grow through. A solid plastic grid was placed in between the mesh layers, which creates an air gap to prevent the exchange of water and solutes between the soil and litter compartments. The two litter compartments were separated by a solid wall from each other, and were filled with beech leaf litter to foster hyphal growth from both root system halves into their respective litter compartments. After 1 yr of growth, we added a ¹⁵N labelled ammonium chloride (NH₄) and amino acid (AA) solution to only one of the two litter compartments to provide an additional N source, accessible to one half of the plant's root system via its associated mycorrhiza. Within 24 h of the N addition we exposed the plant's canopies to ¹³C‐labelled CO₂ using a gas‐tight acrylic glass incubation chamber.

Fig. 2

¹³C and ¹⁵N enrichments (at% excess, APE) of root system halves (a, b), individual fine root segments (c) and mycorrhizal root tips (d) from six beech trees grown in split‐root boxes. Mycorrhizal fungi associated with one half of the root system had access to an additional source of ¹⁵N‐labelled nitrogen (dark blue bars, closed circles) whereas mycorrhizas associated with the other half of the root system had no access to the labelled nitrogen source (light blue bars, open circles), but could have received it through internal redistribution within the plant. Trees were exposed to ¹³C‐labelled CO₂ 24 h after ¹⁵N addition and 24 h before harvest. (a, b) Bars show the mean ¹³C and ¹⁵N enrichment of the root system halves (calculated as the weighted mean of all measured segments of each root system half) of six plant replicates (error bar, ± SE, n = 6). (c) The lines depict linear regressions of carbon and nitrogen isotope enrichment in individual root segments (dark blue line: N‐amended side, P = 0.014, R ² = 0.1416, n = 35; light blue line: N‐free side, P < 0.0001, R ² = 0.39, n = 32). (d) ¹³C and ¹⁵N enrichments of individual root tips of N‐amended and unamended sides. Root tip data in (d) are represented on logarithmic scales. No significant correlation was found for this relationship. The specific root tip analysed using nanoscale secondary ion mass spectrometry (NanoSIMS) is marked in red (N).

Fig. 3

(a) Light microscopy image of a cross‐section of an ectomycorrhizal root tip of beech (Fagus sylvatica) associated with fungi from the genus Thelephora, stained with toluidine blue. Squares refer to the areas shown in Fig. 4. (b) Total CN⁻ secondary ion signal intensity distribution image, visualizing the cellular structure of a consecutive section analysed using NanoSIMS. E, endodermis; HE, extended hyphae; HM, mantle hyphae; HN, Hartig net; PC, plant cortex; VT, vascular tissue. Labels ‘a’, ‘b’ and ‘c’ indicate external hyphae selected for linescan analysis (Fig. 8). Bars, 50 µm.

Fig. 4

Nanoscale secondary ion mass spectrometry (NanoSIMS) images from three selected areas (indicated in Fig. 3) of the root tip cross‐section (shown as an assembled mosaic image in Figs 5, 6). Isotopic label content is displayed as at%¹³C and at%¹⁵N. Colour scales range from the natural isotope abundance (determined on an unlabelled control) to 3 at%¹³C and 20 at%¹⁵N. Image series from left to right: Total CN⁻ signal intensity distribution, visualizing the cellular structure (a, d, g); overlay of the CN⁻ and at%¹³C distribution images (b, e, h), overlay of the CN⁻ and at%¹⁵N images (c, f, i). Colours depicting the natural abundances of ¹³C and ¹⁵N were omitted in the overlay images to allow for a better visualisation of the structural information (NanoSIMS images displaying solely at%¹³C and at%¹⁵N information are provided in Supporting Information Figs S6, S7, with colour‐blind friendly versions in Figs S8, S9). E, endodermis; HE, extended hyphae; HM, mantle hyphae; HN, Hartig net; PC, plant cortex; VT, vascular tissue. Red arrows indicate (a) the septate hyphae structure and (e) a punctual ¹³C enrichment in Hartig net hyphae. Bars, 10 µm.

Fig. 5

Nanoscale secondary ion mass spectrometry (NanoSIMS) visualization of the spatial distribution of ¹³C enrichment within an ectomycorrhizal root tip of beech (Fagus sylvatica) associated with fungi from the genus Thelephora 24 h after the plant has been exposed to a ¹³C‐CO₂ atmosphere and 48 h after the fungi accessed a ¹⁵N‐labelled N source. Shown is an overlay of the total CN⁻ secondary ion signal intensity distribution image (Supporting Information Fig. S10) and the corresponding ¹³C label distribution image (Fig. S6) acquired on a cross‐section of the sampled root tip. The picture consists of 16 individual images (each 50 × 50 µm), assembled as a mosaic. The isotopic label content is presented as at%¹³C, displayed on a false colour scale ranging from the natural abundance value (dark blue, determined on an unlabelled control) to 3 at%¹³C (red). For a better visualization of areas enriched in ¹³C, colouring representing the natural abundance of the isotope is omitted in the overlay image (NanoSIMS images displaying solely at%¹³C information are provided in Fig. S6, with a colour‐blind friendly version in Fig. S8). White arrows indicate external hyphae exhibiting low ¹³C and ¹⁵N enrichment, which typically show ¹⁵N in the centre of the hyphal cross‐section (decoupled from ¹³C enrichment), whereas red arrows indicate those with overall high ¹³C and ¹⁵N enrichment, where ¹³C and ¹⁵N are typically co‐localized in the outer ring of a hyphal cross‐section (cf. Fig. 8). Bar, 50 µm.

Fig. 6

Nanoscale secondary ion mass spectrometry (NanoSIMS) visualization of the spatial distribution of ¹⁵N enrichment within an ectomycorrhizal root tip of beech (Fagus sylvatica) associated with fungi from the genus Thelephora 24 h after the plant has been exposed to a ¹³C‐CO₂ atmosphere and 48 h after the fungi accessed a ¹⁵N‐labelled N source. Shown is an overlay of the total CN⁻ signal intensity distribution image (Supporting Information Fig. S8) and the corresponding ¹⁵N label distribution image (Fig. S7), acquired on a cross‐section of the sampled root tip. The picture consists of 16 individual images, assembled as a mosaic (each 50 × 50 µm). The isotopic label content is presented in terms of at%, displayed on a false‐colour scale ranging from the natural abundance value (dark blue, determined on an unlabelled control) to 20 at%¹⁵N (red). For a better visualization of areas enriched in ¹⁵N, colouring representing the natural abundance of the isotope is omitted in the overlay image (NanoSIMS images displaying solely at%¹⁵N information are provided in Fig. S7, with a colour‐blind friendly version in Fig. S9). White arrows indicate external hyphae exhibiting low ¹³C and ¹⁵N enrichment, which typically show ¹⁵N in the centre of the hyphal cross‐section (decoupled from ¹³C enrichment), whereas red arrows indicate those with overall high ¹³C and ¹⁵N enrichment, where ¹³C and ¹⁵N are typically co‐localized in the outer ring of a hyphal cross‐section (cf. Fig. 8). Bar, 50 µm.

Fig. 7

Spatial correlation of relative ¹³C and ¹⁵N enrichment in plant (green) and fungal (purple) tissues. Each data point represents ¹³C and ¹⁵N enrichment (in at% excess, APE) of one region of interest (ROI) obtained from NanoSIMS images (Supporting Information Figs S6, S7) of a complete cross‐section of an ectomycorrhizal root tip of beech (Fagus sylvatica). Regions of interest were grouped according to tissue type (fungus: external hyphae, hyphal mantle, Hartig net; plant: plant cortex, endodermis, vascular tissue). For each tissue type ROIs were further categorized into lumen (circles) and cell walls (triangles). (a, b) Overview correlations combining lumen and cell wall ROIs of plant cortex and external hyphae, respectively. Lines represent the fit of a logarithmic regression model (fungi: R ² = 0.51, P < 2.2e^{− 16}; plant: R ² = 0.75, P < 2.2e^{− 16}). (c) Segmented linear regression analyses for each tissue type. Significant correlations are depicted as solid lines; nonsignificant correlations depicted as dashed lines. Breakpoints of regressions (in c) are located at the kink of each line. Coefficients of determination (R ²), significance values (P) and slopes of correlations depicted in (c) are provided in Table 2. The axis scales are optimized for the data ranges of each correlation; correlations with fixed axis scales for better comparisons among tissue types are provided in Fig. S5.

Fig. 8

¹³C and ¹⁵N distribution pattern in individual hyphae emanating from an ectomycorrhizal root tip of beech (Fagus sylvatica) associated with fungi from the genus Thelephora 24 h after the plant has been exposed to a ¹³C‐CO₂ atmosphere and 48 h after the fungi accessed a ¹⁵N‐labelled N source. Selected images (a–c) showing representative examples of external hyphae at high, medium and low isotopic enrichments. (d) Breakpoint linear regression analysis showing ¹³C and ¹⁵N enrichment (at% excess, APE) within regions of interest (ROIs) of all external hyphae in the cross‐section of the root tip; the open triangles and the dashed line represent the cell walls; the closed circles and the solid line represent the lumen; (d) also shows the positions of both the cell walls (CW) and lumen (L) of the selected hyphae in the regression analyses: a, relatively low ¹⁵N and almost no ¹³C enrichment, below breakpoint (BP); b, medium enrichment, around the BP; and c, high enrichment, above the BP. The line plots on the right side of each image series show isotopic enrichment profiles across each of the three selected hyphae (solid red line: at%¹³C; dashed blue line: at%¹⁵N) obtained from line‐scan analysis at the positions indicated by the white lines in panels (a)‐I, (b)‐I and (c)‐I. The colour scales at the bottom of the ¹³C and ¹⁵N NanoSIMS images range from blue (natural abundance) to red (high isotopic enrichment). Bars: (a–c) 1 µm.