Surface chemical defence of the eelgrass Zostera marina against microbial foulers

Abstract

Plants rely on both mechanical and chemical defence mechanisms to protect their surfaces against microorganisms. The recently completed genome of the eelgrass Zostera marina, a marine angiosperm with fundamental importance for coastal ecosystems, showed that its re-adaptation from land to the sea has led to the loss of essential genes (for chemical communication and defence) and structural features (stomata and thick cuticle) that are typical of terrestrial plants. This study was designed to understand the molecular nature of surface protection and fouling-control strategy of eelgrass against marine epiphytic yeasts. Different surface extraction methods and comparative metabolomics by tandem mass spectrometry (LC-MS/MS) were used for targeted and untargeted identification of the metabolite profiles of the leaf surface and the whole tissue extracts. Desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) coupled with traditional bioassays revealed, for the first time, the unique spatial distribution of the eelgrass surface-associated phenolics and fatty acids, as well as their differential bioactivity against the growth and settlement of epiphytic yeasts. This study provides insights into the complex chemical defence system of the eelgrass leaf surface. It suggests that surface-associated metabolites modulate biotic interactions and provide chemical defence and structural protection to eelgrass in its marine environment.

Figure 1

Leaf tissue and surface-associated chemistry of Z. marina involved in antifouling defence. (A) Eelgrass meadow in the Baltic Sea, Kiel Fjord. Scale bar = approx. 30 cm. Photo by Stefano Papazian. (B) Cumulative regression model (Sum, purple) comparing the inhibitory antifouling activity of eelgrass extracts obtained by surface dipping extraction (S, red), whole leaf (blue, W), and whole leaf after surface dipping (light-blue, W-S), on the settlement of the marine epiphytic yeast, D. hansenii. (C) Multivariate analysis (PCA 3 components) for comparative LC-MS/MS metabolomics of surface solid-phase (C18), solvent dipping (S), whole leaf (W) and surface-free (W-S) extracts. (D,E) Concentrations of phenolic metabolites detected in the (D) surface extracts (S, C18) and (E) whole leaf extracts (W, W-S), i.e. p-coumaric acid (p-Co), apigenin (A), luteolin (L), apigenin-7-sulfate (AS), luteolin-7-sulfate (LS), caffeic acid (CA), ferulic acid (FeA), rosmarinic acid (RA), zosteric acid (ZA), diosmetin (D), and diosmetin-7-sulfate (DS). Error bars = standard error. (F) Molecular network built in GNPS from spectral data obtained from LC-MS/MS analyses of all extracts. Four main clusters show the dereplication and respective chemical groups annotated as phenolics, phospholipids, and fatty acid esters (Supplementary Table S2).

Figure 2

Spatial distribution of the eelgrass surface metabolites by DESI-IMS. Photograph of eelgrass Z. marina leaf-blade, scale bar = 3.5 mm (A), and DESI-IMS images at 150-μm lateral resolution showing distribution and relative intensity of m/z [M-H]⁻ ions for the metabolites (B) diosmetin, (C) diosmetin-7-sulfate, (D) zosteric acid, (E) rosmarinic acid, (F) apigenin-7-sulfate, (G) palmitic acid, and (H) azelaic acid. Heat-map scaling shows the highest local accumulation points indicated by the respective maximum range on the intensity scale (a.u.). Total scanned surface area = 312 mm² (3.1 cm²). Actual scanned leaf surface = 125 mm² (1.25 cm²).

Figure 3

Specific localization of Z. marina surface metabolites. DESI-IMS showing the distribution on the eelgrass leaf of (A) palmitic acid, and (B) superimposition of myristic acid (red) and diosmetin-7-sulfate (green). (C) Analysis in OpenMSI (https://openmsi.nersc.gov) showing metabolite distribution via RGB-color superimposition of palmitic acid (red), azelaic acid (green), and rosmarinic acid (blue), and respective intensity of m/z ions in the DESI-IMS spectra at the apex (1) and at the margin (2). Images are scaled to the average intensity for each ion (no cut-off). (D) The DESI-IMS spatial-chemical information across four regions of interest (ROI) of the leaf surface, i.e. apex, lower lamina, margin, and midvein (10-sample points), were modelled by chemometrics, with (E) PCA showing ROI cluster intra- and inter-cluster metabolic variation, and (F) respective spectral similarity in OpenMSI.

Figure 4

The major metabolites of Z. marina identified by LC-MS/MS and/or DESI-IMS. (A–H) Phenolic compounds, including the sulfated flavonoids (F-H). (I–K) Aliphatic carboxylic acids, including azelaic acid (K). (L–P) Fatty acids. (Q) Trehalose.

Figure 5

Eelgrass chemical defence against microbial foulers. (A) DESI-IMS scanned leaf-blade showing superimposition of azelaic acid, palmitic acid, and DS, representing three major classes of leaf surface-associated metabolites: FA peroxides (yellow), FAs (red), and phenolic compounds (blue). (B) Model suggesting differential functions of Z. marina surface metabolites. Surface metabolites may collectively confer physical and chemical defence, providing antifouling activity, antimicrobial activity and a protective lipid ‘tunic’ layer. (C) Visual summary of the defence metabolites found in this study for Z. marina, showing concentrations of flavonoids, phenolic acids, and fatty acids in the whole leaf and on the leaf surface, reported as relative abundances (in blue, from low to high abundance as one to three squares). The DESI-IMS leaf surface distribution for each metabolite is reported as relative abundances (light to dark green) on the midvein (central square) or margin (outer squares). The antifouling bioactivity for each of the metabolites tested against growth and settlement of microbial foulers is reported relative to the percentage of inhibition (red) or activation (green) as measured in the bioassay (see Supplementary Tables S7–S9).