ALBERT

Beschreibung: Dose-response experiments for yeast growth or settlement were conducted in 96-well plates impregnated with eelgrass extracts Concentrations of leaf surface extracts were related to naturally occurring concentrations on Z. marina surfaces assuming a leaf area / fresh-weight ratio of 78.99 cm2*g-1. Correspondingly, a 1-fold natural concentration of surface extract was tested when extract obtained from 0.955 cm² leaf surface was impregnated onto 0.955 cm² well surface. Concentrations of tissue extracts were dosed based on the assumption of a dry-weight / fresh-weight proportion of 10%. Correspondingly, a 1-fold natural concentration of tissue extract was tested when extract obtained from 10 mg dry weight was present in the final volume of 100 µl. The yeasts were maintained at 25°C on a shaker in liquid medium containing: 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of glucose, and sea salt 3%. For settlement assays aliquots of yeast liquid cultures were pipetted into the wells. After 2 h, the wells were emptied and cells attached to the walls were stained with Calcofluor white for 10 min. The unattached cells and the excess dye were removed by rinsing with sterile seawater, and the fluorescence of stained cells attached to the wells was measured at 350 nm excitation and 430 nm emission. For growth bioassays, 100 µl medium containing yeast cells at 0.07-0.17 initial OD610 were pipetted into the wells. Plates were incubated at 25°C on a shaker in darkness and OD610 was repeatedly measured over 20 h. Exponential growth curves were fitted with the GraphPad Prism 5.0 software package to OD data time series obtained for each well allowing determination of division rates. Division rates obtained for single wells with addition of compounds were then related to mean division rates obtained for six control wells without such addition.

Beschreibung: 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.