Sugars dominate the seagrass rhizosphere

Seagrasses are among the most efficient sinks of carbon dioxide on Earth. While carbon sequestration in terrestrial plants is linked to the microorganisms living in their soils, the interactions of seagrasses with their rhizospheres are poorly understood. Here, we show that the seagrass, Posidonia oceanica excretes sugars, mainly sucrose, into its rhizosphere. These sugars accumulate to µM concentrations—nearly 80 times higher than previously observed in marine environments. This finding is unexpected as sugars are readily consumed by microorganisms. Our experiments indicated that under low oxygen conditions, phenolic compounds from P. oceanica inhibited microbial consumption of sucrose. Analyses of the rhizosphere community revealed that many microbes had the genes for degrading sucrose but these were only expressed by a few taxa that also expressed genes for degrading phenolics. Given that we observed high sucrose concentrations underneath three other species of marine plants, we predict that the presence of plant-produced phenolics under low oxygen conditions allows the accumulation of labile molecules across aquatic rhizospheres.

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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Data
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Study description
Porewater profiles and sediment samples were collected to describe the microbial ecology of the seagrass rhizosphere. Due to the complexity of the associated datasets, please refer to Tables S1 and S2 for sample sizes and statistical approaches used in this study unless otherwise indicated below.

Research sample
Porewater metabolomics. Porewater samples were collected at multiple locations and multiple time points across a multi-year study exploring the metabolite composition underneath seagrass meadows. Depth profiles consisted of taking 2 mL of porewater every 5 cm from the sediment surface to -30 or -40 cm beneath the meadow. In Sant'Andrea Bay, Elba, Italy, porewater samples were collected underneath the meadow, 1 m and 20 m away from the meadow. Depending on sampling location and month sampled, 6-9 individual depth profiles were collected from each sampling site. Porewater samples from Galanzana Bay, Elba, Italy, were also collected across a 24 hour period using fixed lances to reduced variation based on sampling location: At each time point we collected between 6 and 10 samples, as some samples were lost due to clogging of the porewater lance at the time of sampling.
Dissolved organic carbon. 20 mL of porewater samples were also collected for dissolved organic carbon analysis from underneath, 1 m and 20 m away from a seagrass meadow in Sant'Andrea Bay, Elba, Italy (n=6). From each site, a subset of these samples (n=3) were also analyzed for dissolved organic matter composition.
Seagrass metabolomics. Replicate seagrass samples (n=6 per sampling time point) for metabolomic analysis were collected from Galanzana Bay, Elba, Italy over a 24 hour time period at the same time as the 24 hour samples collected for porewater metabolomics. Seagrass samples were used to measure bulk sucrose concentrations from the seagrass leaves, rhizomes and root tissues, as well as explore the distribution of sucrose within the seagrass root (n=8).
Sediment samples. Sediment samples were collected from Sant'Andrea Bay Elba, Italy for metagenomic and metatranscriptomic analysis. Samples were taken underneath the seagrass meadow, 1 m and 20 m away from the seagrass meadow (n=3 per site).
Metabolic activity experiments. Sediment cores were collected underneath and 1 m away from seagrass meadow from Sant'Andrea Bay, Elba, Italy (n=3). Sediments were incubated under oxic or anoxic conditions, and in the presence or absence of phenolic compounds. 13C-sucrose consumption was monitored over time. Sucrose respiration rates were calculated from these incubations, but not statistically compared.

Sampling strategy
Porewater samples were collected using a steel lance (1 m long, 2 μm inner diameter) outfitted with a wire mesh (63 μm) to prevent the intake of sediment and seagrass, porewater was slowly extracted from sediments into sterile syringes.
Individual seagrass plants were collected by hand and immediately immersed in liquid nitrogen to halt changes in metabolite composition during sampling.

March 2021
Sediment samples for metagenomic and metatranscriptomic analyses were collected using push cores. Directly after collection, cores were sectioned into 5 cm slices and frozen at -20 °C. A subsample of each sediment slice was also preserved in RNAlater (SigmaAldrich) for RNA extraction.
Dissolved organic carbon (DOC) and dissolved organic matter (DOM) samples were collected in parallel with porewater metabolomic samples from inside, at the edge and 20 m outside a P. oceanica seagrass meadow in October 2016. DOC/DOM samples were filtered through pre-combusted (500 °C, 4 h) Whatman GF/F filters (0.7 μm) into 20 mL acid-washed and pre-combusted scintillation vials. Samples were acidified to pH 2 using 25% hydrochloric acid and stored at 4 °C until analysis.
Samples for measuring the rate of sucrose consumption by seagrass sediments were frozen for metabolomic analysis at each sampling time point (0, 3, 6, 12 and 24 hours post 13C-sucrose introduction). Furthermore, samples for cavity ring-down spectroscopy to measure the production of 13CO2 were also collected at each time point by halting biological activity in 3 mL of each sample using 50 μl saturated HgCl2 solution. For each experimental condition 3 replicate incubation bottles were prepared from 3 different sampling cores inside and 1 m away from the seagrass meadow. .
For all samples collected, sample size was chosen according to sizes used in comparable studies.

Data collection
All metabolomic data was collected using either gas chromatography-mass spectrometry or mass spectrometry imaging using instruments at the

Data exclusions
No data were excluded from the analysis.

Reproducibility
All attempts to repeat the experiments were successful.

Randomization
All samples were randomly collected from seagrass habitats without a priori expectations that the sampling would influence the analysis.

Blinding
Blinding was not preformed because it was not relevant to this study. This study was an exploratory investigation into the microbial ecology of seagrass meadows, without a priori expectations that would influence the analysis.
Did the study involve field work?

Yes No
Field work, collection and transport Field conditions Field work was conducted across a wide variety of field sites and time points during this multi-year study.