Secrets of seagrass success

A new clue to the exceptional productivity of Mediterranean seagrass meadows comes from discovery of an intimate symbiosis with nitrogen-fixing bacteria.

The importance of seagrasses. Different species of seagrass are found growing on sediments in shallow coastal and estuarine waters throughout the world. These flowering plants can form dense meadows which have a special ecological role as “ecosystem engineers”, due to their high production through photosynthesis, which provides nursery areas for diverse species of fish, habitats for invertebrates, and a direct food source for turtles, dugongs, manatees, seabirds, and other animals. They also trap sediment and prevent coastal erosion.  The importance of urgent conservation and restoration of seagrass beds for biodiversity and as “blue carbon” sinks — they may sequester CO2 at up to 35 times faster than rainforests — has been highlighted by the Marine Conservation Society, and others at the recent COP26 meeting. 

Posidonia oceanica meadow, showing ecosystem engineering roles and other important ecosystem impacts. Adapted from original image by Dimitris Poursanidis, via Flickr, CC BY-NC-SA 2.0

Symbiotic interactions with rhizosphere bacteria and clams.  Like all plants, seagrasses require a source of fixed nitrogen for the formation of cell materials and growth. Nitrogen fixation (diazotrophy) is a strictly anaerobic and highly energy-dependent process by which nitrogen (N2) is reduced to ammonium (NH4+) which can then be assimilated into cellular material.  In sediments, diazotrophy is mainly carried out by certain groups of heterotrophic bacteria and methanogenic archaea. In seagrass sediments, a very important process of N2 fixation is carried out by sulfate-reducing bacteria during the breakdown of organic material. However, sulfate reduction generates hydrogen sulfide, which is highly toxic to the seagrass plants and other organisms in the sediment. One solution to this problem has been identified in Caribbean seagrass (Thalassia testudinium) beds that contain high numbers of lucinid clams (Codakia orbicularis) whose gills harbour endosymbiotic thioautotrophic bacteria (König et al., 2015). These bacteria oxidize sulfides to sulfate, thus reducing the toxicity of the sediment, while seagrass roots provide oxygen and organic matter, which stimulates growth of the clams and their symbionts. Thus, a three-stage symbiosis forms the foundation of this ecosystem (van der Heide et al., 2012). Unexpectedly, it was found that these clam endosymbionts also fix nitrogen (König et al., 2016), a finding confirmed in studies with another clam species, Loripes lucinales (Petersen et al., 2017). 

A Mediterranean seagrass contains diazotrophic bacteria within its tissues.  The presence of nitrogen-fixing bacteria within the cells of certain plants has been known for decades. In particular, the intimate and highly evolved association between rhizobia and leguminous plants like peas, beans and alfalfa has been extensively studied and manipulated to improve the growth of crops. Close associations between the seagrass rhizosphere and diazotrophic bacteria have been reported for many years but there has been no evidence that the bacteria can live within the plant tissue, as occurs in terrestrial plants. Now, researchers at the Max-Planck Institute for Marine Microbiology, Bremen have described a new bacterial species that lives inside the root tissue of the Mediterranean seagrass Posidonia oceanica (Mohr et al., 2021). Samples of seagrass plants taken from dense meadows off the isle of Elba, Italy were studied in laboratory aquaria using sensitive analytical techniques developed at the Institute, Primary production was measured using microsensors that detect flux of COand O2, and mass spectrometry was used to measure nitrogen fixation by tracer incorporation when incubated in water enriched by bubbling with 15Ngas. Carefully designed manipulations of the incubation setup enabled assessment of transfer of fixed 15N to different parts of the plant. The roots of plants sampled in the summer (when seawater levels of inorganic nitrogen levels are extremely low) showed high levels of 15N2 incorporation accompanied by rapid transfer of 15N labelled compounds to the leaves. Mohr and colleagues calculated that the rates of nitrogen fixation they observed was sufficient to fully explain the net production of the seagrass meadow during the summer, as well as transfer of nitrogenous compounds to the surrounding sediments. 

Identification of the seagrass symbiont. Sediment and root samples from seagrasses showing different levels of nitrogen fixation (due to the season of sampling) were subjected to microbial community analysis, based on amplification of 16S ribosomal RNA genes and metagenomic analysis. This revealed significant differences in the composition of the root microbiome. The key difference is due to the presence of a bacterium belonging to the Gammaproteobacteria. Gene analysis showed that this is closely related to a nitrogen-fixing bacterium called Celerinatantimonas diazotrophica that had previously been isolated from saltmarsh grasses. The authors concluded that the seagrass endosymbiont is a new species, which they have named Candidatus Celerinatantimonas neptuna (a provisional species name to reflect the fact that the bacterium has not yet been cultured or fully characterized). Using fluorescent in situ hybridization (FISH) probes, the Ca. C. neptuna bacteria were shown to be present inside the cells and intercellular spaces of seagrass roots, with very high density occurring in the summer, correlating with high nitrogen fixation rates. The researchers then used nanoSIMS — a specialized mass spectrometry technique pioneered in the MPI lab — that enables the presence of isotopic tracers to be visualized within individual cells. This showed that the bacteria fix nitrogen into organic compounds, which are then transferred to the surrounding plant cells. 

Schematic of the symbiotic interaction between Ca. C. neptuna (magenta) and P. oceanica indicating the transfer of fixed N from N2 fixation (dark red arrows) and plant-derived sugars (black arrows); the potential for further plant growth promotion and defence mechanisms is also indicated. ROS, reactive oxygen species. From Mohr et al. (2021) CC BY 4.0.

Metagenomic analysis reveals the metabolic capabilities of the symbiont. Mohr et al. (2021) then used advanced gene sequencing techniques to obtain the genome and transcriptome of Ca. C. neptuna from nitrogen-fixing plants. All the nif genes encoding subunits of the nitrogenase enzyme system that reduces N2to NH4+ were identified in the genome and shown to be highly transcribed under nitrogen-fixing conditions. Analysis also showed that the two-way exchange of metabolites (amino acids and sugars) between the symbiont and host seems analogous to that observed in rhizobia-legume interactions. 

Evolutionary implications. Seagrasses are thought to have evolved from terrestrial flowering plants (angiosperms) about 100 million years ago, with adaptations to life in the sea evidenced by genomic analysis (Olsen et al. 2019)Mohr et al. (2021) suggest that adaptations of the microbiome likely occurred in a coastal environment, given the close relationship of the Poseidon seagrass symbiont with the Celerinatantimonas species found in saltmarsh grasses. There are over 70 known speciesin four different families, with distinctive structures and growth forms. It will be interesting to determine the role of symbiotic nitrogen fixation in other species, and its influence on their ecological roles.

References

König, S., Gros, O., Heiden, S. et al. (2017). Nitrogen fixation in a chemoautotrophic lucinid symbiosis. Nat Microbiol 2, 16193. https://doi.org/10.1038/nmicrobiol.2016.193

König, S., Gros, O., Heiden, S. et al. (2017) Nitrogen fixation in a chemoautotrophic lucinid symbiosis. Nature Microbiology 2, 16193. https://doi.org/10.1038/nmicrobiol.2016.193

Mohr, W., Lehnen, N., Ahmerkamp, S., Marchant, H. K., Graf, J. S., Tschitschko, B., … & Kuypers, M. M. (2021). Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium. Nature https://doi.org/10.1038/s41586-021-04063-4

Petersen, J. M., Kemper, A., Gruber-Vodicka, H., Cardini, U., Van Der Geest, M., Kleiner, M., … & Weber, M. (2017). Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation. Nature Microbiology 2, 16195. https://doi.org/10.1038/nmicrobiol.2016.195.

Van der Heide, T., Govers, L. L., De Fouw, J., Olff, H., Van der Geest, M., Van Katwijk, M. M., et al. (2012). A three-stage symbiosis forms the foundation of seagrass ecosystems. Science 336, 1353–1472.

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