Plants and microbes often have a symbiotic relationship, relying on each other for nutrients or shelter. Understanding and engineering such symbioses is an essential step in the journey towards tackling global challenges such as food security, carbon capture and ecosystem restoration. Now in The ISME Journal, researchers at the Okinawa Institute of Science and Technology (OIST) have reported in depth on the symbiotic relationships and microbial communities within Azolla ferns, bringing new insights to these important plants.
"Azolla are fascinating. They grow really fast, and they're everywhere, found on almost every continent. With their microbial symbionts, they are great at fixing nitrogen, so have been investigated as biofertilizers and as protein supplements for animal feeds. And interestingly, huge amounts of Azolla spores were found in fossilized samples from around the time of the last global cooling 50 million years ago, when a decrease in CO2 helped cool the atmosphere, so they're under investigation for carbon capture properties too," explained Professor David Armitage, head of the Integrative Community Ecology Unit at OIST and author on this study.
The researchers were interested in the microbial communities within Azolla leaf pockets; tiny hollow chambers within the leaves that hold the symbiotic bacteria. Which bacteria were present in which species of Azolla, what symbiotic relationships were present, and how did symbiotic bacteria differentially evolve compared to their free-roaming cousins? By answering these questions, they hoped to both bring new fundamental understanding to these interesting plants and to open new fields of study for future microbial ecologists and molecular engineers.
Separating the symbionts from microbial nomads
The first question the team set out to answer was exactly which microbes were present and symbiotic within the fern leaf pockets. Previous studies on wild Azolla across the globe had identified many different bacterial species prevalent in these leaf cavities, but here, the team confirmed that only the cyanobacteria Trichormus azollae were true symbionts of these plants.
By taking leaf pocket samples from a wide variety of Azolla species and reconstructing the genomes of the microbial residents, the researchers found that only T. azollae was present in all leaf pockets, indicating it as the only symbiotic bacteria to this plant. "Whilst we did see other bacteria present across some different samples, we believe these are transient visitors to these ferns," confirmed Prof. Armitage.
From here, the researchers decided to study T. azollae in more depth. Cyanobacteria are known to be symbiotic to a wide range of different plants, and adept at producing nitrogen. Therefore, they have potential applications in food security, as the introduction of symbiotic cyanobacteria could support the nutrient supply of essential crops.
The genetic impacts of symbiosis
By comparing the symbiotic T. azollae cyanobacteria to its free-living cyanobacterial relatives, the researchers aimed to identify the genomic impacts of symbiosis. Here, they found the symbiont's genomes in an extreme state of decay, possibly explaining the observation that it is no longer capable of surviving outside of the host plant. "There were more pseudogenes than functioning genes," highlighted Prof. Armitage. "30 to 50% of genes were lost in the symbiotic cyanobacteria compared to the free-roaming variants."
To understand the reasons behind this decay, one can think of natural selection, and the evolutionary pressure exerted on genes conferring important functions. If a mutation on a gene has beneficial or negative effects on its owner's survival, it may be selected for or removed through natural selection. These pseudogenes are thought to arise when selective pressures on the gene have weakened, if, for example, the gene's function is no longer necessary. Mutations accumulate, eventually causing the genes to stop functioning, and become so-called 'pseudogenes'. Over much longer evolutionary time periods, such pseudogenes may potentially be removed, resulting in overall genome reduction.
The team were able to pinpoint which genes associated with particular functions were differentially evolving between the symbionts and their relatives. Genes relating to adhesion, intracellular trafficking, secretion, and vesicular transport had higher predicted expression and abundance in the symbiotic bacteria. These may help the cyanobacteria stick within the leaf pockets and support nitrogen fixation, bringing benefits to the host. Conversely, genes related to defense mechanisms, stress responses, replication and repair appeared to experience relaxed selection or had been degraded to pseudogenes, indicating the stress-free environment of these cavity-dwelling symbionts.
From fundamental insights to food security
Through these results, the researchers hope that the wider scientific community may be able to harness genomic insights to target the genes involved in symbiosis. Prof. Armitage concluded, "Our aim is that this work can act as a blueprint, guiding scientists on how to encourage such symbioses to tackle worldwide concerns like food security, by engineering nitrogen-fixing crops. Plant-microbe symbioses have so many potential impacts if we can just uncover how and why they function, and the example of Azolla is among the most extreme and tight-knit between partners."
Header image by Erika Fukuhara.
