Nitrogen fixation is a crucial part of plant growth and agriculture. Because the element nitrogen is a critical ingredient for proper development of plants, they need a steady supply of it in order to grow healthy and have a high yield of offspring seeds. For most plants, this is done by relying on nitrogen already existing in the soil that is kept in a state of constant flux by the microbial breakdown of deceased organisms. This is formally known as the nitrogen cycle, as many likely learned in high school biology.

The Creation of Nitrogen

But, for a select few groups of plants, they have been able to shake off this reliance on nutrient-rich soil, at least when it comes to nitrogen requirements. These families include several crops species like beans, peas, and lentils found in the legume family Fabaceae. Farmers often choose to use crop rotation with these crops so that they can help enrich the soil with nitrogen again, as the prior season’s crops aren’t left to decay and so the stolen nitrogen wouldn’t naturally be replaced otherwise.

For climates and soils where nitrogen fixing plants aren’t feasible, farmers rely on artificial nitrogen that they supply to the soil before and during planting seasons. This created organic nitrogen is made using the Haber–Bosch process, which revolutionized agriculture around the world after its development in 1910. Now, plants could be given nitrogen for growth no matter where you are and even if you don’t have a ready supply of nitrogen-fixing crops to rotate with.

That process can be intensive though and the growing demand for nitrogen is becoming such that the supply is costly to produce. So a return to using naturally nitrogen-fixing crops or perhaps expanding the number of crops capable of conducting that system would be preferable. To do that, more has to be understood and known about the plant fixation process. Let’s go over the basics first.

A Bacterial Partnership

The method of nitrogen-fixation is conducted thanks to a symbiotic relationship some plants have with bacteria. Specifically the Rhizobium genus and the Frankia genus of bacteria for the legume family. These diverse filamentous bacteria are given housing in specialized plant cells held within nodules in the roots of the plants. Through this, the bacteria are awarded plant-specific nutricious compounds produced via photosynthesis, like sugars, while they convert nitrogen in the atmosphere into organic nitrogen in the form of ammonia (N2 to N3).

In a massive collaboration between universities around the world from Europe to the US to China, Canada, and Argentina, a group of scientists looked into finding out more about the species that had successfully evolved this capability and possibly to find out why it hadn’t spread farther into the plant kingdom. What they found was perplexing, to say the least.

It was thanks to the reorganization of the plant family tree in 1995 that things began to become clearer in nature, but didn’t reveal any direct answers on why they were that way. The phylogenetic reordering showed that the plants capable of nitrogen-fixation were restricted to four orders: Fabales, Fagales, Cucurbitales, and Rosales, together forming what is called the nitrogen-fixing root nodule (NFN) clade of plants.

However, within these orders, only 10 out of the 28 families had any plants capable of NFN synthesis. They were also not spread evenly throughout the evolutionary tree. Even more confusing, they did not fit within a monophyletic group, that is a group capable of tracing their combined lineage back to a common ancestor. Their commonality was back at the beginning of the root of the NFN clade, but then why weren’t these plants also found in the other families?

A secondary confusion is also why and how the split emerged between the two bacterial genera Rhizobium and Frankia. The former is found with the legumes (Fabales) and the Rosales, while the latter had adherents in Rosales and in the Cucurbitales and Fagales. Why is this distribution both combined in the one order and seemingly random for the split in the rest? The simplest explanation the researchers could initially put forward is that some sort of genetic event in the ancestor of the NFN clade as a whole predisposed some of its descendents one way and the rest another. Thus allowing independent evolution of the capability with no connection to the other families multiple times. This is known as the multiple origin hypothesis.

Testing for Answers

But that was just a hypothesis without any data behind it, even if it was based on the most logical answer. The scientists wanted to provide genetic and biological confirmation of this hypothesis that may also shed light on the aforementioned questions. Their tested samples were restricted to only those with available genomes and that were within the four orders that are known to produce nodules and not the outlying species. Since the limited number of already sequenced genomes would introduce a sampling bias in their data, they also sequenced seven genomes from scratch of primary species within the families. They decided to add three more non-nodulating members from the same clade as a control and to further fill out the phylogeny they were investigating.

This left them with samples from the 28 families within the four orders. They were able to determine that the multiple gains of symbiosis claim was correct and, furthermore, the reason why only 10 of the families had nodule-creating species is because the other families appear to have had a loss of function for that ability at some point in their evolutionary history. It should be noted though that they disproved the claim that this multiple gain of function was due to ancient genes that predisposed certain offspring. Therefore, either the genes in question quickly evolved to other forms and were not detected or that the genetic changes involved are more subtle and widespread.

The loss of function, meanwhile, seemed more intriguing and was shown to be connected to a specific gene named nodule inception (NIN) that is required for nodule symbiosis to be possible. But that gene was knocked out or made inactive through mutations in all the families without NFN synthesis. This makes little sense, as one would expect plants with the negative loss of function mutations would be outcompeted by their siblings that can better process nitrogen. The researchers offered a few suggestions on why it may have still been advantageous for the plants to independently lose the gene across the NFN clade.

More Questions and More Research

The straightforward option is that parasitic bacteria may have taken advantage of the symbiosis and invaded the nodules, making many plant species still with them less fit for survival. An event in the soil or environment causing an overabundance of nitrogen may have also made the symbiosis less necessary for most families in the clade. Lastly, other stresses and loss of nutrients could have proven more severe a constraint for growth than nitrogen was, making the loss of the gene have little impact on the problems the plants were having with proper growth.

Clearly, further research is required to tease out which of these is correct, if any of them, along with more research into whether adding nodule capabilities to more species is feasible as a method for increasing nitrogen availability for plant growth. Synthetic biologists are already working on this topic and the new information from this study should prove useful in that endeavor. Once achieved, greater plant growth for all types of plants may be available and the next agricultural revolution will come right along with it.

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Photo CCs: Medicago italica root nodules 2 from Wikimedia Commons

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