Nitrogen-Fixing Nodules Evolved More Than Once in Plants

Feeding the world's expanding population is made possible by nitrogen fertilizers, but their production is energy-intensive, expensive, and detrimental to ecosystems. Some plants, though, have developed the capacity to produce their own nitrogen with the aid of bacteria. How they managed to pull it off repeatedly is clarified by a recent study.

For researchers hoping to breed new plants that can obtain their own nitrogen, this is vital information, according to Lead Author Heather Rose Kates, a Postdoctoral Associate at the Florida Museum of Natural History.

Breeding and crop improvement efforts often focus on a single model species, which can overlook the evolutionary context of traits.”

Heather Rose Kates, Study Lead Author and Postdoctoral Associate, Department of Natural History, Florida Museum

This study suggests that there may be multiple distinct genetic pathways that should be investigated, rather than focusing all of the efforts on understanding how one species produces nitrogen in order to save time.

Only looking at what you could think of as one version of the trait could limit the effectiveness of engineering that trait in other plants.”

Heather Rose Kates, Study Lead Author and Postdoctoral Associate, Department of Natural History, Florida Museum

All life on Earth depends on nitrogen, which can be scarce. Even though nitrogen is a plentiful element in the atmosphere, there is frequently insufficient of it due to fierce competition for it in natural settings. Nitrogen makes up 78% of the air.

It is trapped in a molecular form that is inaccessible to most living things. Diazotrophic microbes are the only cells on Earth that can "fix" nitrogen from the atmosphere.

Some plants have leveraged this to their advantage. Approximately 17,000 plant species establish a mutualistic relationship with diazotrophs. When these microbes infect the roots, the plant develops knob-like structures called nodules around them. Inside the nodules, the bacteria receive sugar for growth and, in return, supply their host plant with nitrogen in a usable form.

The nitrogen-fixing clade is the sole group of closely related plants that share this mutualistic relationship; however, even within this related species, the trait is only sporadically present.

The majority of plants that exhibit nitrogen-fixing symbiosis are classified as legumes or beans; this family comprises crops such as clover, peanuts, and soybeans. A few non-legumes that can develop nodules are birch, rose, and gourd family members.

Due to the genetic complexity involved in nodule production, many researchers have hypothesized that this trait only evolved once in this group of closely related plants. If so, there may be a single switch in the genetic code of the plants that, in species lacking this characteristic—many agricultural crops—could activate the ability to nodulate.

“When a trait involves a lot of genes and also has a high cost to the plant in terms of energy, which we know forming root nodules does, we expect there to be a strong selective pressure against evolving that trait. So, in that context, a single origin hypothesis makes sense,” said Kates.

Kates and her colleagues tested this theory by using genetic data to reconstruct the evolutionary history of nodulating plants and their close relatives. Analyzing DNA sequences from almost 15,000 species, they created the largest scratch-built tree of life for this or any other group.

The volume of data that needed to be analyzed hindered previous attempts to calculate the number of times nodulation evolved. The group worked with so many specimens from the herbarium that they had to create a whole new system for organizing the data.

We had basically two years to assemble 15,000 tissue samples from the nitrogen-fixing clade, sequence them, and build a tree.”

Robert Guralnick, Study Co-Author and Curator, Department of Biodiversity Informatics, Florida Museum

The DNA of many of the specimens had been harmed or deteriorated due to their age—many had been collected almost a century ago.

“But our approaches for extraction and sequencing were tuned for those issues. We were quite surprised about the generally high quality and quantity of recovery and usable genetic data from our samples,” said Guralnick.

According to their study, nodulation underwent a two-step evolutionary process. The group's ancestor acquired the fundamental genetic toolkit required to generate nodules, which it inherited from all of its progeny.

However, further instructions were required to activate the machinery and cause nodules to grow. This second characteristic underwent at least 16 evolutionary changes. Furthermore, plants in the group lost their nodules on ten different occasions, proving that a species' ability to grow nodules is not a permanent trait.

These results imply that nodulation is regulated by a complex circuit breaker rather than a single genetic switch. For nodules to grow, a plant must have several switches turned on.

The researchers identified and sequenced numerous genes involved in the growth of nodules. The next step is to find out precisely which traits these genes code for and how they work.

“The overall goal is to use what we learned from these evolutionary studies to help us understand the underlying genetics and processes involved in nitrogen fixing symbiosis, and then use that information for engineering,” said Pam Soltis, Study Co-Author and Curator, Florida Museum.

Most commercial crops, including rice and wheat, need nitrogen fertilizer because they cannot form nodules. Legumes are the model for many studies on bioengineering the nodulating trait, but Doug Soltis, curator at the Florida Museum and study co-author, suggested this might not be the best strategy.

“Our phylogenetic tree suggests you might want to look at other models. Nitrogen fixation might have evolved differently in legumes than it did in the rose family or birch family, so there may be different roadmaps,” said Doug Soltis, Study Co-Author and Curator, Florida Museum.