The lost ability to fix nitrogen

Case

The lost ability to fix nitrogen

Leguminous plants such as peas and lentils can grow on nitrogen-depleted soil without the addition of fertilizer, while species from related plant families cannot.

Legumes were not the only plants to acquire this characteristic; other plants had it but have since lost it, Vici researcher René Geurts of Wageningen University & Research has discovered. This conclusion has turned this field of research on its head and is a boost for the development of crops that do not need to be fertilized.

Nodule_section - foto: René GeurtsNodule_section - foto: René Geurts

'Plants cannot break down the molecular nitrogen (N2) in the air to make ammonium,' explains René Geurts. 'This is because the two nitrogen atoms are linked with a triple bond, and it takes a lot of energy to pull them apart. But this nitrogen is also one of the most important constituents of the amino acids that make up proteins.' Leguminous, protein-rich plants such as beans, peas and soya have come up with a clever solution: they have formed a symbiotic relationship with rhizobium bacteria. The plants grow special organs called root nodules with which they can absorb these bacteria into their own cells. The bacteria convert molecular nitrogen into ammonium, which the plant puts to use as a nutrient. In exchange, the bacteria get energy in the form of carbohydrates that the plant produces through photosynthesis.

Paraponia nodule_1 photo: René GeurtsParaponia nodule_1 photo: René Geurts

Reducing the use of fertilizers

If we could teach other plants to use nitrogen-fixing bacteria in the same way, we could reduce the use of artificial fertilizers. With this application in mind, researchers all over the world are studying the origins and the precise workings of the symbiotic relationship between leguminous plants and rhizobium bacteria.

From pea to hemp

'We are comparing plants that have this ability with plants that do not,' explains Geurts. This sounds simpler than it is. The family of Leguminosae consists of more than 20,000 species, most of which have been capable of this symbiosis for tens of millions of years. In addition to leguminous plants, there are about 250 other plant species spread over nine families which can also grow nitrogen-fixing root nodules. Geurts focused on a smaller family of plants that evolved somewhat later: he compared two tropical tree genera, Parasponia and Trema, that are both members of the hemp family. 'Parasponia can grow root nodules and fix nitrogen using rhizobium, while Trema cannot. Because they both have a relatively simple genome, this was where we went looking for the genetic causes of this difference.'

Parasponia boom - photo: René GeurtsParasponia boom - photo: René Geurts

Growing trees

Geurts had to overcome a few obstacles, because the research into these two genera had stood still for some twenty years. 'In 2011 we started breeding and propagating the two genera again from scratch. We’re talking about trees here, which don’t usually grow very fast.' But to his surprise, he obtained viable seeds from the trees within six months. 'We have now unravelled the complete genomes of a number of different species of Parasponia and Trema, and manually annotated and compared the results with the help of a small army of students. If you try to do that with bioinformatics alone, you get so many flaws that almost all the differences turn out to be artefacts.'

Disappointment

To his surprise, Geurts found no evidence to suggest that Parasponia had only recently acquired the ability to form root nodules. 'Given the number of unrelated plant families that are able to do this, we would have expected this evolutionary step to have taken place about ten times in parallel, and Parasponia should only have acquired the ability fairly recently. So we were initially very disappointed that we could find no evidence for this in the DNA of Parasponia.'

Parasponia can grow root nodules and fix nitrogen using rhizobium, while Trema cannot.
- René Geurts

A dogma rejected

At the same time, Geurts saw how the Trema plants had lost the genetic information needed to form root nodules. After extensive further research, Geurts concluded that the generally accepted hypothesis was not correct at all. 'The ability to work together with nitrogen-fixing bacteria is much older than we thought. This characteristic was not developed recently and ten times in parallel, but much longer ago and only once, and now the vast majority of species has lost it again. Only ten families have preserved it.'

Why was it lost?

It took some time for the research community to accept this conclusion, says Geurts. 'We are now two years on, and it has become an accepted hypothesis. The next question to answer is why that characteristic, which appears to be so useful to the plant, has been lost? One suggestion is that this has been triggered by changes in the composition of the atmosphere. A plant needs CO2, which it converts into sugars and oxygen through photosynthesis. If the CO2 concentration in the air falls below a certain value, the plant is left with too little substrate for photosynthesis, and so it can no longer provide the bacteria with food. In this case, the symbiotic relationship will simply cost the plant too much energy.'

Repairing the characteristics

In a way, Geurts’ conclusion is good news for the development of plants that can do without artificial fertilizer. 'Instead of adding a new characteristic to plants, we will first try to repair it in plant species that used to have this ability. We will start with Trema because it only recently lost the characteristic. We actually only identified a number of point mutations in the plant’s DNA; the required genes are all still there. By finding out what repairs are required to reactivate the symbiotic interaction, we will hopefully get an idea of how we can adapt the crops of the future to grow root nodules, provide a home to these useful bacteria, and so enable the plants to obtain nitrogen from the air.'

Interview: Sonja Knols