Study identifies mechanisms used by plants to control assimilation of nitrogen

Discovery by scientists in Brazil and at Edinburgh University could lead to the development of crop varieties that are less dependent on nitrogen-based fertilizers.

By Elton Alisson

Agência FAPESP – Researchers at the University of Campinas’s Biology Institute (IB-Unicamp) in Brazil, in collaboration with colleagues at the University of Edinburgh in Scotland, have discovered a significant mechanism used by plants to control nitrogen absorption and assimilation.

The new insight, achieved as part of doctoral research supported by a scholarship from FAPESP, opens up the prospect of the development of plant varieties that are less dependent on nitrogen-based fertilizers, which are widely used in agriculture to boost crop growth and yield.

The study’s main findings are described in an article recently published in Nature Communications. “We identified a new pathway for nitrogen uptake regulation in plants. By modifying this pathway, we were able to increase the plants’ nitrogen assimilation and hence boost their growth. We hope that this discovery will contribute to a reduction in the excessive use of fertilizers that often have an adverse impact on the environment and represent high costs in agricultural production,” said Lucas Frungillo, the first author of the article.

Plants absorb nitrogen from the soil in the form of nitrate, he told Agência FAPESP. Once absorbed by the roots, the nitrate is mostly transported to the aerial part of the plant and then assimilated in the form of amino acids and proteins.

During the assimilation process, nitrate is converted to nitrite in the plant’s leaves in a reaction catalyzed by enzymes called nitrate reductases.

The nitrite is mostly transported to the chloroplasts – organelles in plant cells that are responsible for photosynthesis – and, from there, proceeds along the nitrogen assimilation pathway until amino acids are formed. Nitrite can also be converted into nitric oxide by other enzymatic reactions that take place inside plant cells.

For a long time, according to Frungillo, the nitric oxide-forming reaction along the nitrate assimilation pathway was considered to be a by-product of the nitrogen uptake process.

However, when Frungillo and colleagues studied this process in different varieties of Arabidopsis thaliana, they found that one of the roles played by nitric oxide is to act as a signaling molecule, fine-tuning the amount of nitrogen used for growth by telling the plant’s cells when to limit nitrate absorption and assimilation. A. thaliana is a small herbaceous plant native to Europe, Asia and Africa that belongs to the Brassicaceae, or mustard, family.

“Nitric oxide acts as a signaling mechanism, indicating to the plant whether it has enough nitrogen to develop,” Frungillo said. “It does this by regulating the plant’s nitrate assimilation.”

Signaling strategy

According to Frungillo, the strategy that nitric oxide uses to increase its concentration and act as a signaling mechanism in the plant’s nitrogen assimilation process is regulation of S-nitrosoglutathione reductase 1 (GSNOR1), an enzyme capable of breaking down one of nitric oxide’s bioactive forms.

In other biological models, an increase in the availability of a molecule induces an increase in the activity of an enzyme capable of breaking it down. In the case of nitric oxide, the opposite occurs: an increase in the availability of nitric oxide in the plant leads to a reduction in the activity of GSNOR1.

“Nitric oxide is capable of directly inhibiting the activity of the enzyme GSNOR1 by means of a post-translational modification called S-nitrosylation,” Frungillo said. “In this way, it regulates its own availability in the plant and controls its signaling action, telling the plant when to reduce nitrate assimilation.” Post-translational modifications are processes that change the size, composition, function and location of proteins.

Through genetic engineering, it is possible to increase the number of copies of this enzyme to counteract nitric oxide signaling, reducing the inhibitory impact of the signaler on its breakdown. “This ‘self-promoting’ effect of nitric oxide can be genetically mitigated,” Frungillo said.

To test this hypothesis, during the study, researchers at the University of Edinburgh developed a variety of A. thaliana known as 35S::GSNOR1, which overexpresses GSNOR1.

Genetic and biochemical analyses performed at the laboratory headed by Professor Gary J. Loake and repeated in Brazil showed that the plant succeeded in assimilating a larger amount of nitrogen and therefore grew more than other varieties with normal expression of GSNOR1.

“The plant 35S::GSNOR1 has more copies of the enzyme GSNOR1, which is still regulated by nitric oxide but is far more active in this genotype than in the others that we studied. For this reason, the process of nitric oxide signaling in the plant is reduced, and the plant is able to assimilate more nitrogen and grow a little more,” explained Frungillo, who worked in Loake’s laboratory for five months to perform supplementary experiments and complete the study.

According to the Brazilian researcher, genetic engineering of new plant varieties with altered nitric oxide signaling may be possible to boost plant growth even further.

“By breaking down a bioactive form of nitric oxide, the plant reduces the availability of this signaler and assimilates more nitrogen. Its growth and yield increase as a result,” Frungillo said.

“We expect that the nitrogen assimilation pathway observed in A. thaliana to exist in most other plants as well,” he concluded.

The article “S-nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway” (doi: 10.1038/ncomms640), by Frungillo et al., can be read by subscribers to the journal Nature Communications at www.nature.com/ncomms/2014/141111/ncomms6401/full/ncomms6401.html.