New York University researchers have pinpointed the molecular players responsible for plants "feeling full" once they've taken up enough nitrogen—a finding that could help scientists to develop plants that absorb more nitrogen from the soil and allow farmers to reduce environmentally and economically costly fertilizer application.
"By identifying gene regulators that are sensitive to different levels and types of nitrogen, we uncovered a regulatory factor controlling nitrogen use and a key to improving nitrogen uptake and assimilation into organic nitrogen in plants," said Gloria Coruzzi , the Carroll and Milton Petrie Professor in NYU's Department of Biology and Center for Genomics and Systems Biology and the co-senior author of the study, which appears in the journal The Plant Cell.
Over the past few decades, the use of nitrogen fertilizers has greatly boosted global crop yields. However, plants only absorb around 50 percent of applied fertilizers. A substantial amount leeches into the water supply, which can damage aquatic life and promote harmful algal blooms. The remaining unused nitrogen in the soil contributes to rises in the potent greenhouse gas nitrous oxide, which is 273 times more effective at trapping heat compared to carbon dioxide over a 100-year period.
In addition, producing and transporting fertilizers is costly and threatened by ongoing geopolitical issues; its storage and transport can also be dangerous due to an unstable nitrogen component in fertilizer.
"Improving the efficiency of fertilizer usage would have important environmental, economic, and geopolitical impacts," said Coruzzi, who jointly led this collaborative research with Mariana Obertello of the Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI) in Buenos Aires, Argentina.
To address these global challenges related to nitrogen fertilizers, the researchers unlocked a new regulatory factor involved in a plant's ability to feel full when taking in nitrogen.
"This knowledge may aid the engineering of 'gluttonous' plant varieties that absorb more available nitrogen," said Will Hinckley, a doctoral student in NYU's Department of Biology and the study's lead author.
In the study, the researchers identified plant genes that respond to nitrogen as a function of "N-dose," with the goal of finding condition-specific regulators of nitrogen signals. This enabled them to home in on a protein called HHO5, which they determined to be a key regulator signaling nitrogen satiety. Importantly, HHO5 regulates plant gene expression genome-wide and plant growth in a nitrogen-dose dependent manner.
The researchers also found that different types of nitrogen had unique effects on levels of HHO5 expression, the gene that produces HHO5 protein, encoding a key transcription factor triggering target gene expression genome-wide. They found that expression levels of HHO5 increased when plants contained sufficient organic nitrogen—a form of nitrogen used for long distance transport, and storage, as well as the synthesis of amino acids essential to human nutrition. Once induced by organic nitrogen signals, HHO5 was found to simultaneously play two different roles: promoting expression of genes responding to organic nitrogen and amino acid metabolism, yet at the same time inhibiting the expression of genes involved in the uptake of more inorganic nitrogen from soil—essentially saying, "I'm full."
"Inorganic nitrogen is taken up by plants from soil and assimilated into organic nitrogen. This nitrogen uptake and assimilation process is heavily energy intensive. Therefore, when organic nitrogen sufficiency triggers the HHO5 gene, HHO5 in turn signals for the plant to stop absorbing additional inorganic nitrogen from soil, likely as a means of conserving energy. This became the model of how plants establish nitrogen satiety via HHO5," explained Hinckley.
The researchers dove deeper into the mechanisms of how HHO5 regulates plant genes to better understand how the protein plays two roles at once. They found that when HHO5 acts alone, it turns off the expression of genes involved in the uptake of inorganic nitrogen. However, when HHO5 interacts with a different partner regulatory protein, WRKY21, HHO5 switches into a gene activator of organic nitrogen signaling.
To validate this organic nitrogen feedback mechanism, the NYU scientists engineered plant cells containing high levels of both HHO5 and WRKY21 regulatory proteins using a genomics technique called DoubleTARGET. In DoubleTARGET, the two regulatory proteins of interest—in this case, HHO5 and WRKY21—are each linked to different fluorescent gene markers (green or red) that are then expressed in isolated plant cells; RNA sequencing of these cells reveals target genes in the genome that synergistically respond to these regulatory protein pairs. In this study, the cells with high abundance of both HHO5 and the partner protein WRKY21 displayed enhanced expression of organic nitrogen responsive and defense-related genes.
To further demonstrate that the regulatory protein HHO5 signals the plant to stop absorbing inorganic nitrogen, the researchers in Argentina and NYU collaborated to study plants that lack HHO5 using Arabidopsis—a small flowering plant commonly used as a model in plant biology research. Notably, the plants lacking HHO5 absorbed nearly three times more nitrogen compared to plants with normal HHO5 levels in specific nitrogen conditions.
This finding supports the idea that HHO5 signals organic nitrogen satiety and shuts down uptake of inorganic nitrogen from soil. Moreover, since removal of HHO5 improves nitrogen uptake, this indicates that modifying HHO5 has the potential to lead to tangible improvements in plant nitrogen assimilation and metabolism." said Obertello.
NYU has filed a patent application covering the research and findings described in this paper to enable the improvement in plant nitrogen use efficiency.
In addition to Coruzzi and Hinckley, study authors include Joseph Swift, Samantha Frangos, and Shao-shan Carol Huang for their work done at NYU, as well as Francisco Romei, Jorge P. Muschietti, and Mariana Obertello of INGEBI in Argentina. This work was supported by the National Institutes of Health's National Institute of General Medical Sciences (RO1-GM121753, R35GM138143, and 1T32GM132037-01) to NYU, as well as Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (2015-3052 and 2019-4328) and CONICET (PIP11420100100242) in Argentina. The study appears in The Plant Cell, DOI: 10.1093/plcell/koag201
