HHO5 discovery could make nitrogen fertilizer use more efficient

Farmers lose roughly half of each application of nitrogen fertilizer. A large share runs off into waterways, enters the atmosphere or remains in the ground, increasing both expenses and pollution.

Scientists have identified a plant protein that signals crops to stop taking in nitrogen even when the soil still contains ample supplies.

When the researchers removed this protein, a laboratory plant absorbed almost three times as much nitrogen, suggesting a route towards crops that use fertilizer much more efficiently.

Fertilizer waste is a global problem

Nitrogen fertilizer has boosted harvests for many decades. However, crops take up only about 50% of the nitrogen fertilizer applied to fields. Much of the remainder seeps into water systems, where it can harm aquatic organisms and fuel damaging algal blooms far downstream.

Nitrogen retained in the soil also contributes to nitrous oxide emissions. Across a 100-year timeframe, this gas retains heat 273 times more effectively than carbon dioxide.

Expense creates a third challenge. Producing and transporting fertilizer costs a great deal and is vulnerable to geopolitical pressures, while its unstable nitrogen component can make storage hazardous.

Gloria Coruzzi, the Carroll and Milton Petrie Professor in NYU’s Department of Biology, led the research. She conducted the work alongside Mariana Obertello of INGEBI in Buenos Aires.

“Improving the efficiency of fertilizer usage would have important environmental, economic, and geopolitical impacts,” says Coruzzi.

How plants know they’re full

The researchers sought genes that respond to nitrogen dosage rather than merely detecting its presence. They aimed to identify regulators that operate only at particular nitrogen levels.

They cultivated Arabidopsis, a small flowering plant widely used as a plant-biology model. The team exposed its roots to six doses of nitrogen for two hours before sequencing them.

This investigation identified a protein known as HHO5. It is a transcription factor, meaning a protein that activates or deactivates other genes. HHO5 was found to direct genome-wide gene expression and plant growth according to the nitrogen dose.

The response depended on the nitrogen form as well as its quantity. HHO5 concentrations rose after the plant had accumulated sufficient organic nitrogen, which is used in transport, storage and amino acids.

“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,” says Coruzzi.

Plants enter an energy-saving mode

After activation, HHO5 performed two functions simultaneously. It encouraged genes involved in organic nitrogen and amino-acid metabolism while suppressing genes that draw additional inorganic nitrogen from the soil.

Transforming inorganic nitrogen into organic nitrogen requires considerable energy from the plant. Shutting off further uptake once its reserves are full appears to help preserve that energy.

“This became the model of how plants establish nitrogen satiety via HHO5,” says Will Hinckley, an NYU doctoral student and the study’s lead author.

A second protein alters the response

The protein’s ability to perform both functions depended on its partner. By itself, HHO5 suppresses uptake genes; when paired with a second protein, WRKY21, it becomes an activator.

The researchers tested this using a technique named DoubleTARGET. They tagged two proteins separately with fluorescent markers, expressed them in isolated plant cells, and then sequenced the cells to determine which genes the pair regulates.

Cells with high amounts of both HHO5 and WRKY21 displayed greater expression of organic nitrogen genes and defence genes.

Coruzzi and Hinckley jointly responded by email to questions from Earth.com, saying the dual function was the finding that departed from textbook expectations.

“Classically, transcription factors (TFs) have been seen as either activators of gene expression OR repressors, not both,” they told Earth.com.

They say that identifying WRKY21 as the partner that changes HHO5 from a repressor into an activator unlocked the entire mechanism. While other proteins in the HHO family are recognised for repressing inorganic nitrogen uptake, HHO5 alone activates organic nitrogen responses.

Removing HHO5 increases nitrogen uptake

The last experiment involved eliminating the protein. Teams in Argentina and at NYU examined Arabidopsis plants with no HHO5 at all.

The benefit appeared under conditions most relevant to farming. According to Coruzzi and Hinckley, plants without HHO5 absorbed almost three times more nitrogen when grown in low-nitrogen conditions.

“In simple terms, the plants would have increased nitrogen use efficiency,” they say. “This would save farmers the cost of N-fertilizer, and could also help reduce the environmental impact of excess N use.”

“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,” says Obertello.

NYU has submitted a patent application for the findings, which are intended to improve nitrogen use efficiency in plants.

Greater uptake has disadvantages

The finding has important limitations. The work concerns Arabidopsis grown on agar plates and in hydroponic tanks, rather than maize cultivated in fields.

The mutant plants also bore a cost for their increased uptake. Removing HHO5 reduced root growth, decreased the number of seeds in each silique and lowered seed nitrogen content.

This is significant because fertilizer is intended to produce grain, not larger roots. Nitrogen taken into a plant is beneficial only when it reaches the portions consumed by people.

HHO5 differs from related proteins in one further respect. It is located in phloem cells, the tissue that transports nitrogen through the plant, potentially explaining why removal of this single gene has such a strong effect.

“We were excited to find that HHO5 was specifically expressed in phloem,” Coruzzi and Hinckley say. “Phloem cells are highly specialized, and are important for N transport.”

The study was designed to examine nitrogen dose and form. The researchers say the protein’s position within the plant proved equally important.

Towards more efficient crops

The broader ambition is to develop crops that continue absorbing nitrogen while the soil still has nutrients available. Other research groups are pursuing the same objective through nitrogen-fixing bacteria.

“This knowledge may aid the engineering of ‘gluttonous’ plant varieties that absorb more available nitrogen,” says Hinckley.

The team expresses greater confidence than that wording may imply. Coruzzi and Hinckley say nitrogen-response genes are strongly conserved between Arabidopsis and maize, while HHO5 itself occupies the centre of a nitrogen-response network shared by Arabidopsis and rice.

“We have also shown that increased NUE phenotypes observed in Arabidopsis mutants are conserved in the orthologous mutants of maize,” they say. “So, the results for HHO5 and N-use in Arabidopsis will likely be translatable to maize and rice.”

It remains to be established in field conditions whether the switch operates identically in wheat, rice or maize. The seed penalty observed in Arabidopsis would be the first issue that a crop-based version must address.

The National Institute of General Medical Sciences at the NIH funded the research, with Argentine support provided by ANPCyT and CONICET.