New imaging system reveals soybean roots fixing nitrogen in real time
image: (a) During the introduction of [13N]N2 gas, valve #1 is closed and valve #2 and cyclotron valve are opened, which allows for the [13N]N2 gas to be transferred to the pot through the pot gas-entry port, transported into the soil via the riser tubes and the gas then exits the system by being directed into a waste bag. (b) During evacuation, the cyclotron's [13N]N2 supply line is shut off, valve #2 is closed, and valve #1 is opened. Prior to the initiation of the evacuation of residual gas from the soil, the tubing from the quick push connect on valve #2 is manually disengaged. This will allow for room air to be pulled into the pot through the gas port on the pot lid, flushing out the residual radioactive gas within the soil column through the pot risers, and then discharging it into the waste bag.
Credit: The authors
By coupling positron emission tomography (PET) with [13N]N2 radiotracer gas, the system allows researchers to directly observe and quantify nitrogen fixation within root nodules.
Synthetic nitrogen fertilizers underpin modern crop yields but carry a steep environmental price. Their industrial production consumes vast amounts of fossil fuel and contributes significantly to greenhouse gas emissions. Moreover, excess nitrogen leaches into waterways and releases nitrous oxide, a potent greenhouse gas, undermining long-term agricultural sustainability. Legume-rhizobia symbiosis, however, offers a natural alternative. In this partnership, rhizobial bacteria in root nodules convert atmospheric nitrogen gas into ammonia, which is assimilated into plant tissues. Soybeans account for nearly three-quarters of global biologically fixed nitrogen and can fulfill much of their nitrogen demand through this process. Yet, quantifying SNF has remained a challenge, with existing methods such as acetylene reduction assays and nitrogen-15 tracing limited by destructive sampling or indirect measurements. PET imaging, combined with the short-lived tracer [13N]N2, has shown promise, but until now applications were limited to roots suspended in air rather than natural soil environments.
A study (DOI: 10.1016/j.plaphe.2025.100027) published in Plant Phenomics on 14 March 2025 by Leon Kochian’s team, Global Institute for Food Security, opens new opportunities to evaluate genetic differences in nitrogen fixation efficiency and to support breeding programs aimed at reducing dependence on chemical fertilizers.
In this study, researchers employed PET imaging coupled with a novel gas delivery system (IMP2RIS) to non-invasively visualize and quantify SNF in soybean roots. The method involved introducing the short-lived radiotracer [13N]N2 into the nodulated roots of soybean plants grown in soil-like media, followed by three-dimensional PET scanning to track nitrogen assimilation and translocation. Three soybean genotypes—Dundas, Woodstock, and Gaillard—were chosen based on known differences in nitrogen fixation capacity. The PET imaging protocol was carefully timed relative to the half-life of [13N], with Gaillard and Woodstock imaged after two half-lives and Dundas after four, allowing observation of both immediate fixation in root nodules and subsequent transport of assimilates into the stem. Results clearly distinguished genotypic variation in fixation efficiency: Dundas showed the strongest performance, with basal stem radioactivity concentrations of 28.1 MBq/mm³ and an estimated fixation rate of 41.4 μmol N₂ h⁻¹, compared to intermediate rates in Woodstock (5.2 μmol N₂ h⁻¹, 2.8 MBq/mm³) and lower rates in Gaillard (7.1 μmol N₂ h⁻¹, 4.4 MBq/mm³). Visualizations confirmed rapid assimilation of nitrogen into root nodules across all genotypes, while signals in the basal stem reflected the slower translocation of fixed nitrogen compounds toward the shoots. These findings not only validate IMP2RIS as an efficient system for in-soil PET imaging of SNF but also underscore its ability to capture genotype-specific differences in fixation dynamics. Importantly, this non-destructive, real-time approach complements traditional destructive methods such as ¹⁵N analysis, providing a dynamic perspective on nitrogen uptake and allocation. By enabling quantitative whole-plant assessment of SNF in soil-like conditions, the method offers a powerful tool for breeding programs aimed at enhancing biological nitrogen fixation and reducing reliance on synthetic fertilizers.
The ability to measure nitrogen fixation in living plants without destruction has significant implications. For plant breeders, IMP2RIS provides a functional phenotyping tool to identify soybean cultivars with superior SNF traits. Integrating this approach into breeding pipelines could accelerate the development of crop varieties that require less synthetic fertilizer, lowering production costs while reducing environmental harm.
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References
DOI
Original URL
https://doi.org/10.1016/j.plaphe.2025.100027
Funding information
This research was supported by funding from a Canada Excellence Research Chairs (CERC) Grant to L.K., and from funding from the Global institute for Food Security, and the University of Saskatchewan, to L.K.
About Plant Phenomics
Plant Phenomics is dedicated to publishing novel research that will advance all aspects of plant phenotyping from the cell to the plant population levels using innovative combinations of sensor systems and data analytics. Plant Phenomics aims also to connect phenomics to other science domains, such as genomics, genetics, physiology, molecular biology, bioinformatics, statistics, mathematics, and computer sciences. Plant Phenomics should thus contribute to advance plant sciences and agriculture/forestry/horticulture by addressing key scientific challenges in the area of plant phenomics.