University of Rouen-Normandy Scientists Explore Eco-Friendly Sampling Approach for GC-HRMS

Research led by Valerie Peulon-Agasse, an associate professor from the University of Rouen-Normandy in Rouen, France, sampled root exudates a new ecofriendly method for gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS). Their findings were published in the Journal of Chromatography Open (1).

Fresh green soybean plants with roots | Image Credit: © Олег Мальшаков - stock.adobe.com

Root exudates are substances secreted by the roots of living plants and microbially modified products of these substances (2). They consist of low-molecular-weight organic compounds that are freely and passively released root-cell material and mucilage. Root exudates are complex mixtures of soluble organic substances, which can contain sugars, amino acids, enzymes, and other substances. Exudates can act as signaling molecules, or communication intermediates, between plants and the surrounding rhizosphere microbiome, which can consist of fungi, bacteria, viruses, or nematodes. Understanding, root exudation can be vital for elucidating rhizosphere communication and promoting an understanding of plant-soil interactions.

However, sampling root exudates can be challenging. Though they are produced in soil, most exudate knowledge has been acquired with artificial devices–such as hydroponics, which involves growing plants using mineral nutrient solutions in water without soil–that do not reflect the biological, physical, or chemical properties of soils (3). Further, root exudates’ quantity and composition can vary significantly among plant species, plant ages, root depths, and environmental conditions, as determined by various soil properties or microclimates.

In this study, a sampling method called EcoRoot was investigated for direct soil sampling, specifically searching for root exudates of Poa annua L., which belongs to the Poaceae family. EcoRoot corresponds to the combination of rhizoboxes and sorption traps. As such, root exudate sampling in annual herbaceous plants using sorption traps were evaluated and optimized. First, a screening of sorption traps, including those made of nitrocellulose membranes (CelN), nylons, and glass microfiber (GMF), was performed on a standard mixture containing compounds known to be found in root exudates. Adsorption and desorption processes were optimized, and afterwards, the sampling method’s environmental impact was evaluated using the analytical greenness metric for sample preparation (AGREEprep).

The obtained score was compared with those obtained for hydroponic methodologies, and afterwards soluble root exudates of Poa annua L., which belongs to a family of grasses (Poaceae), were collected from the selected sorption traps into rhizoboxes. Poa annua L. was selected because it is one of the most widespread grasses in the world. Untargeted analysis was performed using gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) after silylation in full scan mode. The detected compounds were compared to those trapped in soil without plants. Finally, untargeted screening allowed for the detection and identification of soluble root exudate compounds and the identification of the first metabolomic fingerprint of P. annua.

The results obtained for the sorption capacities of the different supports showed the traps could both adsorb and desorb compounds. CelN trap use led to the best sorption/desorption yields for most of the targeted compounds, obtaining over 91% for fumaric acid, followed by the GMF and nylon traps. General conclusions could not be reached regarding the influence of contact time between the sorption traps and exudates on recovery; therefore, a contact time of 60 min was selected. With extraction involving an ultrasonic bath, clear trends were observed with higher recovery yields at a 30 min extraction time.

After the sample preparation method was optimized, GC-HRMS screening was performed. Excluding all compounds specific to the soil and CelN traps (or those in common), 516 features remained and were considered. Therefore, the EcoRoot methodology used to assess root exudates was validated. Further investigations are recommended, especially on sample preparation steps that may involve the use of combinations of traps with different compositions or a reduction of the trap size. Specifically, trap size may be reworked to be closer to the root size to avoid as much soil contamination as possible.

References

(1) Bohm, V.; Forey, E.; Chauvat, M.; Cardinael, P.; Peulon-Agasse, V. Evaluation of Trap Discs for Root Exudate Eco-Friendly Sampling Using Rhizoboxes: Application to Untargeted Screening of Organic Compounds by Gas Chromatography Hyphenated with High Resolution Mass Spectrometry. J. Chromatogr. Open 2025, 7, 100205. DOI: 10.1016/j.jcoa.2025.100205

(2) Root Exudate. ScienceDirect 2005. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/root-exudate (accessed 2025-4-16)

(3) Hydroponics. ScienceDirect 2020. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/hydroponics (accessed 2025-4-16)