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bioRxiv · 10.64898/2026.02.13.705714

A transparent window into the rhizosphere: a simplified workflow for spatially resolved soil metabolomics

Subrahmaniam, H. J.·Moritz, P.·Becker, P. J.·Riedner, M.·Thomsen, S.·Meier, I. C.·Jensen, K.
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Abstract

Root exudates play a central role in nutrient cycling, microbial recruitment, and plant-plant interactions, yet most experimental approaches for analyzing exudate chemistry rely on sterile hydroponic systems that poorly represent soil conditions. We present a low-cost, open-source, 3D-printed rhizobox platform and associated workflow that enable non-destructive root imaging and targeted rhizosphere soil sampling for LC-MS based metabolomics under realistic soil conditions. The design integrates a transparent removable window for repeated root observations, a defined soil volume to support spatially explicit sampling, and a blank-informed data-processing pipeline to distinguish plant-derived metabolites from soil and construction material background. We validated the system using the model plants Arabidopsis thaliana (Col-0) and Phragmites australis. We demonstrate reliable plant growth and consistent root development across the imaging window. We also show robust detection of species-specific rhizosphere metabolite profiles, with minimal variation in the vertical or temporal dimensions relative to the strong species effects. We further illustrate the application of the workflow in a factorial experiment manipulating social context (solo vs. conspecific pairs) and short-term heat stress in A. thaliana, showing that the approach is sensitive to treatment-associated changes in metabolite richness, diversity, and chemical composition in soil. The complete protocol, from rhizobox fabrication and assembly to soil extraction, LC-MS acquisition, and data curation can be implemented within 4-6 weeks using standard laboratory equipment and openly available design files. By combining ecological realism with analytical control, this workflow provides a broadly applicable method for quantifying rhizosphere metabolite dynamics across species, treatments, and spatial sampling zones, facilitating experimental studies of below-ground chemical processes in plant ecology.

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Subrahmaniam, H. J., Moritz, P., Becker, P. J., Riedner, M., Thomsen, S., Meier, I. C., Jensen, K.. 2026-02-14. A transparent window into the rhizosphere: a simplified workflow for spatially resolved soil metabolomics. https://doi.org/10.64898/2026.02.13.705714

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