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

Root exudates shape nutrient cycling, microbial recruitment, and plant-plant interactions, yet most approaches for analysing exudate chemistry rely on hydroponic systems that poorly represent soil. We present an open-source workflow built around a 3D-printed rhizobox that enables non-destructive root imaging and targeted rhizosphere soil sampling for LC-MS metabolomics. The workflow combines a frame that prints as a single piece on a consumer printer, sampling of defined soil zones registered to root images, and a blank-informed, feature-specific background filter. The filter is essential because ∼90% of detected features also occur in soil, material or solvent controls. In a validation experiment with Arabidopsis thaliana and Phragmites australis, both species grew along the window, and species identity explained 38% of compositional variation among rhizoboxes, whereas sampling date and depth had no detectable effect. In a factorial experiment with A. thaliana, rhizoboxes holding intraspecific pairs showed higher metabolite richness and diversity and a distinct composition compared with solo plants, whereas short heat stress had no detectable effect. Inner and outer sampling zones of paired boxes did not differ at the replication available, and we give the replication needed to resolve such effects. The protocol can be implemented within 4 to 6 weeks using standard laboratory equipment and openly available design files. Combining ecological realism with analytical control, this workflow offers a practical route to quantifying rhizosphere metabolite dynamics across species, treatments, and defined sampling zones, facilitating experimental study of below-ground processes in plant ecology.