In-situ profiling of spatiotemporal dissolved organic matter dynamics in the rhizosphere

Metabolite secretion from plant roots and their transformations by microbes constitute an important component of the formation of soil organic matter. Root discharge and microbial activity is highly heterogeneous, with microscale hotspots playing an outsized role. Thus, gaining spatial and temporal information of the biochemical interactions that occur within the rhizosphere and other subsurface terrestrial ecosystems is an area of growing interest, and requires the development of sensitive analytical approaches for characterizing metabolites from small volumes of porewaters. Here, we discuss mass spectrometry-based analyses of pore waters collected from novel rhizoboxes fitted with non-invasive micro-dialysis probes that continuously samples pore-waters along single growing Avena sativa roots. Samples are collected every hour over a 3-week time span enabling us characterize metabolites dynamics over diurnal cycles along root tip, zone of elongation and mature root zones. Samples, consisting of less than 100 μL of pore water, are analyzed by two mass spectrometry-based methods. First, 12 Tesla Fourier Transform ion cyclotron resonance mass spectrometry analysis provides accurate masses of secreted metabolites that can be assigned molecular formula. Secondly, micro-flow liquid chromatography coupled with orbitrap mass spectrometry is used to assign retention times and fragmentation spectra to major metabolites to enable identification and quantitative comparison across samples. The abundances of these features are then correlated with independent measurements of dissolved organic carbon and compared against the natural soil background to distinguish those metabolites specific to rhizosphere. Our results demonstrate the utility of mass spectrometry analyses paired with micro-dialysis sampling for characterizing metabolite profiles within the rhizosphere of a living plant.