Abstract
The interplay of rhizosphere components such as root exudates, microbes, and minerals results in small-scale gradients of organic molecules in the soil around roots. The current methods for the direct chemical imaging of plant metabolites in the rhizosphere often lack molecular information or require labeling with fluorescent tags or isotopes. Here, we present a novel workflow using laser desorption ionization (LDI) combined with mass spectrometric imaging (MSI) to directly analyze plant metabolites in a complex soil matrix. Undisturbed samples of the roots and the surrounding soil of Zea mays L. plants from either field- or laboratory-scale experiments were embedded and cryosectioned to 100 μm thin sections. The target metabolites were detected with a spatial resolution of 25 μm in the root and the surrounding soil based on accurate masses using ultra-high mass resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS). Using this workflow, we could determine the rhizosphere gradients of a dihexose (e.g., sucrose) and other plant metabolites (e.g., coumaric acid, vanillic acid). The molecular gradients for the dihexose showed a high abundance of this metabolite in the root and a strong depletion of the signal intensity within 150 μm from the root surface. Analyzing several sections from the same undisturbed soil sample allowed us to follow molecular gradients along the root axis. Benefiting from the ultra-high mass resolution, isotopologues of the dihexose could be readily resolved to enable the detection of stable isotope labels on the compound level. Overall, the direct molecular imaging via LDI-FT-ICR-MS allows for the first time a non-targeted or targeted analysis of plant metabolites in undisturbed soil samples, paving the way to study the turnover of root-derived organic carbon in the rhizosphere with high chemical and spatial resolution.
Highlights
The rhizosphere is a hotspot for microbial activity, organic carbon input, and carbon turnover in soils
The size for the undisturbed soil sample of up to 0.91 cm diameter and 1.0 cm height was chosen after we observed the disintegration of the sample and loss of soil for larger undisturbed soil samples especially during the embedding and cryosectioning step
The sectioning of soil samples required embedding in a mixture of gelatin and carboxymethyl cellulose (CMC)
Summary
The rhizosphere is a hotspot for microbial activity, organic carbon input, and carbon turnover in soils. The interlinked physical, chemical, and biological processes in the rhizosphere combined with the structural heterogeneity of soils are a challenge for the analysis of this complex system. While the direct sampling of root exudates requires artificial – mostly soil-free – conditions (Phillips et al, 2008; Oburger and Jones, 2018) the determination of spatial gradients of small and often polar and mobile molecules in the soil cannot be achieved by ex situ sampling of exudates. The temporal dynamics of root exudation in the soil can be studied by sampling the soil solution (Dessureault-Rompré et al, 2007; Schulz and Vetterlein, 2007; Weidenhamer et al, 2014; Tiziani et al, 2021) and combined with structural information about the root-soil system via X-ray computed tomography (CT) (Lohse et al, 2020). Small-scale spatial heterogeneity of root exudates within a few hundred micrometers cannot be analyzed by these approaches
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