Direct Imaging of Plant Metabolites in the Rhizosphere using Laser Desorption Ionization Ultra-high Resolution Mass Spectrometry

Abstract

<p>The rhizosphere is an important hotspot for microbial activity, organic carbon input, and carbon turnover in soils. The interplay of these rhizosphere components results in small scale gradients of organic molecules in the zone around a root. Mass spectrometric imaging (MSI) can reveal the spatial distribution of individual plant metabolites in the soil, which cannot be achieved using bulk analysis. Using non-fragmenting ionization techniques such as laser desorption ionization (LDI) allows for the detection of intact molecules without the need for labeling with e.g. fluorescent tags.</p><p>Direct MSI for the chemical imaging of intact molecules of the rhizosphere has been recognized as a still existing analytical gap. Here we present a novel method allowing mass spectrometric molecular rhizosphere imaging directly in a complex soil matrix.</p><p>Our novel approach consists of sampling the roots and the surrounding soil of <em>Zea mays </em>plants in either field- or lab-scale experiments using small metal cylinders. After excavation, the loam soil pellets were embedded in gelatin and cryosectioned to 100 µm sections. After selecting regions of interest on the soil section, the root and the soil surrounding the root was analysed using ultra-high resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS).</p><p>Given the large background of soil-derived organic carbon, the high mass resolution and sensitivity of FT-ICR-MS allow distinguishing root-derived molecules from soil organic matter based on their exact masses. We show that our method is capable to recover rhizosphere gradients of a dihexose (C<sub>12</sub>H<sub>22</sub>O<sub>11</sub>, e.g. sucrose, maltose) directly in the soil with a spatial resolution of 25 µm.</p><p>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. Analysing several sections from the same soil pellet allowed to recover 3D molecular gradients from one root segment. Utilizing the potential to easily change the mass window a variety of potential metabolites can be analysed in the same region around the root. Thus the chemical diversity of potential root exudates can be revealed.</p><p>Our workflow enables the study of root-derived organic carbon with high spatial resolution directly in a soil context. For the first time, direct molecular imaging of the rhizosphere via LDI-FT-ICR-MS will allow for a non-target or targeted analysis of complex soil samples.</p><p>Visualizing the root structure via X-ray computed tomography in a soil sample before the embedding would enable a guided sampling approach to analyse molecular distributions at certain parts of the root. Moreover, the molecular LDI-MSI results could be correlated with elemental imaging via laser ablation – inductively coupled plasma – mass spectrometry directly at the same sample position - allowing for an even more detailed insight into chemical processes in the rhizosphere.</p>

Correlative Imaging of the Rhizosphere─A Multimethod Workflow for Targeted Mapping of Chemical Gradients

“…In addition to our workflow, Bandara et al 37 developed a workflow that is suitable to identify bacteria in undisturbed soil. On a similar set of samples, Lohse et al 49 presented a workflow using mass resolution laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry for the direct analysis of the molecular gradients in the rhizosphere. Our workflow can be used with the mentioned approaches as they complement each other and result in a more holistic picture of rhizosphere processes.…”