Background

Previous approaches to studying rhizosphere have often focused on sterile and hydroponic growth conditions (van Dam and Bouwmeester, 2016). These approaches limit our understanding of the multi-trophic nature of the rhizosphere, as they fail to provide information about (microbial) breakdown products of root exudation chemicals and de novo produced chemicals by rhizosphere-inhabiting microbes, even though these may drive plant-soil interactions in response to environmental change, such as pathogen attack or abiotic stress. The challenge arises from understanding and simplifying the complex, and often overwhelming, level of chemical diversity that originates from untargeted analyses of non-sterile soil (Figure 1). Nevertheless, untangling this diversity by identifying chemical networks, their origin and function, is critical for obtaining new insight into the ecology of a much under-explored area of plant and soil science. Although not without its caveats, recent advances in mass spectrometry (MS) technology and statistical-analytical techniques permit powerful exploratory methods to uncover the chemistry of natural soils (Pétriacq et al., 2017; Swenson et al., 2015 and 2018). Such techniques are becoming increasingly accessible.


Figure 1. Rhizochemicals that shape below-ground interactions. Rhizochemicals are compounds that are enriched in the rhizosphere and include a) root exudation chemicals, b) microbial breakdown products of root exudation chemicals, c) de novo synthesized metabolites by rhizosphere-inhabiting microbes. Rhizochemicals can have important signaling activities thereby shaping below-ground interactions between plants and soil microbes.

The protocol presented here describes an adjustable experimental system for sample collection and sample preparation, followed by data acquisition using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF). Subsequent statistical analysis allows separating chemical signatures by retention time and mass to charge (m/z) ratios between plant-free soil and plant-containing soil, which ultimately identifies profiles of rhizosphere-enriched chemical (i.e., rhizochemicals). These profiles can be further annotated using chemical databases, such as METLIN (Smith et al., 2005). Moreover, during mass spectrometry examination, running a library of relevant standards can increase the accuracy of this approach and allow focus of the analysis on specific metabolite classes.

The protocol outlined here, and originally used in Pétriacq et al. (2017), was initially developed for the analysis of rhizosphere chemistry in the model plant species Arabidopsis thaliana (Arabidopsis; Figures 2 and 3) and has been adapted for maize. For other plant species, a similar experimental design can be adapted and implemented, but method optimization is advised. Particularly, we recommend further optimization when insufficient separation is obtained between rhizochemical signatures from the plant-containing soil samples and the plant-free soil samples (see Figure 4). These adjustments involve i) testing different soils, ii) using different extraction solutions (e.g., with varying MeOH concentrations), iii) increasing root biomass in collection tubes, iv) adjusting soil moisture, v) increasing the time of sample extraction from the collection tubes, and vi) varying MS settings. Further attention should be paid to the fact that extraction solutions with high MeOH concentrations can cause damage to microbial and/or root cells, in which case specific assays can be used to assess the extent of cell damage, as detailed in Petriacq et al. (2017). If cell damage occurs, we recommend using a lower MeOH concentration in the extraction solution and/or shorter extraction times. If insufficient sample yield is obtained with shorter extraction times, a pressurized collection system can be used, as is illustrated in Figure 2D.