Background: Plants exude a plethora of compounds to communicate with their environment. Although much is known about above-ground plant communication, we are only beginning to fathom the complexities of below-ground chemical communication channels. Studying root-exuded compounds and their role in plant communication has been difficult due to the lack of standardized methodologies. Here, we develop an interdisciplinary workflow to explore the natural variation in root exudate chemical composition of the model plant Arabidopsis thaliana. We highlight key challenges associated with sampling strategies and develop a framework for analyzing both narrow- and broad-scale patterns of root exudate composition in a large set of natural A. thaliana accessions.
Methods: Our method involves cultivating individual seedlings in vitro inside a plastic mesh, followed by a short hydroponic sampling period in small quantities of ultrapure water. The mesh makes it easy to handle plants of different sizes and allows for large-scale characterization of individual plant root exudates under axenic conditions. This setup can also be easily extended for prolonged temporal exudate collection experiments. Furthermore, the short sampling time minimizes the duration of the experiment while still providing sufficient signal even with small volume of the sampling solution. We used ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for untargeted metabolic profiling, followed by tentative compound identification using MZmine3 and SIRIUS 5 software, to capture a broad overview of root exudate composition in A. thaliana accessions.
Results: Based on 28 replicates of the Columbia genotype (Col-0) compared with 10 random controls, MZmine3 annotated 354 metabolites to be present only in Col-0 by negative ionization. Of these, 254 compounds could be annotated by SIRIUS 5 software.
Conclusions: The methodology developed in this study can be used to broadly investigate the role of root exudates as chemical signals in plant belowground interactions.
Keywords: Arabidopsis thaliana; chemical analysis; interdisciplinary; natural variation; root exudates.
Plants exude many compounds to communicate with their surroundings. For decades, our understanding of this chemical communication was limited to studying the aboveground parts of plants, as roots are hidden within the soil, which makes them difficult to study. We are only beginning to comprehend the complexities and importance of below ground communication channels (including plant-microbes, plant-insects, and plant-plants). Identifying the chemical compounds plant exude belowground (called root exudates) is important for us to fully comprehend their potential roles in a plant´s life. Here, we developed a simplified and easy-to-manage setup for collecting and analyzing root exudates from individual Arabidopsis thaliana plants.
Copyright: © 2023 Subrahmaniam HJ et al.