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Unraveling the Secrets of Plant Roots: Simplified Method for Large Scale Root Exudate Sampling and Analysis in Arabidopsis Thaliana

Open Research Europe(2023)

Department of Ecoscience | Department of Chemistry | Department of Molecular Biology and Genetics - Plant Molecular Biology

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Abstract
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.
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Key words
root exudates,natural variation,Arabidopsis thaliana,interdisciplinary,chemical analysis,eng
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要点】:本文提出了一种简化的大规模植物根际分泌物采样与分析方法,用于研究模式植物拟南芥根际分泌物的化学组成及其在植物地下通讯中的作用。

方法】:通过在体外培养单个幼苗于塑料网格中,并在小量的超纯水中进行短期水培采样,结合使用超高效液相色谱-四级杆飞行时间质谱(UHPLC-QTOF-MS)进行无目标代谢组学分析,以及MZmine3和SIRIUS 5软件进行代谢物注释。

实验】:实验通过对拟南芥哥伦比亚生态型(Col-0)的28个重复样本与10个随机对照样本进行比较,使用负离子模式在MZmine3中注释了354种仅在Col-0中存在的代谢物,其中254种通过SIRIUS 5软件得到注释。