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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol describes how to assess the impact of xenobiotics on alfalfa via nontargeted metabolomic analysis.

Abstract

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in secretion of root exudates is usually an external detoxification strategy for plants under stress conditions. This protocol aims to introduce general guidelines for the collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate (DEHP) on metabolite production. First, alfalfa seedlings are grown under DEHP stress in a hydroponic culture experiment. Second, the plants are transferred to centrifuge tubes containing 50 mL of sterilized ultrapure water for 6 h to collect root exudates. The solutions are then freeze-dried in a vacuum freeze dryer. The frozen samples are extracted and derivatized with bis(trimethylsilyl)) trifluoroacetamide (BSTFA) reagent. Subsequently, the derivatized extracts are measured using a gas chromatograph system coupled with a time-of-flight mass spectrometer (GC-TOF-MS). The acquired metabolite data are then analyzed based on bioinformatic methods. Differential metabolites and significantly changed metabolism pathways should be deeply explored to reveal the impact of DEHP on alfalfa in view of root exudates.

Introduction

Di(2-ethylhexyl) phthalate (DEHP) is a synthetic chemical compound that is widely used in various plastics and polymers as a plasticizer to improve their plasticity and strength. In the past few years, an increasing number of studies have suggested that DEHP is an endocrine disruptor and has adverse effect on the respiratory, nervous, and reproductive systems of humans and other animals1,2,3. Considering its health risk, the United States Environmental Protection Agency, European Union, and Environmental Monitoring Center of China have all classified DEHP in the list of prior....

Protocol

The aim of this protocol is to provide a general pipeline, from a hydroponic culture experiment to metabolomic analysis, quantifying the effect of DEHP on alfalfa root exudates.

1. Hydroponic culture experiment

NOTE: This protocol presents an example of an alfalfa hydroponic culture experiment designed to obtain alfalfa (Medicago sativa) seedlings under the stress of different concentrations of DEHP. Three treatments were set up: the control without any additions, and the nutrient solution spiked with 1 mg kg-1 and 10 mg kg-1 of di(DEHP. The concentrations of DEHP wer....

Representative Results

In this experiment, alfalfa root exudates were collected, extracted, and analyzed according to the above methods (Figure 1). Three treatment groups were set up: control, low concentration of DEHP (1 mg L−1), and high concentration of DEHP (10 mg L−1).

A total of 778 peaks were detected in the chromatograph of the control, of which 314 metabolites could be identified according to the mass spectra. As shown in F.......

Discussion

This protocol provides general guidance on how to collect and measure the root exudates of alfalfa under DEHP stress, as well as how to analyze the metabolome data. Close attention needs to be paid to some critical steps in this protocol. In hydroponic culture experiments, alfalfa seedlings were hydroponically cultured in glass bottles filled with nutrient solutions with different concentrations of DEHP. The glass bottles should be protected from light by covering them with aluminum foil throughout the culture period, in.......

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was jointly supported by the National Natural Science Foundation of China (41877139), the Major Projects of the National Natural Science Foundation of China (41991335), the National Key Research and Development Program of China (2016YFD0800204), the Natural Science Foundation of Jiangsu Province (No. BK20161616), the "135" Plan, and the Frontiers Program of the Chinese Academy of Sciences (ISSASIP1615).

....

Materials

NameCompanyCatalog NumberComments
AdonitolSIGMA≥99%
Alfalfa seedsJiangsu Academy of Agricultural Sciences (Nanjing, China)
Analytical balanceSartoriusBSA124S-CW
BSTFAREGIS Technologieswith 1% TMCS, v/v
CentrifugeThermo Fisher ScientificHeraeus Fresco17
Chromatographic columnAgilentDB-5MS (30 m × 250 μm × 0.25 μm)
Di(2-ethylhexyl) phthalateDr. Ehrenstorfer
FAMEsDr. Ehrenstorfer
Gas chromatography(GC)Agilent7890A
Grinding instrumentShanghai Jingxin Technology Co., LtdJXFSTPRP-24
Mass spectrometer(MS)LECOPEGASUS HT
MethanolCNW TechnologiesHPLC
Methoxyaminatio hydrochlorideTCIAR
Microcentrifuge tubeEppendorfEppendorf Quality1.5 mL
OvenShanghai Yiheng Scientific Instrument Co., LtdDHG-9023A
PyridineAdamasHPLC
R softwarestatistical analysis software (pathway enrichment, topology)
SIMCA16.0.2 statistical analysis software (OPLS-DA etc)
Ultra low temperature freezerThermo Fisher ScientificForma 900 series
UltrasoundShenzhen Fangao Microelectronics Co., LtdYM-080S
Vacuum dryerTaicang Huamei biochemical instrument factoryLNG-T98

References

  1. Yin, X. H., Zeb, R., Wei, H., Cai, L. Acute exposure of di(2-ethylhexyl) phthalate (DEHP) induces immune signal regulation and ferroptosis in oryzias melastigma. Chemosphere. 265, 129053 (2021).
  2. Seyoum, A., Pradhan, A.

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AlfalfaRoot ExudatesDi 2 ethylhexyl PhthalateDEHPXenobioticsMetabolomicsMedicago SativaSeed SterilizationGerminationRoot Exudate CollectionMetabolite ProfilingExtractionCentrifugation

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