Collection of Alfalfa Root Exudates to Study the Impact of Di(2-ethylhexyl) Phthalate on Metabolite Production

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 animals^1,2,3. 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....

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.......

Materials

Name	Company	Catalog Number	Comments
Adonitol	SIGMA		≥99%
Alfalfa seeds	Jiangsu Academy of Agricultural Sciences (Nanjing, China)
Analytical balance	Sartorius	BSA124S-CW
BSTFA	REGIS Technologies		with 1% TMCS, v/v
Centrifuge	Thermo Fisher Scientific	Heraeus Fresco17
Chromatographic column	Agilent	DB-5MS (30 m × 250 μm × 0.25 μm)
Di(2-ethylhexyl) phthalate	Dr. Ehrenstorfer
FAMEs	Dr. Ehrenstorfer
Gas chromatography(GC)	Agilent	7890A
Grinding instrument	Shanghai Jingxin Technology Co., Ltd	JXFSTPRP-24
Mass spectrometer(MS)	LECO	PEGASUS HT
Methanol	CNW Technologies		HPLC
Methoxyaminatio hydrochloride	TCI		AR
Microcentrifuge tube	Eppendorf	Eppendorf Quality	1.5 mL
Oven	Shanghai Yiheng Scientific Instrument Co., Ltd	DHG-9023A
Pyridine	Adamas		HPLC
R software			statistical analysis software (pathway enrichment, topology)
SIMCA16.0.2			statistical analysis software (OPLS-DA etc)
Ultra low temperature freezer	Thermo Fisher Scientific	Forma 900 series
Ultrasound	Shenzhen Fangao Microelectronics Co., Ltd	YM-080S
Vacuum dryer	Taicang Huamei biochemical instrument factory	LNG-T98