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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • 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 priority pollutants. Soil has been considered as an important sink of DEHP in the environment, due to the application of plastic mulching and organic fertilizers, irrigation with wastewater, and sludge farm application4. As expected, DEHP has been ubiquitously detected in farmland soils, the content of which even reaches up to milligrams per kilogram of dried soil in some regions in China5,6. DEHP can enter plants mainly via the roots and undergo biomagnification at different trophic levels in soil ecosystems7. Therefore, significant concern has been raised about DEHP-induced stress in plants over recent decades.

Plants are usually vulnerable to DEHP exposure. DEHP stress has been observed to exert an adverse effect on seed germination and normal metabolism, thereby inhibiting plant growth and development8,9. For example, DEHP can induce oxidative damage to mesophyll cells, decrease the contents of chlorophyll and osmolytes, and elevate antioxidative enzyme activities, eventually resulting in a decline in the yield and quality of edible plants10,11. However, most of the previous studies on the response of plants to DEHP stress have focused on oxidative stress and physiological and biochemical characteristics. The corresponding mechanisms associated with plant metabolism are less-studied. Root exudates is a generic term describing compounds that plant roots secrete and release into the environment. They have been considered as the interaction media between plants and rhizosphere soil, playing an important role in supporting plant growth and development12. It has been well known that root exudates account for approximately 30%-40% of all photosynthetic carbon13. In polluted environments, root exudates are involved in improving the tolerance of plants to the stress of pollutants through metabolism or external exclusion14. As a consequence, a deep understanding of the response of plant root exudates to pollution stress may help reveal the underlying mechanisms associated with cell biochemistry and biological phenomena15.

Metabolomics technology provides an efficient strategy for measuring a large number of small molecule metabolites simultaneously within cells16,17, tissues18, and even exudates of organisms19, including sugars, organic acids, amino acids, and lipids. Compared with traditional or classical chemical analysis methods, the metabolomics approach greatly increases the number of metabolites that can be detected20, which can help identify metabolites in a higher-throughput way and identify key metabolic pathways. Metabolomics has been widely used in the research field of biological response in stress environments, such as heavy metals21, emerging pollutants22, and nanoparticles19. Most of these studies on plants have focused on the metabolic changes in interior plant tissues, whereas few have been reported on the response of root exudates to environmental stress. Therefore, the aim of this study is to introduce general guidelines for the collection of alfalfa root exudates to study the impact of DEHP on metabolite production. The results will provide a method guidance for the follow-up study of plant metabolomics by DEHP.

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 were set according to the real content of DEHP in soil23. Each treatment had six replicates.

  1. Sterilize alfalfa seeds with 0.1% sodium hypochlorite for 10 min and 75% ethyl alcohol for 30 min.
    1. Rinse the sterilized seeds several times with distilled water and then germinate on moist filter paper in a sterile Petri dish at 30 °C in the dark.
  2. Transfer 20 uniform, germinated, big-plump seeds onto an engraftment basket in a culture bottle filled with nutrient solution, composed of (in µM): Ca(NO3)2, 3,500; NH4H2PO4, 1,000; KNO3, 6,000; MgSO4, 2,000; Na2Fe-ethylenediaminetetraacetic acid (EDTA), 75; H3BO3, 46; MnSO4, 9.1; ZnSO4, 0.8; CuSO4, 0.3; and (NH4 )6Mo7O24, 0.02. Adjust the solution pH to 7.0 using 0.1 M KOH. Renew all solutions weekly.
  3. Place all the culture bottles in a controlled growth chamber with a light intensity of 150-180 µmol m-2 s-1 with a photoperiod of 16 h each day, at 27 °C and 20 °C representing day (16 h) and night (8 h), respectively.
  4. Transfer 15 uniform alfalfa seedlings to a new glass bottle for culture experiments under 1 mg kg-1 and 10 mg kg-1 DEHP stress after 2 weeks. Wrap the glass bottles with aluminum foil and parafilm to prevent photolysis and volatilization of the DEHP. To apply the same conditions, also wrap the control bottles with aluminum foil and parafilm. Supplement the nutrient solution daily to maintain the liquid level.
  5. Randomly place and rotate the bottles every 2 days to ensure consistent growth conditions for the alfalfa seedlings.
  6. After 7 days of cultivation, remove the alfalfa seedlings from the bottles and wash with ultrapure water several times, preparing for the collection of root exudates.

2. Collection, extraction, and metabolomic analysis of root exudates

NOTE: This protocol is divided into three parts: a collection experiment, an extraction experiment, and metabolomic analysis of the root exudates. The goal of the collection experiment is to transfer the metabolites secreted in plant samples to the solution system for subsequent extraction.

  1. Collection experiment
    1. Transfer 10 uniform alfalfa seedlings to centrifuge tubes filled with 50 mL of sterilized deionized water. Submerge the roots in water to collect root exudates for 6 h; keep the tubes upright. Perform at least six replicates for each treatment.
    2. Wrap the centrifuge tubes with aluminum foil to protect the roots from light.
    3. Remove the plants and freeze-dry the collected liquid for metabolite profiling.
  2. Extraction experiment
    1. Add 1.8 mL of extraction solution (methanol:H2O = 3:1, V/V) to the tubes and vortex for 30 s.
    2. Apply ultrasound waves to the tubes for 10 min in an ice water bath.
    3. Centrifuge the samples at 4 °C and 11,000 × g for 15 min.
    4. Carefully transfer 200 µL of supernatant into a 1.5 mL microcentrifuge tube. Take 45 µL of supernatant from each sample and mix it into quality control (QC) samples at a final volume of 270 µL, which is used for calibration of the metabolome data of samples.
    5. Freeze-dry the extracts in a vacuum concentrator. Continue drying with 5 µL of the internal standard (ribonucleol).
    6. After evaporation in a vacuum concentrator, add 30 µL of methoxyamination hydrochloride (dissolved in pyridine at a concentration of 20 mg mL-1) to the tubes and incubate the tubes at 80 °C for 30 min. Then, add 40 µL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) reagent (with 1% trimethylchlorosilane [TMC], V/V) to the samples and place the tubes at 70 °C for 1.5 h for derivatization.
    7. Cool the samples to room temperature and add 5 µL of fatty acid methyl esters (FAMEs) (in chloroform) to the QC samples.
  3. Metabolomic analysis
    1. Inject 1.0 µL of the derivatized extracts into a gas chromatograph system coupled to a time-of-flight mass spectrometer (GC-TOF-MS) for metabolomic profiling analysis using a splitless mode.
      1. Use a capillary column (30 m x 250 µm x 0.25 µm) for the separation of root exudates, with helium as a carrier gas at a flow rate of 1.0 mL min-1. Set the injection temperature to 280 °C, and maintain the transfer line temperature and ion source temperature at 280 °C and 250 °C, respectively.
      2. For separation, use the following oven program: 1 min isothermal heating at 50 °C, a 10 °C/min-1 oven ramp to 310 °C, and a final isothermal heating at 310 °C for 8 min.
      3. Perform electron collision mode with -70 eV of energy. Obtain mass spectra using full scan monitoring mode with a mass scan range of 50-500 m/z at a rate of 12.5 spectra/s.
    2. Filter individual peaks to remove noise. The deviation value is filtered based on the interquartile range.
    3. Fill the missing values with half of the minimum values, standardize, and normalize the data.
    4. Import the final data in .csv format into statistical analysis software for multivariate analysis.
    5. Look up the metabolites in Kyoto Encyclopedia of Genes and Genomes (KEGG) database (a database resource for understanding high-level functions and utilities of the biological system), and classify the metabolites into different categories, such as carbohydrates, acids, lipids, alcohols, and amines. Use statistical analysis software to construct a pie chart to indicate the percentage of each category in all the root exudates.
    6. Apply supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) to demonstrate the differences among groups.
    7. Screen significantly changed metabolites as differential metabolites based on a variable importance in projection (VIP) > 1 and p < 0.05 (Student's t test).
    8. Use the metabolome data to construct heat maps with the statistical analysis software and use the fold changes under different treatments to construct histograms.
    9. Look up the differential metabolites in the KEGG database and Pubchem and compile the metabolic pathways containing the differential metabolites. Perform pathway enrichment analysis or topology analysis.

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

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