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.