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

This protocol describes how to assess the impact of xenobiotics on alfalfa while non-targeted metabolomics analysis. The technique is very simple and convenient for operation and more types of root exudates can be identified. Which helps construct the metabolomic fingerprinting of plant root exudates.

Begin the medicago sativa or alfalfa seeds'sterilization by rinsing the seeds with 0.1%sodium hypochlorite for 10 minutes. Followed by a wash with 75%ethyl alcohol for 30 minutes. Next, rinse the sterilized seeds several times with distilled water.

And then germinate them on the moist filter paper placed in a sterile petri dish. At 30 degree Celsius in the dark. After germination, transfer 20 uniformly germinated big plump seeds onto an engraftment basket in a culture bottle filled with nutrient solution.

Place all the culture bottles in a growth chamber having controlled conditions. After two weeks, transfer 15 uniform alfalfa seedlings to a new glass bottle to expose them to one milligram per kilogram and 10 milligrams per kilogram DEHP stress for the culture experiment. Wrap the treatment and control glass bottles with aluminum foil and parafilm to prevent photolysis and volatilization of the DEHP.

Supplement the nutrient solution daily to maintain the liquid level. Randomly place and rotate the bottles every two days to ensure consistent growth conditions for the alfalfa seedlings. After seven days of cultivation, remove the alfalfa seedlings from the bottles.

And wash them with ultrapure water several times to prepare them for the collection of root exudates. Begin the root exudate collection experiment by transferring 10 uniform alfalfa seedlings to centrifuge tubes filled with 50 milliliters of sterilized deionized water. To collect root exudates, keep the tubes upright for six hours with the roots submerged in water.

Once done, wrap the centrifuge tubes with aluminum foil to protect the roots from light. Remove the plants and freeze dry the collected liquid for metabolite profiling. Proceed with the extraction experiment by adding 1.8 milliliters of extraction solution to the freeze dried samples.

In vortex for 30 seconds. Next, apply the ultrasound waves to the tubes for 10 minutes in an ice water bath. Before centrifuging the samples.

Carefully transfer 200 microliters of supernatant into a 1.5 milliliter micro centrifuge tube. Aspirate 45 microliters of supernatant from the centrifuge tube and mix it into quality control or QC samples at a final volume of 270 microliters. Once done, freeze dry the extracts in a vacuum concentrator.

Once drying is complete, add five microliters of internal standard ribonuclease and continue drying. After evaporation, add 30 microliters of methoxyamination hydrochloride solution and incubate the tubes at 80 degrees Celsius for 30 minutes. Next, add 40 microliters of BSTFA reagent to the samples before placing the tubes at 70 degrees Celsius for 1.5 hours for derivatization.

Once the incubation is over and the derivatized sample reaches room temperature, add five microliters of fatty acid methyl esters dissolved in chloroform to the derivatized samples. Using a capillary column measuring 30 meters by 250 micrometers by 0.25 micrometer, separate one microliter of the root exudates with a carrier gas helium at a flow rate of 1.0 milliliter per minute. Set the injection temperature to 280 degrees Celsius.

And maintain the transfer line temperature at 280 degrees Celsius. Set the oven program for separation. After separating the extracts, perform mass spectrometry in electron collision mode with an energy of minus 70 electron volts.

Maintain the ion source temperature at 250 degrees Celsius. Obtain mass spectra using full scan monitoring mode with a mass scan range of 50 to 500 mass by charge at a rate of 12.5 spectra per second. 778 peaks were detected in the control sample's chromatograph.

Of which 314 metabolites were identified according to the mass spectra. The acids were further subdivided into fatty acids, amino acids, organic acids and phenolic acids. In addition, some common substances generally present in the root exudates were also detected in alfalfa root exudates including pyrimidines, hydroxy pyrimidines, flavonoids, phenols, ketones, pyrimidines and diterpenes.

A heat map was plotted based on the VIP score to visualize the variation in differential metabolites among different DEHP treatments. Compared to the control samples, the DEHP exposure significantly changed the content of 50 metabolites and alfalfa root exudates. Mainly including some carbohydrates and low molecular weight organic acids.

The metabolic pathways analysis showed that DEHP significantly inhibited the metabolism of carbohydrates which are the product of photosynthesis indicating that DEHP can suppress the photosynthesis of alfalfa to a certain extent DEHP was seen to promote the metabolism of fatty acids which help resist stress from DEHP. At least the seed root exudates should be performed for each treatment to ensure accurate metabolomic data analysis. We can determine differential metabolize and further investigate the important rules in the environmental behavior of contaminants.

The interactions between plants and the wider field biological community can be deciphered mostly by using this method.