Name: collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate on metabolite production
Uploaded: 2023-06-02T12:45:00
Description: Watch this Scientific Journal Video about Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production at JoVE.com

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 &#34;135&#34; Plan, and the Frontiers Program of the Chinese Academy of Sciences (ISSASIP1615).

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 ...

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adonitol</td>
			<td>SIGMA</td>
			<td></td>
			<td>&#8805;99%</td>
		</tr>
		<tr>
			<td>Alfalfa seeds</td>
			<td>Jiangsu Academy of Agricultural Sciences (Nanjing, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>BSA124S-CW</td>
			<td></td>
		</tr>
		<tr>
			<td>BSTFA</td>
			<td>REGIS Technologies</td>
			<td></td>
			<td>with 1% TMCS, v/v</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>Heraeus Fresco17</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>Agilent</td>
			<td>DB-5MS (30 m &#215; 250 &#956;m &#215; 0.25 &#956;m)</td>
			<td></td>
		</tr>
		<tr>
			<td>Di(2-ethylhexyl) phthalate</td>
			<td>Dr. Ehrenstorfer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FAMEs</td>
			<td>Dr. Ehrenstorfer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatography(GC)</td>
			<td>Agilent</td>
			<td>7890A</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding instrument</td>
			<td>Shanghai Jingxin Technology Co., Ltd</td>
			<td>JXFSTPRP-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass spectrometer(MS)</td>
			<td>LECO</td>
			<td>PEGASUS HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CNW Technologies</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>Methoxyaminatio hydrochloride</td>
			<td>TCI</td>
			<td></td>
			<td>AR</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Eppendorf Quality</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd</td>
			<td>DHG-9023A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Adamas</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>R software</td>
			<td></td>
			<td></td>
			<td>statistical analysis software (pathway enrichment, topology)</td>
		</tr>
		<tr>
			<td>SIMCA16.0.2&#160;</td>
			<td></td>
			<td></td>
			<td>statistical analysis software (OPLS-DA etc)</td>
		</tr>
		<tr>
			<td>Ultra low temperature freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Forma 900 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound</td>
			<td>Shenzhen Fangao Microelectronics Co., Ltd</td>
			<td>YM-080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dryer</td>
			<td>Taicang Huamei biochemical instrument factory</td>
			<td>LNG-T98</td>
			<td></td>
		</tr>
	</tbody>
</table>

collection of alfalfa root exudates to study the impact of di(2-ethylhexyl) phthalate on metabolite production

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.

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&#8722;1), and high concentration of DEHP (10 mg L&#8722;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...

Watch this Scientific Journal Video about Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production at JoVE.com

Collection of Alfalfa Root Exudates to Study the Impact of Di2-ethylhexyl Phthalate on Metabolite Production

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.

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

Root exudates are the main media of information communication and energy transfer between plant roots and the surrounding environment. The change in ...

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