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 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.
<ol>
	<li>Sterilize alfalfa seeds with 0.1% sodium hypochlorite for 10 min and 75% ethyl alcohol for 30 min.
	<ol>
		<li>Rinse the sterilized seeds several times with distilled water and then germinate on moist filter paper in a sterile Petri dish at 30 &#176;C in the dark.</li>
	</ol>
	</li>
	<li>Transfer 20 uniform, germinated, big-plump seeds onto an engraftment basket in a culture bottle filled with nutrient solution, composed of (in &#181;M): 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.</li>
	<li>Place all the culture bottles in a controlled growth chamber with a light intensity of 150-180 &#181;mol m-2 s-1 with a photoperiod of 16 h each day, at 27 &#176;C and 20 &#176;C representing day (16 h) and night (8 h), respectively.</li>
	<li>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.</li>
	<li>Randomly place and rotate the bottles every 2 days to ensure consistent growth conditions for the alfalfa seedlings.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Collection experiment
	<ol>
		<li>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.</li>
		<li>Wrap the centrifuge tubes with aluminum foil to protect the roots from light.</li>
		<li>Remove the plants and freeze-dry the collected liquid for metabolite profiling.</li>
	</ol>
	</li>
	<li>Extraction experiment
	<ol>
		<li>Add 1.8 mL of extraction solution (methanol:H2O = 3:1, V/V) to the tubes and vortex for 30 s.</li>
		<li>Apply ultrasound waves to the tubes for 10 min in an ice water bath.</li>
		<li>Centrifuge the samples at 4 &#176;C and 11,000 &#215; g for 15 min.</li>
		<li>Carefully transfer 200 &#181;L of supernatant into a 1.5 mL microcentrifuge tube. Take 45 &#181;L of supernatant from each sample and mix it into quality control (QC) samples at a final volume of 270 &#181;L, which is used for calibration of the metabolome data of samples.</li>
		<li>Freeze-dry the extracts in a vacuum concentrator. Continue drying with 5 &#181;L of the internal standard (ribonucleol).</li>
		<li>After evaporation in a vacuum concentrator, add 30 &#181;L of methoxyamination hydrochloride (dissolved in pyridine at a concentration of 20 mg mL-1) to the tubes and incubate the tubes at 80 &#176;C for 30 min. Then, add 40 &#181;L of bis(trimethylsilyl)trifluoroacetamide (BSTFA) reagent (with 1% trimethylchlorosilane [TMC], V/V) to the samples and place the tubes at 70 &#176;C for 1.5 h for derivatization.</li>
		<li>Cool the samples to room temperature and add 5 &#181;L of fatty acid methyl esters (FAMEs) (in chloroform) to the QC samples.</li>
	</ol>
	</li>
	<li>Metabolomic analysis
	<ol>
		<li>Inject 1.0 &#181;L 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.
		<ol>
			<li>Use a capillary column (30 m x 250 &#181;m x 0.25 &#181;m) 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 &#176;C, and maintain the transfer line temperature and ion source temperature at 280 &#176;C and 250 &#176;C, respectively.</li>
			<li>For separation, use the following oven program: 1 min isothermal heating at 50 &#176;C, a 10 &#176;C/min-1 oven ramp to 310 &#176;C, and a final isothermal heating at 310 &#176;C for 8 min.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Filter individual peaks to remove noise. The deviation value is filtered based on the interquartile range.</li>
		<li>Fill the missing values with half of the minimum values, standardize, and normalize the data.</li>
		<li>Import the final data in .csv format into statistical analysis software for multivariate analysis.</li>
		<li>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.</li>
		<li>Apply supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) to demonstrate the differences among groups.</li>
		<li>Screen significantly changed metabolites as differential metabolites based on a variable importance in projection (VIP) &#62; 1 and p &#60; 0.05 (Student&#39;s t test).</li>
		<li>Use the metabolome data to construct heat maps with the statistical analysis software and use the fold changes under different treatments to construct histograms.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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.

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 order to prevent DEHP from photolysis and assure the uniformity of DEHP concentrations in all culture solutions25,26. DEHP concentrations were set as 1 mg L-1 and 10 mg L-1, according to the concentrations usually found in soils contaminated with DEHP27,28. During the collection of root exudates, the centrifuge tubes still need to be wrapped with aluminum foil to protect the roots from light. Here, the collection time was set to be 6 h. If the collection time is too short, the concentration of some low-abundance root exudates might be too low to be detected, so the composition of the root exudates might not reflect the real response of alfalfa seedlings under DEHP stress. If the collection time is too long, the collected root exudates might be degraded to a certain extent by microbes in the culture systems. In this case, some microbial metabolites might also be detected, changing the composition of the root exudates29. Moreover, at least six replicates should be performed for each treatment so as to ensure accurate metabolome data analysis.
Only some certain types of root exudates, such as amino acids, fatty acids, phenolic compounds, and other organic acids, can be determined using conventional targeted analytical methods (i.e., searching for known compounds)30,31,32, using standard analytical methods, such as spectrophotometry, high-pressure liquid chromatography (HPLC), ion chromatography (IC), gas chromatography-tandem mass spectrometry (GC-MS/MS), or liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). With the development of instrumental analysis techniques, nontargeted metabolomic analysis has emerged based on GC-TOF-MS, liquid chromatography-high resolution mass spectrometry (LC-HR-MS), and nuclear magnetic resonance (NMR)33. Compared with other conventional analytical methods of root exudates, nontargeted metabolomic analysis greatly expands the number of detected root exudates, which facilitates deeply understanding plant metabolic responses to environmental stress.
The technique in the current study has some limitations, especially in the quantitative analysis of metabolites. Nontargeted metabolomic analysis only demonstrates the relative quantitative relationships among different metabolites, rather than accurate concentrations. This is unfavorable for investigations into the environmental behaviors or ecological effects of root exudates34. For the growth media of alfalfa seedlings, hydroponic culture experiments were performed in the current study, which have the advantages of easy control and easy operation. However, the artificial hydroponic growth environment is different from the real soil environment, leading to possible underestimation of the gross exudation rates due to metabolite reuptake by roots35. Compared with hydroponic culture, sand culture is a relatively more convincing method because it is closer to the real soil environment, facilitating the collection of root exudates in a realistic environment36. Therefore, work is under progress to establish a method of sand culture or even real soil culture to make the experimental results more convincing, which will help us conduct more in-depth research, showing better results of practical significance for explaining the toxicity response.
Based on the nontargeted metabolic analysis, we can not only explore the metabolic response of plants to environmental stress37, but also determine differential metabolites and further investigate the important role of the differential metabolites in the environmental behavior of contaminants38. Previous studies have shown that pelargonic acid level can be used as an indicator of root membrane damage when plants are under stress39. Unsaturated fatty acids (palmitic acid) were also found to increase membrane fluidity40, which may protect alfalfa root membranes from DEHP damage. Some flavonoids inhibited by DEHP can participate in the interaction between legumes and microorganisms. Moreover, the interactions between plants and the rhizosphere biological community may be more deeply deciphered with nontargeted metabolic analysis, especially under the stress of contaminants41. More types of root exudates will be identified42 with the development of analytical instruments for nontargeted metabolic analysis, which is helpful for constructing the metabolomic fingerprinting of plant root exudates43.

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><tbody><tr><td>Adonitol</td><td>SIGMA</td><td></td><td>&amp;ge;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 &amp;times; 250 &amp;mu;m &amp;times; 0.25 &amp;mu;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&amp;nbsp;</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 Figure 2, these metabolites could be classified into six types based on the relative abundance: carbohydrates (28.6%), acids (15.58%), lipids (13.87%), alcohols (3.91%), amines (0.92%), and others (37.12%). Metabolites that accounted for less than 0.5% were grouped as other substances (Figure 2A). The acids were further subdivided into fatty acids (56.09%), amino acids (26.62%), organic acids (13.95%), and phenolic acids (3.34%) (Figure 2B). In addition, some common substances in the root exudates of most plants could also be detected in alfalfa root exudates, including pyrimidines, hydroxy pyridines, flavonoids, phenols, ketones, pyrimidines, flavonoids, and diterpenes.
A heat map was plotted to visualize the variation in differential metabolites among different DEHP treatments, based on the VIP score (Figure 3). Compared with the control, the exposure to DEHP significantly changed the content of 50 metabolites in alfalfa root exudates, mainly including some carbohydrates and low-molecular weight organic acids. Five types of carbohydrates (lyxose, digitoxose, erythrose, trehalose, and fructose 2, 6-bihosphate) were upregulated in the presence of DEHP, and two of these (lyxose and digitoxose) were significantly increased as the concentration of DEHP increased. In addition, five metabolites were downregulated in the presence of DEHP, including monosaccharides such as D-talose and glucose, disaccharides such as maltose, cellobiose, and trehalose, and sugar alcohols such as D-arabitol. Carbohydrate content has been considered as an indicator of plant physiological status24. Therefore, the decrease in monosaccharide and disaccharide levels herein indicated physiological stress caused by DEHP stress. Compared with carbohydrates, DEHP exerted a greater effect on acid metabolism in alfalfa seedlings. Under exposure to DEHP, the contents of 11 acid metabolites were significantly increased, mainly including 2-amino-2-norbornanecarboxylic acid, 5-hydroxyindole-2-carboxylic acid, 3-hydroxy-L-proline, pelargonic acid, and palmitic acid. At the same time, DEHP also inhibited the metabolism of some flavonoids in alfalfa seedlings, including 4&#39;, 5-dihyrroxy-7-methoxyisoflavone and neohesperidin.
The metabolic pathways influenced by DEHP are described in Figure 4. DEHP significantly inhibited the metabolism of carbohydrates, such as some monosaccharides and disaccharides, which are products of photosynthesis. Therefore, DEHP can suppress the photosynthesis of alfalfa to a certain extent. Moreover, DEHP can promote the metabolism of fatty acids, which are helpful for plants to resist stress from DEHP. The major metabolic pathways influenced by DEHP were carbohydrate metabolism and fatty acid metabolism, while amino acid metabolism, lipid metabolism, and the tricarboxylic acid (TCA) cycle were affected to a much lesser extent.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64470/64470fig01.jpg" /> 
Figure 1: A flow chart of nontargeted metabolomic analysis for alfalfa root exudates. BSTFA represents bis(trimethylsilyl)trifluoroacetamide (BSTFA) reagent (with 1% trimethylchlorosilane [TMC], V/V). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64470/64470fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64470/64470fig02.jpg" /> 
Figure 2: Classification of metabolites. (A) Classification of the known metabolites and (B) acids. The percentage of each type of material is divided by the sum of the peak area of each category by the sum of the peak area of all the substances in the control. Other substances were those at &#60;0.5%. Other acids were those at &#60;0.5%. This figure has been modified from Wang et al.25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64470/64470fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64470/64470fig03.jpg" /> 
Figure 3: Heatmap of hierarchical clustering analysis for root exudates (VIP &#62; 1, p &#60; 0.05) of alfalfa seedlings with different DEHP treatments. Red and green represent high and low abundance, respectively. ACK represents the control; 1+AD represents the treatment with 1 mg L&#8722;1&#160;DEHP; 10+AD represents the treatment with 10 mg L&#8722;1&#160;DEHP. This figure has been modified from Wang et al.25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64470/64470fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64470/64470fig04.jpg" /> 
Figure 4: Relationships between the disturbance of metabolic pathways and the alterations in biological endpoints (1 mg L&#8722;1 DEHP, 10 mg L&#8722;1 DEHP). The metabolic pathways were established based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The metabolites in green text were the metabolites detected in the present work. The signs &#34;red boxes&#34; and &#34;blue boxes&#34; in parentheses indicate that metabolites increased (p &#60; 0.05) or decreased (p &#60; 0.05) contributions to the biological endpoints, respectively. The figure has been made readable by separating the metabolites roughly into carbohydrate, fatty acid, and protein metabolism, as shown by the green, red, and black rectangular boxes, respectively. ACK represents the control; 1+AD represents the treatment with 1 mg L&#8722;1 DEHP; 10+AD represents the treatment with 10 mg L&#8722;1 DEHP. This figure has been modified from Wang et al.25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64470/64470fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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Nanyang Technological University

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1.6K Views.  Chinese Academy of Sciences. Dit protocol beschrijft hoe de impact van xenobiotica op alfalfa kan worden beoordeeld tijdens niet-gerichte metabolomics-analyse. De techniek is zeer eenvoudig en handig voor bediening en meer soorten wortelexsudaten kunnen worden geïdentificeerd. Dat helpt bij het construeren van de metabolomische fingerprinting van exsudaten van plantenwortels.Begin de sterilisatie van de medicago sativa of alfalfazaden door de zaden gedurende 10 minuten te spoelen met 0,1% natriumhypochloriet. Gev...

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

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The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme ...

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is imperative to investigate the accumulation and translocation of PFAAs within plants. Long-distance transport is an important pathway for PFAAs transferred from the plant leaves to the edible tissues through the phloem. However, it was previously difficult to assess the translocation potential of organic contamination in a short-term exposure period. The split-root experiment provides a solution to effectively uncover the long-distance translocation of PFAAs using a hydroponic experiment, which, in this study, was carried out in two 50 mL centrifuge tubes (A and B), of which centrifuge tube A had 50 mL of one-quarter strength Hoagland sterile nutrient solution, while centrifuge tube B had the same amount of nutrient concentration, and the target PFAAs (perfluorooctane sulfonic acid, PFOS, and perfluorooctane acid, PFOA) added at a given concentration. A whole wheat root was manually separated into two parts and inserted carefully into tubes A and B. The concentration of PFAAs in the roots, shoots of wheat, and solutions in tubes A and B were evaluated using LC-MS/MS, respectively, after being cultured in an incubator for 7 days and harvested. The results suggested that PFOA and PFOS experience a similar long-distance transport process through the phloem from the shoot to the root and could be released into the ambient environment. Thus, the split-root technique can be used to evaluate the long-distance transport of different chemicals.

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is ...

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized in&#160;the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

The present protocol describes a simple and efficient method for the identification of 2,4-dibromophenol metabolites in plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Biology

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

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

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