We gratefully acknowledge financial support from the Natural Science Foundation of China (NSFC 21737003), Chinese Universities Scientific Fund (No. 2452021103), and Chinese Postdoctoral Science Foundation (No. 2021M692651, 2021M702680).

Wheat seeds, Triticum aestivum L., were procured (see Table of Materials) and used for the present study.
1. Wheat seedling germination and hydroponic culture
<ol>
	<li>Select similar-sized wheat seeds and disinfect them for 15 min with 8% (w/w) hydrogen peroxide solution.</li>
	<li>Rinse the disinfected seeds with deionized water thoroughly, and then place them on humid filter paper in the dark at room temperature to germinate for 5 days.</li>
	<li>Select approximately nine germinated seedlings of uniform size and transfer them to plastic beakers with 250 mL of nutrient solution (1/4 strength of Hoagland&#39;s solution; its chemical composition is shown in Table 1). 
	NOTE: Out of the nine seeds, three each were selected for blank, PFOA, and PFOS, respectively.</li>
	<li>Cultivate the seedlings in growth chambers for 7 days before exposure with a cycle of 14 h at 22 &#176;C and 10 h at 27 &#176;C.</li>
</ol>
2. The root splitting experiment
<ol>
	<li>Carry out the seedling cultivation in two 50 mL centrifuge tubes (A and B). 
	NOTE: In centrifuge tube A, 50 mL of sterile 1/4 strength Hoagland&#39;s solution was present, and the same amount of nutrient solution was present in centrifuge tube B.
	<ol>
		<li>Dissolve the commercial PFOA and PFOS (see Table of Materials) in methanol and dilute them with the sterile nutrient solution as the stock solution. Then, add the stock solution to tube B at a PFOA/PFOS concentration of 100 &#181;g/L.</li>
		<li>Perform the treatments in triplicates with a blank control to monitor the background contamination. A schematic diagram of the split-root exposure experiments is shown in Figure 1.</li>
	</ol>
	</li>
	<li>Separate the whole roots of the wheat seedling using tweezers into two equal parts so that the roots are still hooked to the same shoot and carefully insert them into tubes A and B, respectively.</li>
	<li>Seal the two tubes with aluminum foil and culture them in an incubator for 7 days. Maintain the same incubation conditions as stated in step 1.4.</li>
	<li>Collect the wheat seedlings after 7 days of culture and separate the wheat into three parts: shoots and roots cultured in the spiked solution of PFAAs and unspiked solution, respectively, using sterilized scissors.</li>
	<li>Freeze-dry the plant samples in a lyophilizer at &#8722;55 &#176;C for 48 h.</li>
	<li>Homogenize and weigh the root and shoot samples. Collect the spiked and unspiked solution samples.</li>
</ol>
3. Extraction of PFOA and PFOS from plant tissues
<ol>
	<li>Add 2 mL of sodium carbonate buffer (0.25 mol/L), 1 mL of tetrabutylammonium hydrogen sulfate (0.5 mol/L), and 5 mL of methyl tert-butyl ether (see Table of Materials) to a 15 mL polypropylene tube, including the homogenized root or shoot.</li>
	<li>Shake the tube at 250 rpm for 20 min and centrifuge at 2,000 x g for 10 min at room temperature to obtain the supernatant organic phase. Perform the extraction process twice.</li>
	<li>Mix the collected extracts, vaporize to dryness in a gentle nitrogen (N2) stream, and then reconstitute with 5 mL of methanol and vortex them, maintaining the same speed for approximately 30 s.</li>
	<li>Condition the pesticarb cartridge (see Table of Materials) with 5 mL of 0.1% NH4OH in methanol, 5 mL of water, and 5 mL of methanol.</li>
	<li>Add the 5 mL of extracting methanol solution through the pesticarb cartridge (500 mg/6 mL) to remove the pigment, elute the cartridge with 5 mL of methanol, and collect in the same tubes.</li>
	<li>Evaporate the collected 10 mL of methanol solution to almost dryness and reconstitute with 200 &#181;L of methanol, followed by vortexing and centrifugation at 10,000 x g for 20 min at room temperature.</li>
</ol>
4. Sample preparation from the nutrient solution
<ol>
	<li>Condition with 5 mL of methanol and 5 mL of water to activate the polar enhanced polymer (PEP) extraction cartridge (60 mg/g, 3 mL) (see Table of Materials).</li>
	<li>Add 1 mL of the spiked solution or 50 mL of the unspiked solution samples (step 2.6) through the cartridge, respectively.</li>
	<li>Elute the target PFAAs with 10 mL of methanol, evaporate the extract with gentle N2, and then reconstitute with 200 &#181;L of methanol for analysis.</li>
</ol>
5. Instrumental analysis
<ol>
	<li>Use an ultra-performance liquid chromatography UPLC coupled with tandem mass spectrometry (LC-MS/MS) for quantification of the target PFAAs in multi-reaction mode (MRM) and negative electrospray ionization (ESI-) (see Table of Materials).</li>
	<li>Inject 10 &#181;L of samples and separate the target PFAAs using a C18 liquid chromatographic column (1.7 &#181;m, 2.1 mm x 50 mm, see Table of Materials), and use 2 mM ammonium acetate in water (phase A) and methanol (phase B) as the mobile phase for UPLC, with a flow rate of 0.3 mL/min. Maintain the column temperature at 50 &#176;C. 
	NOTE: The ion transitions of PFOA and PFOS are 413 to 369 and 499 to 80, respectively. The gradient elution program and the LC-MS/MS instrumental parameters for quantification of the target PFAAs are listed in Table 2.</li>
	<li>Process the data with the data analysis software (see Table of Materials).</li>
</ol>

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The authors have nothing to disclose.

To ensure the accuracy of this method, careful operation must be taken to ensure that the spiked solution in tube B does not contaminate the unspiked solution in tube A. The given concentration of target PFAAs in the present study was relatively higher than their concentration in the real environment, ensuring to monitor target PFAAs in wheat and unspiked solution using LC-MS/MS. 
There are limitations to this method. Since only one wheat seedling was used in each treatment group and the root ...

Perfluoroalkyl acids (PFAAs) are widely utilized in various commercial and industrial products due to their excellent physicochemical properties, including surface activity and thermal and chemical stability1,2,3. Perfluorooctane sulfonic acid (PFOS) and perfluorooctane acid (PFOA) are the two most important PFAAs used worldwide4,5,6, although these compounds were listed in the international Stockholm Convention in 2009 and 20197,8, respectively. Due to their persistence and widespread use, PFOS and PFOA have been widely detected in various environmental matrices. The concentrations of PFOA and PFOS in surface water from different worldwide rivers and lakes are 0.15-52.8 ng/L and 0.09-29.7 ng/L, respectively9. Due to the use of groundwater or reclaimed water for irrigation and also using biosolids as fertilizer, PFOA and PFOS are widely present in the soil, ranging between 0.01-123 &#181;g/kg and 0.003-162 &#181;g/kg, respectively10, which could introduce a large amount of PFAAs into plants and pose potential risks to human health. The PFAA (C4-C8) concentrations in agricultural soil and grain (wheat and maize) show a positive linear correlation11. Therefore, it is imperative to investigate the accumulation and translocation of PFAAs within plants.
The translocation of PFAAs in plants firstly occurs from the roots to the aboveground tissues, and the translocation of PFAAs from the roots to the edible tissues is regarded as long-distance transport12,13. Previous studies have detected bisphenol A, nonylphenol, and natural estrogens in vegetables and fruits14, which implies that these chemicals might migrate via the phloem. Hence, uncovering the translocation of PFAAs in plants is important to assess their potential risk. However, the accumulation and translocation of PFAAs are impacted by their bioavailability in the soil, so it is not easy to evaluate the translocation ability of target PFAAs in plants. Additionally, hydroponic experiments are generally limited by several factors, making it more difficult to acquire the edible tissues of plants. Typically, the phloem was collected directly from plants to observe the translocation of organic compounds through long distances in plants, whereas it is difficult to acquire phloems from plant seedlings15. Hence, a simple and effective method, the split-root technique, was introduced to study the translocation of PFAAs in plants during relatively short-term exposure. As for the split-root investigation, the roots in one plant seedling are separated into two parts; one part is put into the nutrient solution containing target PFAAs (tube A), and the other is placed in the nutrient solution in the absence of PFAAs (tube B). After exposure for several days, the PFAAs in tube B are measured by LC-MS/MS. The concentration of PFAAs in tube B discloses the translocation potential of PFAAs through the phloem within plants16,17,18.
The split-root experiment has been reported for studying the long-distance translocation of many compounds in plants, such as CuO nanoparticles17, steroid estrogens18, and organophosphate esters16. These studies provided evidence that these compounds could transfer via the phloem to the edible parts of plants. However, whether PFAAs could aid in translocation in plants and the impact of compound properties need to be further explored. Based on these reports, the split-root experiment was conducted in the present study to disclose the long-distance transport of PFAAs in wheat.

<table><tbody><tr><td>ACQUITY UPLC BEH C18 column</td><td>Waters, Milford, MA</td><td></td><td>Liquid chromatographic column</td></tr><tr><td>Cleanert PEP cartridge</td><td>Bonna- Angel Technologies, China</td><td></td><td>Solid phase extraction column</td></tr><tr><td>Clearnert Pesticarb cartridge</td><td>Bonna- Angel Technologies, China</td><td></td><td>Solid phase extraction column</td></tr><tr><td>LC-MS/MS(Waters Acquity UPLC i-Class Coupled to Xevo TQ-S)</td><td>Waters, Milford, MA</td><td></td><td>Liquid chromatography and mass spectrometry</td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>Boyikang Instrument Ltd., Beijing, China</td><td>FD-1A50</td><td>Freeze-dried sample</td></tr><tr><td>Masslynx</td><td>Waters, Milford, MA</td><td></td><td>data analysis software</td></tr><tr><td>Methyl tert-butyl ether</td><td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td><td></td><td>use for extracting target compounds from plant tissues</td></tr><tr><td>MPFAC-MXA</td><td>Wellington Laboratories (Ontario, Canada)</td><td>PFACMXA0518</td><td>the internal standards</td></tr><tr><td>PFAC-MXB</td><td>Wellington Laboratories (Ontario, Canada)</td><td>PFACMXB0219</td><td>mixture of PFAA calibration standards</td></tr><tr><td>PFOA</td><td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td><td>335-67-1</td><td>a represent PFAAs</td></tr><tr><td>PFOS</td><td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td><td>2795-39-3</td><td>a represent PFAAs</td></tr><tr><td>Sodium carbonate buffer</td><td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td><td></td><td>use for extracting target compounds from plant tissues</td></tr><tr><td>Tetrabutylammonium hydrogen sulfate</td><td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td><td></td><td>use for extracting target compounds from plant tissues</td></tr><tr><td>Wheat seeds</td><td>Chinese Academy of Agricultural Sciences (Beijing,China)&amp;nbsp;</td><td></td><td>&lt;em&gt;Triticum aestivum L.&lt;/em&gt;</td></tr></tbody></table>

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.

The split-root experiment investigated the long-distance transport of PFAAs in wheat. As shown in Figure 2A,C, both PFOA and PFOS could be taken up by the wheat root and transferred to the shoot. PFOS and PFOA were not detected in the wheat root and solution in tube A of the blank control. It was found that PFOS and PFOA were detected in the wheat roots cultured in the unspiked solution, with a concentration of 0.26 ng/g &#177; 0.02 ng/g and 0.64 ng/g &#177; 0.05 ng/g dry we...

Watch this Scientific Journal Video about Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique at JoVE.com

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.

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 ...

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1.5K Views.  Northwest A&F University. The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.

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

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Biochemistry

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

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.

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.

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

Evaluation of Wild Peanut Species for Resistance to Aflatoxin Accumulation

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance.
Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes&#160;(stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins.
We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Author Spotlight: Quantification of Aflatoxins and Phytoalexins in Peanut Seeds to Identify Genetic Resistance Against Aspergillus

We demonstrate a medium-throughput method for quantification of aflatoxins and stilbenoid phytoalexins in single peanut seeds using ultra performance liquid chromatography. This method was specifically developed for the analyses of wild Arachis species challenged by the aflatoxigenic Aspergillus species.

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

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Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

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

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds