This study was supported by the National Natural Science Foundation of China (21976160) and Zhejiang Province Public Welfare Technology Application Research Project (LGF21B070006).

1. Differentiation of carrot callus
NOTE: Autoclave all equipment used here and perform all operations in a UV-sterilized ultra-clean workbench.
<ol>
	<li>Vernalize the seeds by immersing the uniform carrot seeds (Daucus carota var. sativus) into deionized water at 4 &#176;C for 16 h.</li>
	<li>Surface-sterilize the vernalized seeds with 75% ethanol for 20 min, and then rinse three times with sterile deionized water under aseptic conditions.</li>
	<li>Further sterilize the seeds with 20% H2O2 for 20 min, and wash them with sterilized deionized water six times under aseptic conditions.</li>
	<li>Aseptically germinate the seeds by sowing them on hormone-free MS medium (pH 5.8, autoclaved at 121 &#176;C for 20 min) containing 1% agar-gel, and incubating at 26 &#176;C with a 16 h photoperiod (350 &#181;mol/m2s) for 15 days.</li>
	<li>Obtain the explants by cutting the hypocotyl and cotyledon of the seedlings into small pieces (0.5 cm).</li>
	<li>Transform the explants to Petri dishes (two to four explants per dish) containing 15-20 mL of aseptic MS medium supplemented with auximone (2,4-dichlorophenoxyacetic acid; 1 mg/L) and phytokinin (6-benzylaminopurine; 0.5 mg/L) under aseptic conditions.</li>
	<li>Incubate the explants in the dark at 26 &#176;C for 3-4 weeks to induce the callus.</li>
	<li>Separate the callus tissues (around 1 cm diameter) formed from the initial explants using a sterile scalpel and forceps. 
	NOTE: The newly formed callus tissues are white to creamy yellow in color and loosely attach to the initial explants.</li>
</ol>
2. 2,4-dibromophenol treatment
<ol>
	<li>Dissolve 1 &#181;g of 2,4-dibromophenol in 10 mL of aseptic liquid MS medium (the final concentration of 2,4-dibromophenol is 100 ppb, pH 5.6-7.0).</li>
	<li>Add 3 g of the fresh carrot callus (step 1.8) into glass flasks containing the prepared 2,4-dibromophenol solution (from step 2.1) under aseptic conditions. Consider this as the 2,4-dibromophenol treatment. 
	NOTE: The glass flasks were autoclaved and sealed using paraffin film.</li>
	<li>Include a medium control containing the 2,4-dibromophenol solution only (prepared in step 2.1) to evaluate the abiotic degradation of 2,4-dibromophenol.</li>
	<li>Include a blank control containing the carrot callus only (no 2,4-dibromophenol solution) to check for any potential contamination.
	<ol>
		<li>Prepare the blank control containing carrot by adding 3 g of fresh collected carrot callus only into 10 mL of sterile MS medium.</li>
	</ol>
	</li>
	<li>Incubate the 2,4-dibromophenol treatment and the medium and blank controls at 130 rpm and 26 &#176;C in the dark in an incubator for 120 h.</li>
	<li>Remove the glass flasks from the incubator to collect the samples from the 2,4-dibromophenol treatment and controls after 120 h of incubation. 
	NOTE: All samples were prepared in triplicate.</li>
</ol>
3. Sample preparation
<ol>
	<li>Separate the callus carefully from the MS medium by filtration with glass fiber filters (0.45 &#181;m) for the 2,4-dibromophenol treatment and the controls. Collect the callus after washing with ultrapure water three times.</li>
	<li>Freeze-dry the collected callus with liquid nitrogen, and subsequently homogenize the collected callus (0.2 g) with a high-throughput tissue grinder at 70 Hz for 3 min.</li>
	<li>Spike the homogenized callus by adding 50 &#181;L of 25 mg/L surrogate 4-n-NP-d4 with a glass microsyringe and subsequently vortexing for 1 min.</li>
	<li>Sonicate the samples with 5 mL of methanol/water (1:1, v/v) in an ultrasonicator (150 W, 40 kHz) filled with ice water for 30 min, to extract 2,4-dibromophenol and the metabolites.</li>
	<li>Centrifuge the suspensions at 8,000 x g at 4 &#176;C for 10 min, and collect the supernatants by pipetting.
	<ol>
		<li>Repeat the extraction processes for the callus sample three times and combine the extracts.</li>
	</ol>
	</li>
	<li>Pass the extracts through hydrophilic lipophilic balanced solid phase extraction (HLB SPE) cartridges with a flow rate of 1 mL/min. 
	NOTE: The HLB SPE cartridges were pretreated sequentially with 6 mL of methanol and 6 mL of water to remove any interferences.</li>
	<li>Elute the analytes by passing 6 mL of methanol through the HLB SPE cartridges. Then, concentrate the obtained eluents to 1 mL under a gentle stream of nitrogen gas for instrumental analysis.</li>
	<li>Inject 10 &#181;L of resultant eluents into the UPLC-Q-TOF-MS for 2,4-dibromophenol and their metabolites analysis15.
	<ol>
		<li>Filter all the samples with a 0.22 &#181;m nylon membrane before instrumental analysis.</li>
	</ol>
	</li>
</ol>
4. Instrumental analysis
NOTE: 2,4-dibromophenol and their metabolites&#39; analyses were performed on an ultra-performance liquid chromatograph (UPLC) in combination with a micrOTOF-QII mass spectrometer equipped with an electrospray ionization (ESI), operating in the positive and negative ion mode.
<ol>
	<li>Open the column heater door and install the UPLC column by connecting the column inlet to the injection valve and column outlet to the mass spectrometer&#39;s inlet. 
	NOTE: A C18 column (50 mm x 2.1 mm; 1.7 &#181;m particle size) was used for the separation of analytes at 40 &#176;C.</li>
	<li>Connect mobile phase A (ultrapure water) and mobile phase B (chromatography-grade methanol) to the instrument by inserting the end of the solvent tubes A and B into the corresponding solvent bottles, respectively.
	<ol>
		<li>Filter all mobile phases (500 mL for each) through a 0.22 &#181;m filter, and sonicate for more than 30 min.</li>
	</ol>
	</li>
	<li>In the software window, click on Instrument | Inlet Method to edit the conditions for the liquid chromatogram.
	<ol>
		<li>Set the gradient conditions of mobile phase B as follows: a flow rate of 1.0 mL/min; 0-0.5 min, 5%; 0.5-3.5 min, 5% to 50%; 3.5-6.5 min, 50% to 100%; 6.5-7 min, 100%; 7-10 min, 100% to 5%.</li>
		<li>Set the injection rate of the samples into the UPLC-Q-TOF-MS as 0.2 mL/min. 
		NOTE: The injection of the sample is programmed using a fully automatic sampler.</li>
	</ol>
	</li>
	<li>In the software window, select MS Method and then set up the parameters of Q-TOF-MS: a drying gas (N2) flow rate of 8 L/min, a temperature of 300-350 &#176;C; a capillary voltage of 4,500 V; a collision energy of 5-45 V; and a full scan range of 40-800 Da.</li>
	<li>Place the sample vials in the corresponding locations of the sample trays by serial number, and re-insert the sample trays into the sample chamber. 
	NOTE: Keep the sample trays flat and make sure that the door of the sample chamber is closed.</li>
	<li>In the software window, select File | New to create a database. Name the database.</li>
	<li>Load the sample program created above by selecting MS File | Inlet file | Inject Volume.</li>
	<li>Save the database in the sample folder of the project by clicking File | Save.</li>
	<li>Select Run | Start in the software main window, and then select Acquire Sample Data and click OK in the window of the start sample list run to collect data. 
	NOTE: The real-time chromatogram can be viewed by clicking Chromatogram | Real-time Update during the data acquisition process.</li>
	<li>Process the data in the software by selecting the target data row and clicking the Chromatogram window to view the MS scan chromatogram.</li>
	<li>In the Chromatogram window, click Display | TIC | ScanWaveDS | Add trace | OK to get the daughter scan mass spectra.</li>
	<li>Identify the metabolites by comparing the chromatograms of the 2,4-dibromophenol treatment and the controls.</li>
	<li>Elucidate the metabolite candidates by the retention time, mass, and fragmentation patterns16,17. 
	NOTE: The error of mass accuracy between the experimental m/z values of the parent ions of the metabolite candidates to their corresponding theoretical m/z should be less than 10 ppm.</li>
</ol>

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The authors have nothing to disclose.

This protocol was developed to efficiently identify the biotransformation of xenobiotics in plants. The critical step of this protocol is the culture of the plant callus. The most difficult part is the differentiation and maintenance of the plant callus, because the plant callus is easily infected and developed to plant tissues. Therefore, it is important to make sure that all equipment used is autoclaved, and all operations are performed under aseptic conditions. The differentiation and maintenance of the plant callus s...

An increasing number of organic pollutants have been discarded into the environment due to anthropogenic activities1,2, and soil is considered a major sink for these contaminants3,4. The contaminants in the soil can be taken up by plants and potentially transferred to higher trophic-level organisms along food chains, by directly entering the human body through crop consumption, consequently leading to unintended exposure5,6. Plants utilize different pathways to metabolize xenobiotics for detoxification7; elucidating the metabolism of xenobiotics is important, as it controls the actual fate of contaminants in plants. As the metabolites can be excreted by leaves (to the atmosphere) or the roots, determining the metabolites in the very early phases of exposure hence provides the possibility to test an extended number of metabolites8. However, studies using intact plants require long-term experiments and complex sample preparation protocols that can be affected by various factors.
Plant callus cultures, therefore, are a good alternative for studying the metabolism of xenobiotics in planta, as they can greatly shorten the treatment time. These cultures exclude microbial interference and photochemical degradation, simplify the matrix effect of intact plants, standardize the cultivation conditions, and require less experimental effort. Plant callus cultures have been successfully applied as an alternative approach in metabolic studies of triclosan9, nonylphenol10, and tebuconazole8. These studies showed that the metabolic patterns in callus cultures were similar to those in intact plants. This study proposes a method for the efficient and accurate identification of metabolites of xenobiotics in plants without complex and time-consuming protocols. Here, we use plant callus cultures in combination with high-resolution mass spectrometry for the analysis of metabolites with low-intensity signals11,12.
To this end, carrot (Daucus carota var. sativus) callus suspensions were exposed to 100 &#181;g/L of 2,4-dibromophenol for 120 h in a shaker at 130 rpm and 26 &#176;C. 2,4-dibromophenol was chosen due to its disruptive endocrine activity13 and widespread occurrence in soil14. The metabolites were extracted and analyzed by high-resolution mass spectrometry. The protocol proposed here can investigate the in planta metabolism of other types of organic compounds that can be ionized.

<table><tbody><tr><td>2,4-dichlorophenoxyacetic acid</td><td>WAKO</td><td>1 mg/L</td><td></td></tr><tr><td>20% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co., Ltd.</td><td>10011218-500ML</td><td></td></tr><tr><td>4-&lt;em&gt;n&lt;/em&gt;-NP, &gt;99%</td><td>Dr. Ehrenstorfer GmbH</td><td></td><td></td></tr><tr><td>4-n-NP-d&lt;sub&gt;4&lt;/sub&gt;</td><td>Pointe-Claire</td><td></td><td></td></tr><tr><td>6-benzylaminopurine</td><td>WAKO</td><td>0.5 mg/L</td><td></td></tr><tr><td>75% ethanol</td><td>Sinopharm Chemical Reagent Co., Ltd.</td><td>1269101-500ML</td><td></td></tr><tr><td>7890A-5975&amp;nbsp;gas chromatography</td><td>Agilent</td><td></td><td></td></tr><tr><td>ACQULTY ultra-performance&amp;nbsp;liquid chromatography</td><td>Waters</td><td></td><td></td></tr><tr><td>Amber glass vials</td><td>Waters</td><td></td><td></td></tr><tr><td>Artificial climate incubator</td><td>Ningbo DongNan Lab Equipment Co.,LTD</td><td>RDN-1000A-4</td><td></td></tr><tr><td>Autoclaves</td><td>STIK</td><td>MJ-Series</td><td></td></tr><tr><td>C18 column</td><td>ACQUITY UPLC BEH</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>DB-5MS capillary column</td><td>Agilent</td><td></td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>40071190-4L</td><td></td></tr><tr><td>Freeze dryer</td><td>SCIENTZ&amp;nbsp;</td><td></td><td></td></tr><tr><td>High-throughput tissue grinder</td><td>SCIENTZ&amp;nbsp;</td><td></td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>MicrOTOF-QII mass spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Milli-Q system</td><td>Millipore</td><td>MS1922801-4L</td><td></td></tr><tr><td>Murashige &amp;amp; Skoog medium</td><td>HOPEBIO</td><td>HB8469-7</td><td></td></tr><tr><td>N-hexane</td><td>Sigma-Aldrich</td><td>H109658-4L</td><td></td></tr><tr><td>Nitrogen blowing instrument&amp;nbsp;</td><td>AOSHENG</td><td>MD200-2</td><td></td></tr><tr><td>NP isomers, &gt;99%</td><td>Dr. Ehrenstorfer GmbH</td><td></td><td></td></tr><tr><td>Oasis HLB cartridges</td><td>Waters</td><td>60 mg/3 mL</td><td></td></tr><tr><td>Research plus</td><td>Eppendorf</td><td></td><td>100-1000 &amp;micro;L</td></tr><tr><td>Seeds of Little Finger carrot (Daucus carota var. sativus)&amp;nbsp;</td><td>Shouguang Seed Industry Co., Ltd</td><td></td><td></td></tr><tr><td>Shaking&amp;nbsp;Incubators</td><td>Shanghai bluepard instruments Co.,ltd.</td><td>THZ-98AB</td><td></td></tr><tr><td>Solid phase extractor</td><td>AUTO SCIENCE</td><td></td><td></td></tr><tr><td>Ultrasound machine</td><td>ZKI</td><td>UC-6</td><td></td></tr><tr><td>UV-sterilized ultra-clean workbench</td><td>AIRTECH</td><td></td><td></td></tr></tbody></table>

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 steps of the protocol are depicted in Figure 1. Following the protocol, we compared the chromatogram of the carrot callus extract from the 2,4-dibromophenol treatment to the controls, and found eight distinct peaks that are present in the 2,4-dibromophenol treatment but absent in the controls (Figure 2). This indicates that a total of eight metabolites of 2,4-dibromophenol (M562, M545, M661, M413, M339, M380, M424, and M187) were successfully detected in the...

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Elucidating the Metabolism of 2,4-Dibromophenol 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 ...

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JoVE Journal

Environment

777 Views.  Zhejiang University of Technology. Planteneeltcultuur biedt een oplossing voor de nauwkeurige, efficiënte en snelle evaluatie van metabole afbraak van xenobiotica in planten. Planteneeltcultuur kan microbioom- of schimmelinterferentie en fotochemische afbraak uitsluiten, het matrixeffect van intacte planten vereenvoudigen, de teeltomstandigheden standaardiseren, de behandelingsduur verkorten en minder experimentele inspanning vereisen. De hier voorgestelde methode kan inzicht geven in het opnamegedrag en de metabolische mecha...

Video: Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Biochemistry

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 ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

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

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 ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

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 ...

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, ...

Primary Organ Identification in Medicinal Plants Using 13C6&#45;Glucose Labeling

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Author Spotlight: Plant Primary Organs Profiling Using 13C6&#45;Glucose Labeling and LC&#45;MS

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

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