This work was supported by the National Key Research &#38; Development Program (2016YFD0200900) of China, the National Natural Scientific Foundation of China (No. 31772076 and No. 31270728), China Postdoctoral Science Foundation (2018M630700), and Open Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20180111).

1. Tea callus culture
NOTE: Sterile leaves were derived from in vitro-grown plantlet lines first developed in the research group17. All procedures up to section 5 were carried out in a sterile laminar flow hood, except for the culture time in an incubator.
<ol>
	<li>Adjust the pH of the two media (Murashige and Skoog [MS] basal medium and Gamborg&#39;s B5 liquid medium) to 5.8 prior to autoclaving (121 &#176;C, 20 min).</li>
	<li>Cut along the middle vein of a sterile leaf using scissors, and then subdivide each half into small pieces of about 0.3 cm x 0.3 cm in a petri dish.</li>
	<li>Place the sterile explants (the small leaf pieces) onto MS basal media containing the plant hormones 2,4-D (1.0 mg L-1) and KT (0.1 mg L-1). Six explants can be placed in a 300 mL flask containing 100 mL of MS basal media.</li>
	<li>&#160;Culture the above leaf explants at a constant temperature of 25 &#176;C in the dark. After 28 days, select the first generation of induced callus and transfer to fresh flask (a subculture). Acquire the loose and friable callus after 3&#8722;4 subcultures.</li>
</ol>
2. Tea cell suspension culture
<ol>
	<li>Cut the vigorous, friable and loose calluses from the solid medium into small pieces (range here 0.5&#8722;2 mm) using a sterile surgical blade under sterile conditions.</li>
	<li>Weigh about 3 g of the small pieces of callus. Place the callus into a 150 mL flask containing 20 mL of B5 liquid media supplemented with 2,4-D (1.0 mg L-1) and KT (0.1 mg L-1).</li>
	<li>Culture the liquid cell suspension at a constant temperature (25 &#177; 1 &#176;C) in a shaking incubator at 120 rpm in the dark.</li>
	<li>After 7 to 10 days of culturing, remove the culture flasks and let them stand for a few minutes.</li>
	<li>Take all the supernatant as seed material for subculture to fresh medium (subculture ratio of suspension mother liquid to fresh medium ranged between 1:1 and 1:2). Remove the precipitated, large calluses.</li>
	<li>Obtain the final well-grown cell suspension culture after 3&#8722;4 subculture cycles of 28 days each.</li>
</ol>
3. Triphenyl tetrazolium chloride assay of cell viability
<ol>
	<li>Kill a sample of living cells at 100 &#176;C for 10 min as a control cell before viability staining.</li>
	<li>Centrifuge all cell suspension culture for 8 min at 6000 x g. Remove the supernatant before suspending the cells in 2.5 mL of phosphate-buffered saline (PBS) buffer (pH 7.3), and shake it for 1 min by hand.</li>
	<li>Add 2.5 mL of the 0.4%triphenyl tetrazolium chloride (TTC) solution and shake by hand again.</li>
	<li>Incubate the mixture for 1 h in a standing incubator (30 &#176;C).</li>
</ol>
4. Treatment and sampling of tea cell suspension cultures with insecticides
<ol>
	<li>Add an aliquot of 400 &#181;L of filter-sterilized stock solution (500 &#181;g mL-1) of four neonicotinoids (thiamethoxam, acetamiprid, imidacloprid, and imidaclothiz) or two organophosphates (dimethoate and omethoate) into the cell suspension cultures, respectively. 
	NOTE: If the aim is to compare xenobiotic behaviors, use the same mother batch of cell suspensions to test the different compounds.</li>
	<li>Culture the samples of cell suspensions with insecticides at constant temperature (25 &#177; 1 &#176;C) and shaking incubator speed (120 rpm). Take the samples (see step 4.3 or 4.4) on 0, 3, 5 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 days.</li>
	<li>To test a sample containing a neonicotinoid, remove a 1 mL aliquot of the homogeneous cell culture, place it into a 1.5 mL plastic centrifuge tube, and centrifuge at 4000 x g for 2 min.
	<ol>
		<li>Pass the supernatants through a 0.22-&#181;m pore-size filter membrane before analysis by high-performance liquid chromatography-ultraviolet (HPLC-UV) and ultra-high performance liquid chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry (Table of Materials).</li>
	</ol>
	</li>
	<li>To test a sample containing an organophosphate, remove a 500 &#181;L aliquot of the cell culture and place into a 35 mL centrifuge tube or a 1.5 mL plastic centrifuge tube (prepare the latter sample like that of neonicotinoid).
	<ol>
		<li>Add 0.1 g of sodium chloride and 5 mL of acetone/ethyl acetate (3:7, v/v) into the 35 mL centrifuge tube of the 500 &#181;L samples.</li>
		<li>Vortex the mixtures for 1 min, and then allow them to rest for 10 min.</li>
		<li>Take 2.5 mL of the supernatant into a 10 mL glass tube and evaporate to near-dryness using a nitrogen evaporator at 40 &#176;C.</li>
		<li>Dissolve the residue with 1 mL acetone, vortex for 1 min, pass it through a 0.22-&#181;m filter membrane before analysis by gas chromatography-flame photometric detector (GC-FPD).</li>
	</ol>
	</li>
</ol>
5. Sample preparation of intact tea plant with insecticides
NOTE: The intact tea plant trial was conducted in a hydroponic system using tea seedlings grown in 50 mL of a nutrient solution (30 NH4+, 10 NO3-, 3.1 PO4-, 40 K+, 20 Ca2+, 25 Mg2+, 0.35 Fe2+, 0.1 B3+, 1.0 Mn2+, 0.1 Zn2+, 0.025 Cu2+, 0.05 Mo+, and 10 Al3+, in mg L-1)18. An experimental greenhouse was under a light-dark cycle (12 h of light and 12 h of darkness) at 20 &#176;C at Anhui Agricultural University.
<ol>
	<li>Put five plants in a 4 L plastic pot for 15 days.</li>
	<li>Add 0 ppm (control) or 100 ppm of thiamethoxam or dimethoate into plastic pots, respectively.</li>
	<li>Prepare the intact plant sample according to the previous method, except for presoaking19, and then analyze with mass spectrometry for an accurate mass spectrum.</li>
</ol>
6. Instrument analysis
<ol>
	<li>HPLC analysis of the metabolic behavior of neonicotinoids
	<ol>
		<li>Use an HPLC-UV (Table of Materials) to detect the content and metabolic products of thiamethoxam and acetamiprid at a wavelength of 254 nm, and of imidacloprid and imidaclothiz at 270 nm in samples from section&#160;4.3. 
		NOTE: The HPLC-UV condition was the same as the previous study19.</li>
	</ol>
	</li>
	<li>GC analysis of the metabolic behavior of organophosphates
	<ol>
		<li>Detect the content of dimethoate and omethoate in samples from section&#160;4.4 by a GC-FPD using a chiral column (Table of Materials).</li>
		<li>Use nitrogen as the carrier gas and set the flow rate at 1.0 mL min-1.</li>
		<li>Set the initial temperature to 120 &#176;C, and hold it for 5 min. Increase the temperature to 150 &#176;C at 30 &#176;C min-1 and hold for 3 min. Increase to 170 &#176;C at 10 &#176;C min-1 and hold for 7 min. Finally increase to 210 &#176;C at 30 &#176;C min-1 and then hold for 5 min.</li>
		<li>Set the injection temperature to 200 &#176;C in splitless mode; Set the detector temperature to 250 &#176;C.</li>
		<li>Set the injection volume to 1 &#181;L.</li>
	</ol>
	</li>
	<li>UPLC-QTOF analysis of the insecticide metabolites in cell culture
	<ol>
		<li>Detect the metabolites of the insecticides in cell culture (samples from section&#160;4.3) using UPLC-QTOF with a C18 column (Table of Materials).</li>
		<li>Set the flow rate to 0.2 mL min-1. Set the injection volume to 10 &#181;L.</li>
		<li>For the neonicotinoid-treated samples, set the initial mobile phase to 85% A (5 mM ammonium formate water) and 15% B (acetonitrile). Over 10 min, increase mobile phase B to 38% and return to 15% over 1 min, hold for 9 min.</li>
		<li>For the organophosphate-treated samples, set the initial mobile phase to 55% A (0.1% formic acid water) and 45% B (acetonitrile). Over 5 min, increase mobile phase B to 70%, then return to 45% of B over 0.5 min, hold for 2.5 min.</li>
		<li>Set the QTOF operation parameters as follows: gas temperature, 325 &#176;C; drying gas (nitrogen), 10 L min-1; sheath gas temperature, 350 &#176;C; sheath gas flow, 11 L min-1; capillary voltage, 4000 V; nozzle voltage, 1000 V; fragmentor voltage, 100 V for neonicotinoid insecticides or 110 V for organophosphorus insecticides; skimmer voltage, 65 V; operating in positive ion mode.</li>
		<li>Set the instrument to the full scan spectrum and target MS/MS mode.</li>
		<li>Process the data using accurate mass tools; Infer the metabolites with no standard products from the MS/MS annotation as well as the literature12,15,20,21,22.</li>
	</ol>
	</li>
	<li>UPLC-Orbitrap analysis of the insecticide metabolites in intact plant extract
	<ol>
		<li>Detect the metabolites of insecticides in intact plant extract using UPLC-Orbitrap mass spectrometry (Table of Materials).</li>
		<li>Set the mass spectrometry (Table of Materials) operation parameters as follows: sheath gas pressure, 35 arb; gas temperature, 300 &#176;C; nozzle voltage, 3.5 KV; capillary temperature, 350 &#176;C.</li>
		<li>Set the elution programs as the above (steps 6.3.3 and 6.3.4) for UPLC-QTOF analysis of cell culture.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article presents the detailed process of establishing a model of pesticide metabolism in tea plant tissue, including the selection of explants, the determination of cell viability, and the establishment of a tea cell suspension culture with high metabolic activity. Any parts of a plant tissue could be used to initiate callus in a sterilized environment25. Tea leaves were chosen for callus initiation in this study, not only because leaves to tend to be less contaminated than the parts below gr...

Tea is one of the most widely consumed non-alcoholic beverages in the world1,2. Tea is produced from the leaves and buds of the woody perennial Camellia sinensis L. Tea plants are grown in vast plantations and are susceptible to numerous insect pests3,4. Organophosphorus and neonicotinoid insecticides are often used as systemic insecticides5 to protect tea plants from pests such as whiteflies, leaf hoppers, and some lepidopteran species6,7. After application, these insecticides are absorbed or translocated into the plant. Within the plant, these systemic insecticides may be transformed through hydrolysis, oxidation or reduction reactions by plant enzymes. These transformation products can be more polar and less toxic than the parent compounds. However, for some organophosphates, the bioactivities of some products are higher. For example, acephate is metabolized into the more toxic methamidophos8,9, and dimethoate into omethoate10,11. Plant metabolic studies are thus important for determining the fate of a pesticide within a plant12.
Plant tissue cultures have been proven to be a useful platform for investigating the pesticide metabolism, with the identified metabolites similar to those found in intact plants13,14,15. The use of tissue cultures, particularly cell suspension cultures, has several advantages. Firstly, experiments can be carried out free of microorganisms, thus avoiding the interference of pesticide transformation or degradation by microbes. Secondly, tissue culture provides consistent materials for use at any time. Thirdly, the metabolites are easier to extract from tissue cultures than from intact plants, and tissue cultures often have fewer interring compounds and lower complexity of compounds. Finally, tissue cultures can more easily be used to compare a series of pesticides metabolism in a single experiment16.
In this study, a cell suspension derived from the leaves of sterile-grown tea plantlet was successfully established. The tea cell suspension culture was then used to compare the dissipation behaviors of six systemic insecticides.
This detailed protocol is intended to provide some guidance so that researchers can establish a plant tissue culture platform useful for studying the metabolic fate of xenobiotics in tea.

<table><tbody><tr><td>Acetamiprid (99.8%)</td><td>Dr. Ehrenstorfer</td><td>46717</td><td>CAS No: 135410-20-7</td></tr><tr><td>Acetonitrile (CAN, 99.9%)</td><td>Tedia</td><td>AS1122-801</td><td>CAS No: 75-05-8</td></tr><tr><td>Agar</td><td>Solarbio Science &amp;amp; Technology</td><td>A8190</td><td>CAS No: 9002-18-0</td></tr><tr><td>Clothianidin (99.8%)</td><td>Dr. Ehrenstorfer</td><td>525</td><td>CAS No: 210880-92-5</td></tr><tr><td>Dimethoate (98.5%)</td><td>Dr. Ehrenstorfer</td><td>109217</td><td>CAS No: 60-51-5</td></tr><tr><td>Imidacloprid (99.8%)</td><td>Dr. Ehrenstorfer</td><td>91029</td><td>CAS No: 138261-41-3</td></tr><tr><td>Imidaclothiz (99.5%)</td><td>Toronto Research Chemical</td><td>I275000</td><td>CAS No: 105843-36-5</td></tr><tr><td>Kinetin (KT, &gt;98.0%)</td><td>Solarbio Science &amp;amp; Technology</td><td>K8010</td><td>CAS No: 525-79-1</td></tr><tr><td>Omethoate (98.5%)</td><td>Dr. Ehrenstorfer</td><td>105491</td><td>CAS No: 1113-02-6</td></tr><tr><td>Polyvinylpolypyrrolidone (PVPP)</td><td>Solarbio Science &amp;amp; Technology</td><td>P8070</td><td>CAS No: 25249-54-1</td></tr><tr><td>Sucrose</td><td>Tocris Bioscience</td><td>5511</td><td>CAS No: 57-50-1</td></tr><tr><td>Thiamethoxam (99.8%)</td><td>Dr. Ehrenstorfer</td><td>20625</td><td>CAS No: 153719-23-4</td></tr><tr><td>Triphenyltetrazolium Chloride (TTC, 98.0%)</td><td>Solarbio Science &amp;amp; Technology</td><td>T8170</td><td>CAS No: 298-96-4</td></tr><tr><td>2,4-Dichlorophenoxyacetic Acid (2,4-D, &gt;98.0%)</td><td>Guangzhou Saiguo Biotech</td><td>D8100</td><td>CAS No: 94-75-7</td></tr><tr><td>chiral column</td><td>Agilent CYCLOSIL-B</td><td>112-6632</td><td>Chromatography column (30 m &amp;times; 0.25 mm &amp;times; 0.25 &amp;mu;m)</td></tr><tr><td>Gas chromatography (GC)</td><td>Shimadu</td><td>2010-Plus</td><td>Paired with Flame Photometric Detector (FPD)&amp;nbsp;&amp;nbsp;</td></tr><tr><td>High-performance liquid chromatography (HPLC)</td><td>Agilent</td><td>1260</td><td>Paired with Ultraviolet detector (UV)</td></tr><tr><td>HSS T3 C18 column</td><td>Waters</td><td>186003539</td><td>Chromatography column (100 mm &amp;times; 2.1 mm &amp;times; 1.8 &amp;mu;m)</td></tr><tr><td>Ultra-high-performance liquid chromatography (UPLC)</td><td>Agilent</td><td>1290-6545</td><td>Tandem quadrupole time-of-flight mass spectrometer (QTOF)</td></tr><tr><td>Ultra-high-performance liquid chromatography (UPLC)</td><td>Thermo Scientific</td><td>Ultimate 3000-Q Exactive Focus</td><td>Connected to a Orbitrap mass spectrometer</td></tr></tbody></table>

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.

The induction of callus from leaves harvested from field-grown tea trees and from leaves excised from tea plantlets grown in vitro in a sterile environment was compared by measuring contamination, browning, and induction after 28 days of cultivation on MS media (Figure 1A). Callus growth was recorded at 20, 37, 62 and 90 days of culture (Figure 1B). The callus derived from the in vitro-grown leaves showed more vigorous growth tha...

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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.

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 ...

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Research

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Biochemistry

7.7K Views.  Anhui Province Key Lab of Analysis and Detection for Food Safety. Thee is een van de meest populaire drankjes over de hele wereld. Pesticiden worden soms gebruikt om theeplanten te beschermen tegen ongedierte. Sommige systemische pesticiden kunnen worden gemaakt voor dit door enzymen in theeboom.En vervolgens geïntegreerd door oxidatie, hydrolyse, of vermindering reacties. Plant cel suspensie culturen kunnen worden gebruikt als een, gemakkelijk om plant metabolisme gedrag van pesticiden te bestuderen. Deze methode heeft verschillende voordelen.

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

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 ...

Environment

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as 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 ...

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

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, ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

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

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