Name: measuring rates of herbicide metabolism in dicot weeds with an excised leaf assay
Uploaded: 2015-09-07T15:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay at JoVE.com

We thank Wendy Zhang, Austin Tom, Jacquie Janney, Erin Lemley, and Brittany Janney for assistance with plant growth and extractions, Dr. Anatoli Lygin for assistance with chromatographic analyses, and Syngenta Crop Protection for funding.

1. Plant Material, Growth Conditions, and Vegetative Cloning
Note: Three waterhemp populations were investigated in this research: MCR (from McLean County, IL), ACR (from Adams County, IL), and WCS (from Wayne County, IL) ( Table 1).
<ol>
	<li>Collect and suspend waterhemp seeds in 0.1 g L-1 agar:water solution at 4 &#176;C for at least 30 days to enhance germination. Note: Some waterhemp populations are dormant, but this step helps to overcome dormancy and make seed germinate more uniformly.</li>
	<li>Germinate seeds of each waterhemp population (as in Table 1) in 12 x 12 cm trays containing a potting medium in the greenhouse.
	<ol>
		<li>Maintain greenhouse conditions at 28/22 &#176;C day/night temperatures within a 16/8 hr photoperiod12.</li>
		<li>Supplement natural light in the greenhouse with light from mercury halide lamps to provide a minimum of 500 &#181;mol m-2 sec-1 photon flux at the plant canopy level.</li>
	</ol>
	</li>
	<li>Transplant emerged seedlings when they are 2 cm tall into 80 cm3 pots in the greenhouse.</li>
	<li>Transplant seedlings into 950 cm3 pots containing a 3:1:1:1 mixture of potting mix:soil:peat:sand when seedlings are 4 cm tall. Add 5 g of slow-release fertilizer granules to each pot and mix with this soil mixture.</li>
	<li>Cut and remove the shoot apical meristem containing the three youngest leaves from 7 cm tall plants to eliminate apical dominance. 
	Note: This step ensures that each plant will grow enough 3 cm axillary shoots to clone.</li>
	<li>Excise five axillary shoots (3 cm in length) when seedlings are about 14 cm tall from each waterhemp population (as in Table 1). Remove the majority of the leaves on these axillary shoots to decrease water loss upon further handling, but retain the two youngest leaves to allow further growth upon transplanting. 
	Note: Handle carefully and try not to damage the shoots when removing the leaves. Label each plant with a stake.</li>
	<li>Transplant axillary shoots into 80 cm3 pots (one seedling per pot) into potting medium, which is the same medium used for germination. Potting medium should be saturated initially until roots form in the growth chamber, then maintain constant moisture and place pots in the growth chamber for 7 days to establish roots. 
	Note: Plants are very sensitive at this point and should not be stressed.
	<ol>
		<li>Maintain growth chamber conditions at 28/22 &#176;C day/night temperatures within a 16/8 hr photoperiod12.</li>
		<li>A combination of incandescent and fluorescent bulbs can be used to provide light in the growth chamber and should deliver at least 550 &#181;mol m-2 sec-1 photon flux at plant canopy level12.</li>
	</ol>
	</li>
	<li>Transplant again into 950 cm3 pots (one seedling per pot) containing a 3:1:1:1 mixture of potting mix:soil:peat:sand (also include 5 g slow-release fertilizer granules) when cloned plants are 4 cm tall, then transfer back to the greenhouse.</li>
	<li>Clone the plants a second time by using the same method described above (i.e., repeat steps from 1.5 to 1.8, and then proceed to step 2.1). For a typical time-course study, generate clones from at least six independent plants from each waterhemp population and record them as lines MCR1-6, ACR1-6 and WCS1-6.
	<ol>
		<li>Establish at least six distinct &#8220;parent&#8221; clonal lines in order to repeat the experiment and adequately represent genetic variability within each waterhemp population.</li>
		<li>Depending on how many time points will be analyzed, generate enough sub-clones from each &#8220;parent&#8221; clonal line to conduct a time-course study.</li>
	</ol>
	</li>
</ol>
2. Conducting a Metabolism Study with Excised Waterhemp Leaves
<ol>
	<li>Excise the third-youngest leaves (2 to 3 cm in length; include 0.5 cm of attached petiole) when cloned waterhemp plants are 10 to 12 cm tall for herbicide metabolism and DT50 analysis.</li>
	<li>Cut leaf petioles from each waterhemp plant a second time (ensuring that 0.3 cm of the petiole remains) under water, and place these cut ends into 1.5 ml plastic vials (one leaf per vial)15. 
	Note: Cutting a second time under water prevents air bubbles from forming and plugging xylem vessels.
	<ol>
		<li>Firstly, partially immerse excised leaves with cut petiole in pre-incubation buffer (200 &#181;l) of 0.1 M Tris-HCl (pH 6) for 1 hr.</li>
		<li>Secondly, move each leaf to a new 1.5 ml vial containing an incubation buffer (200 &#181;l) of 0.1 M Tris-HCl (pH 6) plus 150 &#181;M [URL-14C] herbicide, and incubate at 28 &#176;C for 1 hr to allow for herbicide absorption into the leaves. 
		Note: Both 14C-mesotrione and 14C-primisulfuron-methyl were used in our studies at specific activities of about 20 mCi mmol-1. Both diffusion and uptake through the xylem transpiration stream contribute to the amount of herbicide taken up per leaf. 
		Note: The effect of metabolic inhibitors on herbicide metabolism can be determined by modifying steps 2.2.1 and 2.2.2 above. Add 100 &#181;M of the inhibitor to the pre-incubation buffer and let incubate for 2 hr, then transfer to incubation buffer with herbicide and again add 100 &#181;M inhibitor. For example, malathion and tetcyclacis inhibit certain cytochrome P450 activities in plants15-17.</li>
		<li>Wash the immersed portion of excised leaves with deionized water and transfer excised leaves to a quarter-strength Murashige and Skoog (MS) salts solution (500 &#181;l) for 0 hr, 5 hr, 11 hr, 23 hr, or 35 hr, respectively, in the growth chamber for the time-course metabolism study.</li>
	</ol>
	</li>
	<li>Harvest by removing leaves from incubation solution at the appropriate time point and manually grind each leaf tissue in liquid nitrogen using a polypropylene tube (15 ml) and glass pestle.</li>
	<li>Extract radiolabeled herbicide and its metabolites from each leaf with 7 ml&#160;of 90% acetone with a laboratory tissue homogenizer in the same polypropylene tube as above. Rinse tissue homogenizer with an additional 7 ml&#160;of 90% acetone, and continue to homogenize until tissue is thoroughly pulverized (usually about 2 min). Note: 90% acetone can be used for the extraction of most herbicides from plant tissues. However, if this method is unable to extract more than 90% of the radiolabeled compounds absorbed, then 90% methanol or 90% acetonitrile can be substituted as the extraction solvent.</li>
	<li>Label 90% acetone extracts (14 ml&#160;total) and store at -4 &#176;C for 16 hr to allow for additional extraction to occur. 
	Note: Following this extraction of treated leaves, the amount of extractable radioactivity is typically at least 98% of radiolabeled compounds absorbed by the treated leaf. Non-extractable radioactivity (bound residues) in excised leaves averaged only 0.3% across all time points in our previous study12, but this amount can vary according to the herbicide used and time point examined.</li>
	<li>Centrifuge samples at 5,000 x&#160;g for 10 min and concentrate supernatants at 40 &#176;C with a rotary evaporator until a final volume of 0.5 ml&#160;is obtained.&#160;Transfer this volume to a 2.0 ml plastic tube.</li>
	<li>Add acetonitrile:water (1:1, by volume) to adjust the final volume of the plant extracts to 1.25 ml&#160;and re-centrifuge at 10,000 x&#160;g for 10 min to remove any particulates.</li>
	<li>Measure total radioactivity in an aliquot taken from each sample (dpm &#181;l-1) by liquid scintillation spectroscopy (LSS)12 and normalize amounts of [14C-labeled] compounds injected for HPLC analysis (for example, 10,000 total dpm/sample/run) so y-axes will be uniform among samples when graphing results.</li>
</ol>
3. HPLC Analysis of Herbicide Metabolism in Excised Leaves
Note: Resolve total extractable radioactivity into parent herbicide and herbicide metabolites using the following RP-HPLC conditions12.
<ol>
	<li>Perform RP-HPLC with a C18 column (4.6 x 250 mm, 5 &#181;m particle size for analytical HPLC) at a flow rate of 1 ml&#160;min-1 for most non-polar herbicides. 
	Note: C4 or C12 RP-HPLC columns can also be used to optimize separation of the parent herbicide from its metabolites. A pre-column filter and/or guard column may also be used to protect and extend the life of the RP column.
	<ol>
		<li>Use the following mobile phase for RP-HPLC: Eluent A, 0.1% formic acid (v/v) in water, and Eluent B 100% HPLC-grade acetonitrile. 
		Note: HPLC-grade methanol can also be used as Eluent B for resolving more polar compounds and metabolites, but be aware that retention times may also change. 
		Note: The elution profile used to resolve mesotrione or primisulfuron-methyl from their polar metabolites in our research 12 is: Step 1, A:B (4:1, v/v) to A:B (3:2, v/v) in a linear gradient (12 min); Step 2, A:B (3:2, v/v) to A:B (3:7, v/v) in a linear gradient (5 min); Step 3, A:B (3:7, v/v) to A:B (1:9, v/v) in a linear gradient for 2 min (comprising a total of 19 min). 
		Note: Gradients and isocratic steps may need to be adjusted based on the physical properties of the parent material in order to optimize resolution.</li>
		<li>Follow the above linear gradient steps with A:B (1:9, v/v) to A:B (4:1, v/v) in a linear gradient (3 min) and A:B (4:1, v/v) isocratic step (for at least 2 min) to re-equilibrate the RP column before injecting the next extract12.</li>
	</ol>
	</li>
	<li>Detect and visualize radiolabeled compounds with a Flow Scintillation Analyzer. Record the amount of parent herbicide remaining in each sample as a percentage of total radioactivity in each sample (detected and quantified by the Flow Scintillation Analyzer according to the manufacturer&#8217;s instructions) to determine rates of herbicide metabolism in each waterhemp population during the time course.</li>
</ol>
4. Statistical Procedures
<ol>
	<li>Arrange treatments in a completely randomized design (or other suitable arrangement for statistical analysis).&#160;</li>
	<li>Perform two independent experiments with each treatment, comprised of three biological replicates for each experiment. The two independent experiments include six biological replicates in total. If the experiment effect is non-significant (&#945; = 0.05), combine and analyze data from each independent experiment. If the experiment effect is significant, then analyze each experiment separately. 
	Note: Include the first three biological replicates in Experiment 1 and the next three biological replicates in Experiment 2. Each biological replicate represents a distinct &#8220;parent&#8221; clonal line derived from each population (MCR1-6, ACR1-6, and WCS1-6) so that each time-course analysis utilizes genetically-identical plants. The two independent experiments include six biological replicates in total, which allows for adequate representation of genetic variability and determination of a median DT50 for each waterhemp population.</li>
	<li>Analyze the data with non-linear least squares regression analysis and fit them with a simple first-order curve in order to estimate DT50s. The equation below describes the model: 
	<img alt="Equation 1" src="/files/ftp_upload/53236/53236eq1.jpg" /></li>
	<li>where in this model y represents the percentage of the parent herbicide remaining at time t, &#181;i is the DT50 for each waterhemp population i, and the parameter C0&#160;is the estimated amount of parent herbicide present at t = 012.</li>
</ol>

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The authors have nothing to disclose.

The excised-leaf method described herein has been used previously in researching primisulfuron metabolism in maize leaves15, but our results demonstrate that this protocol is also effective, accurate, and reproducible for measuring herbicide metabolism in a dicot weed species12. A major advantage of the excised leaf technique compared with whole-plant studies is that an excised leaf is independent of whole-plant translocation patterns of postemergence, systemic herbicides or differences in herbicide...

Herbicide resistance in weeds presents a serious threat to the global production of food and fiber1,2. Currently, thousands of resistant populations and biotypes from over a hundred weed species worldwide have been documented and studied3. A major mechanism that confers herbicide resistance in plants is the alteration of herbicide target-site genes and proteins, including genetic mutations that affect herbicide-protein binding kinetics or amplification of the target-site gene2. Metabolic detoxification via elevated activities of cytochrome P450 monooxygenase(P450) or glutathione S-transferase (GST) enzymes is another mechanism that confers herbicide resistance in weeds, which is distinct in several ways from target-site-based mechanisms2. Metabolic-based resistance has significant ramifications for whether plant fitness costs (a.k.a. fitness penalties) may result from the herbicide-resistance mechanism, as well as regarding the potential for a single detoxification mechanism to confer cross- or multiple-herbicide resistance in weed populations1,2,4. Generally, herbicide metabolism in plants can be divided into three distinct phases5. Phase I involves herbicide conversion or activation such as P450-mediated hydroxylation of aromatic rings or alkyl groups, or by N- or O-dealkylation reactions, leading to increased polarity and partial herbicide detoxification5,6. Newly introduced functional groups in Phase I can provide linkage sites for conjugation to reduced glutathione by GSTs or to glucose by UDP-dependent glycosyltransferases in Phase II5,7. For example, the major initial metabolite of primisulfuron-methyl in maize is hydroxy-primisulfuron-methyl8, which can be further metabolized to hydroxy-primisulfuron-glucoside (Phase II) and then transported to the vacuole for long-term storage or further metabolic processing5,6 (Phase III).
Waterhemp (Amaranthus tuberculatus) is a difficult-to-control, dicot annual weed species that hinders the production of maize (Zea mays), soybean (Glycine max), and cotton (Gossypium hirsutum) in the United States. The high degree of genetic diversity of waterhemp is facilitated by its dioecious biology and long-distance wind pollination, and a single female waterhemp plant can produce up to a million seeds9. These seeds are small and easily spread, which naturally endow waterhemp with an effective dispersal mechanism. Waterhemp displays continuous germination throughout the growing season9, and its seeds are able to germinate after several years of dormancy. Waterhemp is a C4 plant that possesses a higher growth rate than most broadleaf weeds in arable cropping systems10. In addition, numerous waterhemp populations are resistant to multiple families of herbicides3.
A population of waterhemp (designated MCR) from Illinois is resistant to 4-hydroxy-phenylpyruvate dioxygenase (HPPD)-inhibiting herbicides11, such as mesotrione, as well as to atrazine and acetolactate synthase (ALS)-inhibiting herbicides, including primisulfuron-methyl, due to non-target-site based mechanisms12,13. A different population of waterhemp designated ACR14, which is primisulfuron-methyl-resistant (due to a mutation in the ALS gene) and atrazine-resistant but sensitive to mesotrione, and a waterhemp population designated WCS14 that is sensitive to primisulfuron-methyl, mesotrione, and atrazine were used in comparison with MCR in our prior research12 and current experiments (summarized in Table 1). Initial studies did not detect alterations in the HPPD gene sequence or expression levels, or reduced mesotrione uptake, in the MCR population when compared with mesotrione-sensitive populations12. However, metabolism studies with whole plants demonstrated significantly lower levels of parent mesotrione herbicide in MCR compared with ACR and WCS, which correlated with previous phenotypic responses to mesotrione11,12.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Waterhemp Population</td>
			<td>Abbreviation</td>
			<td>Phenotype to Mesotrione</td>
			<td>Mesotrione Resistance Mechanism</td>
			<td>Phenotype to Primisulfuron</td>
			<td>Primisulfuron Resistance Mechanism</td>
		</tr>
		<tr>
			<td>McLean County-Resistant</td>
			<td>MCR</td>
			<td>Resistant</td>
			<td>Metabolism*</td>
			<td>Resistant</td>
			<td>Metabolism</td>
		</tr>
		<tr>
			<td>Adams County-Resistant</td>
			<td>ACR</td>
			<td>Sensitive</td>
			<td>-</td>
			<td>Resistant</td>
			<td>Target-site mutation in ALS14</td>
		</tr>
		<tr>
			<td>Wayne County-Sensitive</td>
			<td>WCS</td>
			<td>Sensitive</td>
			<td>-</td>
			<td>Sensitive</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
* Non-target-site resistance mechanisms, other than enhanced metabolism, may also confer mesotrione resistance in the MCR population12.
Table 1: Description of waterhemp populations from Illinois used in this study.
In addition to determining rates of herbicide metabolism in intact waterhemp seedlings, a different experimental approach was developed and employed in our previous research to investigate metabolism by using an excised waterhemp leaf assay12 as well as various P450 inhibitors (e.g., tetcyclacis and malathion). This method was adapted specifically for waterhemp from a previous investigation of primisulfuron-methyl metabolism in excised maize leaves15, since the excised leaf assay had not yet been reported for conducting herbicide metabolism research in a dicot plant. The organophophosate insecticide malathion has been frequently used for in vivo and in vitro herbicide-metabolism research to indicate P450 involvement16. For example, tolerance and rapid metabolism of mesotrione in maize are due to P450-catalyzed ring hydroxylation, which was verified when malathion increased maize sensitivity to mesotrione17. Similarly, malathion inhibited metabolism of the ALS inhibitor primisulfuron-methyl in excised maize leaves15. A major advantage of the excised leaf technique is that data generated are independent of whole-plant translocation patterns, an important factor to consider when assessing metabolism of systemic, postemergence herbicides in plants. Consequently, this method allows quantitative and qualitative metabolic analyses to focus on a single treated leaf12.
A vegetative cloning strategy, in combination with the excised leaf protocol, was previously utilized in waterhemp to conduct metabolism studies12. Due to the outcrossing nature of waterhemp (separate male and female plants), and large degree of genetic diversity within dioecious Amaranthus species9, this protocol ensured that genetically-identical waterhemp seedlings were analyzed within the time-course experiments. This article demonstrates the utility of the excised leaf method for measuring rates of herbicide metabolism in a dicot weed (waterhemp). The quantity of parent herbicide remaining was determined at each time point (Figure 1) by non-linear least squares regression analysis, and was fit with a simple first-order curve in order to estimate the time for 50% of absorbed herbicide to degrade (DT50). Representative chromatograms from reversed-phase high performance liquid chromatography (RP-HPLC) are displayed for ALS-resistant and -sensitive waterhemp populations, which indicate the disappearance of parent herbicide and concomitant formation of polar metabolite(s) during a time-course study (Figure 2). The focus of our article is to describe and demonstrate the utility of the excised leaf assay in combination with a vegetative cloning method for determining precise and reproducible rates of herbicide metabolism in dicot plants, using uniformly ring-labeled (URL-14C) herbicides in three waterhemp populations that differ in their whole-plant responses to HPPD- and ALS-inhibiting herbicides (Table 1).

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td>for pre-germinating seeds</td></tr><tr><td>Potting medium</td><td>Sun Gro Horticulture</td><td>49040233</td><td>for plant growth</td></tr><tr><td>Nutricote</td><td>Agrivert&amp;nbsp;</td><td>TOTAL BLEND 13-13-13 T100</td><td>slow-release fertilizer</td></tr><tr><td>Growth chamber E15</td><td>Controlled Environments Limited</td><td>20207</td><td>plant culturing</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td>buffer for excised leaves</td></tr><tr><td>HCl (concentrated)</td><td>Fisher Scientific</td><td>A144500</td><td>adjust pH of buffer</td></tr><tr><td>Murashige and Skoog (MS) salts&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M0404</td><td>incubation of excised leaves</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452-4</td><td>leaf washes after incubation</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td>plant extractions</td></tr><tr><td>Acetonitrile (HPLC grade)</td><td>Macron Fine Chemicals</td><td>MKH07610</td><td>HPLC mobile phase</td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Mallinckrodt Analytical</td><td>MK259205</td><td>acidify mobile phase pH</td></tr><tr><td>Micro-centrifuge</td><td>Eppendorf</td><td>5417R</td><td>1.5 or 2.0 ml tubes</td></tr><tr><td>Centrifuge (temperature controlled)</td><td>Eppendorf</td><td>5810R</td><td>15 or 50 ml tubes</td></tr><tr><td>Polypropylene centrifuge tube</td><td>Corning Inc.</td><td>430790</td><td>15 ml, sterile</td></tr><tr><td>Rotary evaporator</td><td>B&amp;Uuml;CHI</td><td>R200</td><td>concentrate plant samples</td></tr><tr><td>Liquid scintillation spectrometry (LSS)</td><td>Packard Instruments</td><td>104470</td><td>quantify &lt;sup&gt;14&lt;/sup&gt;C</td></tr><tr><td>High-performance liquid chromatography</td><td>Perkin Elmer</td><td>N2910401</td><td>resolve herbicide metabolites</td></tr><tr><td>Flow scintillation analyzer&amp;nbsp;</td><td>LabLogic System</td><td>1103303</td><td>for HPLC analysis of &lt;sup&gt;14&lt;/sup&gt;C</td></tr><tr><td>Hypersil Gold C&lt;sub&gt;18&lt;/sub&gt; column&amp;nbsp;</td><td>Thermo-Scientific</td><td>03-050-522&amp;nbsp;</td><td>reversed phase</td></tr><tr><td>Ultima-Flo M cocktail</td><td>Perkin Elmer</td><td>6013579</td><td>for Flow-scintillation analyzer</td></tr><tr><td>Scintillation Cocktail (ScintiVerse BD)</td><td>Fisher Scientific</td><td>SX18</td><td>for LSS; biodegradable</td></tr><tr><td>Laboratory homogenizer</td><td>Kinematica</td><td>CH-6010&amp;nbsp;</td><td>homogenize leaf samples</td></tr></tbody></table>

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.

Large differences in rates of mesotrione metabolism were detected between either WCS or ACR and MCR (Figure 1). At each time point, MCR had metabolized mesotrione more rapidly than the two mesotrione-sensitive populations, WCS and ACR, which correlates with previous whole-plant phenotypic responses11. By cloning enough plants from a single parental plant from each population, herbicide metabolism time-course analyses are uniform and reproducible due to the lack of genetic vari...

Watch this Scientific Journal Video about Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay at JoVE.com

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

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 ...

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Research

JoVE Journal

Environment

11.9K Views.  University of Illinois. 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.

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

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Biochemistry

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Bioengineering

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 ...

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, ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Immunology and Infection

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Biology

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

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

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