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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+herbicide+responses+among+Illinois+waterhemp+(Amaranthus+rudis+and+A.+tuberculatus)+populations.">Variable herbicide responses among Illinois waterhemp (Amaranthus rudis and A. tuberculatus) populations.</a> Crop Protection. 21 (9), 707-712 (2002).</li><li>Jalaludin, A., Yu, Q., Powles, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+resistance+across+glufosinate,+glyphosate,+paraquat+and+ACCase-inhibiting+herbicides+in+an+Eleusine+indica+population.">Multiple resistance across glufosinate, glyphosate, paraquat and ACCase-inhibiting herbicides in an Eleusine indica population.</a> Weed Research. 55 (1), 82-89 (2015).</li><li>Iwakami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+CYP81A12+and+CYP81A21+are+associated+with+resistance+to+two+acetolactate+synthase+inhibitors+in+Echinochloa+phyllopogon.">Cytochrome P450 CYP81A12 and CYP81A21 are associated with resistance to two acetolactate synthase inhibitors in Echinochloa phyllopogon.</a> Plant Physiology. 165, 618-629 (2014).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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&#160;</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&#160;</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&#220;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 14C</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&#160;</td>
			<td>LabLogic System</td>
			<td>1103303</td>
			<td>for HPLC analysis of 14C</td>
		</tr>
		<tr>
			<td>Hypersil Gold C18 column&#160;</td>
			<td>Thermo-Scientific</td>
			<td>03-050-522&#160;</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&#160;</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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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

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

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 ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

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 ...