Name: measuring rates of herbicide metabolism in dicot weeds with an excised leaf assay
Uploaded: 2015-09-07T15:00:00
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 s...

<ol>
	<li>Yu, Q., Powles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism-based+herbicide+resistance+and+cross-resistance+in+crop+weeds:+A+threat+to+herbicide+sustainability+and+global+crop+production.">Metabolism-based herbicide resistance and cross-resistance in crop weeds: A threat to herbicide sustainability and global crop production.</a> Plant Physiology. 166, 1106-1118 (2014).</li><li>Powles, S. B., Yu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+in+action:+plants+resistant+to+herbicides.">Evolution in action: plants resistant to herbicides.</a> Annual Reviews in Plant Biology. 61, 317-347 (2010).</li><li>Heap, I., 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=Global+perspective+of+herbicide-resistant+weeds.">Global perspective of herbicide-resistant weeds.</a> Pest Management Science. 70 (9), 1306-1315 (2014).</li><li>D&#233;lye, C., 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=Non-target-site-based+resistance+should+be+the+centre+of+attention+for+herbicide+resistance+research:+Alopecurus+myosuroides+as+an+illustration.">Non-target-site-based resistance should be the centre of attention for herbicide resistance research: Alopecurus myosuroides as an illustration.</a> Weed Research. 51 (5), 433-437 (2011).</li><li>Kreuz, K., Tommasini, R., Martinoia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+enzymes+for+a+new+job.+Herbicide+detoxification+in+plants.">Old enzymes for a new job. Herbicide detoxification in plants.</a> Plant Physiology. 111, 349-353 (1996).</li><li>Riechers, D. E., Kreuz, K., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detoxification+without+intoxication:+herbicide+safeners+activate+plant+defense+gene+expression.">Detoxification without intoxication: herbicide safeners activate plant defense gene expression.</a> Plant Physiology. 153, 3-13 (2010).</li><li>Siminszky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cytochrome+P450-mediated+herbicide+metabolism.">Plant cytochrome P450-mediated herbicide metabolism.</a> Phytochemistry Reviews. 5 (2-3), 445-458 (2006).</li><li>Fonn&#233;-Pfister, R., 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=Hydroxylation+of+primisulfuron+by+an+inducible+cytochrome+P450-dependent+monooxygenase+system+from+maize.">Hydroxylation of primisulfuron by an inducible cytochrome P450-dependent monooxygenase system from maize.</a> Pesticide Biochemistry and Physiology. 37 (2), 165-173 (1990).</li><li>Steckel, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dioecious+Amaranthus+spp.:+here+to+stay.">The dioecious Amaranthus spp.: here to stay.</a> Weed Technology. 21 (2), 567-570 (2007).</li><li>Horak, M. J., Loughin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+analysis+of+four+Amaranthus+species.">Growth analysis of four Amaranthus species.</a> Weed Science. 48 (3), 347-355 (2000).</li><li>Hausman, N. E., 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=Resistance+to+HPPD-inhibiting+herbicides+in+a+population+of+waterhemp+(Amaranthus+tuberculatus)+from+Illinois,+United+States.">Resistance to HPPD-inhibiting herbicides in a population of waterhemp (Amaranthus tuberculatus) from Illinois, United States.</a> Pest Management Science. 67 (3), 258-261 (2011).</li><li>Ma, R., 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=Distinct+detoxi&#64257;cation+mechanisms+confer+resistance+to+mesotrione+and+atrazine+in+a+population+of+waterhemp.">Distinct detoxi&#64257;cation mechanisms confer resistance to mesotrione and atrazine in a population of waterhemp.</a> Plant Physiology. 163, 363-377 (2013).</li><li>Guo, J., 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=Non-target-site+resistance+to+ALS+inhibitors+in+waterhemp+(Amaranthus+tuberculatus).">Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus).</a> Weed Science. in press, (2015).</li><li>Patzoldt, W. L., Tranel, P. J., Hager, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+waterhemp+(Amaranthus+tuberculatus)+biotype+with+multiple+resistance+across+three+herbicide+sites+of+action.">A waterhemp (Amaranthus tuberculatus) biotype with multiple resistance across three herbicide sites of action.</a> Weed Science. 53 (1), 30-36 (2005).</li><li>Kreuz, K., Fonn&#233;-Pfister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbicide-insecticide+interaction+in+maize:+malathion+inhibits+cytochrome+P450-dependent+primisulfuron+metabolism.">Herbicide-insecticide interaction in maize: malathion inhibits cytochrome P450-dependent primisulfuron metabolism.</a> Pesticide Biochemistry and Physiology. 43 (3), 232-240 (1992).</li><li>Correia, M. A., Ortiz de Montellano, P. R., Ortiz de Montellano, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytochrome P450: Structure, Mechanism, and Biochemistry. , 247-322 (2005).</li><li>Hawkes, T. R., 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=Mesotrione:+mechanism+of+herbicidal+activity+and+selectivity+in+corn.">Mesotrione: mechanism of herbicidal activity and selectivity in corn.</a> Proceedings of the Brighton Crop Protection Conference &#8211; Weeds. 2, 563-568 (2001).</li><li>Patzoldt, W. L., Tranel, P. J., Hager, A. 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 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 ...

measuring-rates-herbicide-metabolism-dicot-weeds-with-an-excised-leaf

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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 ...