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

Summary

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.

Abstract

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 –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.

Introduction

Herbicide resistance in weeds presents a serious threat to the global production of food and fiber^1,2. 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 populations^1,2,4. 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 detoxification^5,6. 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 II^5,7. 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 processing^5,6 (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 mechanisms^12,13. 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 mesotrione^11,12.

Waterhemp Population	Abbreviation	Phenotype to Mesotrione	Mesotrione Resistance Mechanism	Phenotype to Primisulfuron	Primisulfuron Resistance Mechanism
McLean County-Resistant	MCR	Resistant	Metabolism*	Resistant	Metabolism
Adams County-Resistant	ACR	Sensitive	-	Resistant	Target-site mutation in ALS14
Wayne County-Sensitive	WCS	Sensitive	-	Sensitive	-

* 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).

Protocol

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).

1. Collect and suspend waterhemp seeds in 0.1 g L^-1 agar:water solution at 4 °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.
2. Germinate seeds of each waterhemp population (as in Table 1) in 12 x 12 cm trays containing a potting medium in the greenhouse.
   1. Maintain greenhouse conditions at 28/22 °C day/night temperatures within a 16/8 hr photoperiod¹².
   2. Supplement natural light in the greenhouse with light from mercury halide lamps to provide a minimum of 500 µmol m^-2 sec^-1 photon flux at the plant canopy level.
3. Transplant emerged seedlings when they are 2 cm tall into 80 cm³ pots in the greenhouse.
4. 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.
5. 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.
6. 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.
7. 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.
   1. Maintain growth chamber conditions at 28/22 °C day/night temperatures within a 16/8 hr photoperiod¹².
   2. A combination of incandescent and fluorescent bulbs can be used to provide light in the growth chamber and should deliver at least 550 µmol m^-2 sec^-1 photon flux at plant canopy level¹².
8. 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.
9. 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 MCR_1-6, ACR_1-6 and WCS_1-6.
   1. Establish at least six distinct “parent” clonal lines in order to repeat the experiment and adequately represent genetic variability within each waterhemp population.
   2. Depending on how many time points will be analyzed, generate enough sub-clones from each “parent” clonal line to conduct a time-course study.

2. Conducting a Metabolism Study with Excised Waterhemp Leaves

1. 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.
2. 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.
   1. Firstly, partially immerse excised leaves with cut petiole in pre-incubation buffer (200 µl) of 0.1 M Tris-HCl (pH 6) for 1 hr.
   2. Secondly, move each leaf to a new 1.5 ml vial containing an incubation buffer (200 µl) of 0.1 M Tris-HCl (pH 6) plus 150 µM [URL-¹⁴C] herbicide, and incubate at 28 °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 µM 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 µM inhibitor. For example, malathion and tetcyclacis inhibit certain cytochrome P450 activities in plants^15-17.
   3. 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 µl) for 0 hr, 5 hr, 11 hr, 23 hr, or 35 hr, respectively, in the growth chamber for the time-course metabolism study.
3. 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.
4. Extract radiolabeled herbicide and its metabolites from each leaf with 7 ml of 90% acetone with a laboratory tissue homogenizer in the same polypropylene tube as above. Rinse tissue homogenizer with an additional 7 ml 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.
5. Label 90% acetone extracts (14 ml total) and store at -4 °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.
6. Centrifuge samples at 5,000 x g for 10 min and concentrate supernatants at 40 °C with a rotary evaporator until a final volume of 0.5 ml is obtained. Transfer this volume to a 2.0 ml plastic tube.
7. Add acetonitrile:water (1:1, by volume) to adjust the final volume of the plant extracts to 1.25 ml and re-centrifuge at 10,000 x g for 10 min to remove any particulates.
8. Measure total radioactivity in an aliquot taken from each sample (dpm µl^-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.

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.

1. Perform RP-HPLC with a C₁₈ column (4.6 x 250 mm, 5 µm particle size for analytical HPLC) at a flow rate of 1 ml 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.
   1. 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.
   2. 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.
2. 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’s instructions) to determine rates of herbicide metabolism in each waterhemp population during the time course.

4. Statistical Procedures

1. Arrange treatments in a completely randomized design (or other suitable arrangement for statistical analysis). 
2. 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 (α = 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 “parent” clonal line derived from each population (MCR_1-6, ACR_1-6, and WCS_1-6) 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.
3. 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:
   
4. where in this model y represents the percentage of the parent herbicide remaining at time t, µ_i is the DT₅₀ for each waterhemp population i, and the parameter C₀ is the estimated amount of parent herbicide present at t = 0¹².

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	A1296	for pre-germinating seeds
Potting medium	Sun Gro Horticulture	49040233	for plant growth
Nutricote	Agrivert	TOTAL BLEND 13-13-13 T100	slow-release fertilizer
Growth chamber E15	Controlled Environments Limited	20207	plant culturing
Tris base	Fisher Scientific	BP152-500	buffer for excised leaves
HCl (concentrated)	Fisher Scientific	A144500	adjust pH of buffer
Murashige and Skoog (MS) salts	Sigma-Aldrich	M0404	incubation of excised leaves
Methanol	Fisher Scientific	A452-4	leaf washes after incubation
Acetone	Sigma-Aldrich	179124	plant extractions
Acetonitrile (HPLC grade)	Macron Fine Chemicals	MKH07610	HPLC mobile phase
Formic acid	Mallinckrodt Analytical	MK259205	acidify mobile phase pH
Micro-centrifuge	Eppendorf	5417R	1.5 or 2.0 ml tubes
Centrifuge (temperature controlled)	Eppendorf	5810R	15 or 50 ml tubes
Polypropylene centrifuge tube	Corning Inc.	430790	15 ml, sterile
Rotary evaporator	BÜCHI	R200	concentrate plant samples
Liquid scintillation spectrometry (LSS)	Packard Instruments	104470	quantify 14C
High-performance liquid chromatography	Perkin Elmer	N2910401	resolve herbicide metabolites
Flow scintillation analyzer	LabLogic System	1103303	for HPLC analysis of 14C
Hypersil Gold C18 column	Thermo-Scientific	03-050-522	reversed phase
Ultima-Flo M cocktail	Perkin Elmer	6013579	for Flow-scintillation analyzer
Scintillation Cocktail (ScintiVerse BD)	Fisher Scientific	SX18	for LSS; biodegradable
Laboratory homogenizer	Kinematica	CH-6010	homogenize leaf samples