Glyphosate-Resistant and Conventional Canola (Brassica napus L

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Glyphosate-Resistant and Conventional Canola (Brassica napus L.) Responses to Glyphosate and AMPA Treatment Elza Alves Correa, Franck E. Dayan, Daniel Owens, Agnes M. Rimando, and Stephen O. Duke J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00446 • Publication Date (Web): 19 Apr 2016 Downloaded from http://pubs.acs.org on April 21, 2016

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Journal of Agricultural and Food Chemistry

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Glyphosate-Resistant and Conventional Canola (Brassica napus L.) Responses to

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Glyphosate and AMPA Treatment

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Elza Alves Corrêa,† Franck E. Dayan,§ Daniel K. Owens, § Agnes M. Rimando,§

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and Stephen O. Duke§

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†

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Rua Nelson Brihi Badur, 430. 11900-000, Registro/SP, Brazil

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§

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38677, United States

UNESP, Universidade Estadual Paulista “Júlio de Mesquita Filho” Campus de Registro/SP.

USDA, ARS, Natural Products Utilization Research Unit, P. O. Box 1848, University, MS

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ABSTRACT: Glyphosate-resistant (GR) canola contains two transgenes that impart resistance

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to the herbicide glyphosate: 1) the microbial glyphosate oxidase gene (gox) encoding the

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glyphosate oxidase enzyme (GOX) that metabolizes glyphosate to aminomethylphosphonic

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acid (AMPA) and 2) cp4 that encodes a GR form of the glyphosate target enzyme 5-

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enolpyruvylshikimic acid-3-phosphate synthase. The objectives of this research were to

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determine the phytotoxicity of AMPA to canola, the relative metabolism of glyphosate to

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AMPA in GR and conventional non-GR (NGR) canola, and AMPA pool sizes in glyphosate-

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treated GR canola. AMPA applied at 1.0 kg ha-1 was not phytotoxic to GR or NGR. At this

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AMPA application rate, NGR canola accumulated a higher concentration of AMPA in its

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tissues than GR canola. At rates of 1 and 3.33 kg ae ha-1 of glyphosate, GR canola growth was

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stimulated. This stimulatory effect is similar to that of much lower doses of glyphosate on

ACS Paragon Plus Environment


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NGR canola. Both shikimate and AMPA accumulated in tissues of these glyphosate-treated

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plants. In a separate experiment in which young GR and NGR canola plants were treated with

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non-phytotoxic levels of 14C-glyphosate, very little glyphosate was metabolized in NGR plants,

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whereas most of the glyphosate was metabolized to AMPA in GR plants at 7 days after

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application. Untreated leaves of GR plants accumulated only metabolites (mostly AMPA) of

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glyphosate, indicating that GOX activity is very high in the youngest leaves. These data

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indicate that more glyphosate is transformed to AMPA rapidly in GR canola, and that the

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accumulated AMPA is not toxic to the canola plant.

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KEYWORDS: aminomethylphosphonic acid, AMPA, Brassica napus, canola, 5-

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enolpyruvylshikimic acid-3-phosphate synthase, EPSPS, glyphosate, glyphosate oxidase

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Received: January 28, 2016


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INTRODUCTION

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Glyphosate (N-(phosphonomethyl)glycine) is the most used herbicide worldwide.1 Most of

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glyphosate’s success has been due to widespread adoption of transgenic, glyphosate-resistant

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(GR) crops.1, 2 More than 80% of all transgenic crops are GR crops.2, 3 Commercially grown

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GR crops include maize, soybean, cotton, alfalfa, sugar beet, and canola. All of these crops

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contain a transgene encoding a GR form of 5-enolpyruvylshikimic acid-3-phosphate synthase

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(EPSPS), the enzyme target of glyphosate. In the majority of GR crops this transgene encodes

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a resistant EPSPS from the soil microbe Agrobacterium sp. (the cp4 transgene).4 When EPSPS

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is inhibited by glyphosate in plant tissues, shikimate accumulates rapidly due to blockage of the

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shikimate pathway.1, 2 Shikimate accumulation is a commonly used biomarker for glyphosate

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activity in plants.5

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GR canola is grown in the US, Canada and Australia, where economic benefits to

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farmers growing it have been documented.6 In the case of canola, a transgene (goxv247) from

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the soil bacterium Ochrobactrum anthropi, that encodes the enzyme glyphosate oxidoreductase

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(GOX) is used along with the cp4 EPSPS transgene.4 GOX converts glyphosate to glyoxylate

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and aminomethylphosphonic acid (AMPA),7 a compound that is much less toxic to plants than

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glyphosate.8-10 The microbial GOX gene was modified by mutagenesis and selection for

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variants with better enzymatic efficiency, as well as codon-optimized for better expression in

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plants.11 The transgene for GOX alone apparently does not impart adequate resistance to

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glyphosate.12 However, the cp4 gene provides sufficient glyphosate resistance for other GR

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crops, and the reason(s) why goxv247 was used only with GR canola has not been revealed.

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Both soybean, with only cp4, and canola, with cp4 and goxv247, are both about 50-fold more

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resistant to glyphosate than the untransformed crop.13



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AMPA accumulates in leaves of glyphosate-treated GR canola and conventional, non-

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GR (NGR) canola, although the levels are higher in GR canola.13, 14 AMPA also accumulates

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in GR soybean, which does not have the GOX transgene.9, 15-20 Soybean, canola, and some

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other species apparently have an enzyme that acts like GOX, resulting in some accumulation of

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AMPA when treated with glyphosate.7 The plant enzyme that converts glyphosate to AMPA

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may be a glycine oxidase, as this enzyme from microbes can generate AMPA from

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glyphosate.21 Bacterial glycine oxidase has recently been modified to achieve better catalytic

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activity on glyphosate.22-24 Under some circumstances, glyphosate-treated GR soybean can

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apparently produce enough AMPA with its non-transgenic GOX for the levels to be

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phytotoxic.9

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Thus, the question arises as to why the AMPA produced by the transgenic GOX in canola

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does not cause phytotoxicity. AMPA may not be as phytotoxic to canola as to soybean, or

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AMPA accumulation may be limited by rapid degradation of AMPA in canola. The research

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objectives of this paper were to determine 1) the phytoxicity of AMPA to GR and NGR canola;

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2) the metabolism of glyphosate in GR and NGR canola; and 3) the AMPA pool sizes in

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glyphosate-treated GR canola.

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MATERIALS AND METHODS

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Phytotoxicity of Glyphosate and AMPA. Greenhouse experiments were conducted

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with the following two canola varieties: Hyola 440, a conventional NGR variety, and Hyola

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514RR, a GR variety containing the cp4 transgene encoding a GR EPSPS and a goxv247

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transgene encoding a glyphosate oxidase. Although not isogenic, these two varieties have very


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similar traits in field trial evaluations (https://www.ag.ndsu.edu/varietytrials/williston-rec/2006-

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trial-results/Canola_Conventional_Variety08.pdf). Five canola seeds were planted in 15-cm

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diameter plastic pots containing a 1:1 (v/v) mixture of metro-mix and soil conditioner and then

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thinned to one plant per pot after emergence. The greenhouse was maintained at 25/20°C (±

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3°C) day/night temperature with natural light supplemented by sodium vapor lamps to provide

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a 13-h photoperiod. Plants were sub-irrigated with water as needed to maintain adequate soil

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moisture. Canola plants at the five to six-leaf (38 days old) growth stage were used for

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treatment.

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Plants were treated with a foliar application of AMPA or glyphosate. Spray solutions

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were prepared using technical grade glyphosate-isopropylammonium (>95% purity, Chem

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service, West Chester, PA) and technical grade AMPA (99% purity, Sigma-Aldrich, St. Louis,

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MO) mixed with 0.2% v/v Tween 20. The herbicide was applied with a Generation III Spray

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Booth equipped with a model TeeJet EZ 8002 nozzle with conical pattern and 80 degree spray

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angle. The height from nozzle to soil level was 40 cm for all the experiments. The spray head

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traveled over the plants at 1.5 km h-1 and the apparatus delivered the equivalent of 180 L ha-1.

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Glyphosate was applied at 1.00 and 3.33 kg a.e. ha-1 to GR canola and AMPA at 0.25 and 1.0

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kg ha-1 was applied to both GR and NGR canola. Control plants and Tween 20-treated plants

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were included as appropriate controls.

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At 14 days after treatment (DAT), canola plants were excised at the soil surface and

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the leaf number four from the base of the plant in a given treatment was sampled for

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chlorophyll content. Reduced chlorophyll accumulation is a secondary effect of glyphosate.25

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Chlorophyll was determined by the method of Hiscox and Israelstam.26 The plant shoot fresh

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and dry weights were measured.



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Treatments were arranged in a randomized complete block design with four replications. Data were subjected to an analysis of variance (LSD) test at P=0.05. Metabolism and Translocation of [14C]-Glyphosate in Young Canola Leaves.

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Labeled glyphosate [phosphonomethyl-14C] with a specific activity of 1,850-2,220 MBq/mmol

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was purchased from American Radiochemicals Inc. (St. Louis, MO 63146). Canola plants

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were grown to the 5-leaf stage in the greenhouse under the same conditions as in the first

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experiment. Then, 50 µL of 45.2 µM [14C]-glyphosate in 0.25% Tween 20 (4.18 kBq or

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250,000 dpm) was applied to the surface of the second, third and fourth true leaf, as numbered

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beginning with the oldest leaf near the base of the plant, to have enough radioactivity to follow

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glyphosate metabolism with a radioisotope detector. Application of [14C]-glyphosate was

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achieved by pipetting the solution directly onto the chosen leaf and then carefully coating the

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entire surface using the edge of a pipette tip. Plants were kept under continuous light in the

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laboratory for 7 DAT. Leaves were collected in triplicate per treatment after 1 day and at the

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end of the experiment. Leaves were removed from the stem for each time point. The treated

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leaves were washed three times in separate 100 mL volumes of ddH2O, and each wash

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monitored by liquid scintillation counting to confirm the removal of unabsorbed [14C]-

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glyphosate. Plant samples were wrapped in aluminum foil, and dried in a 65°C air flow oven

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for 48 h. The dry weights were recorded and stored at −20°C until further analysis.

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Prior to analysis, leaf samples were ground to a powder under liquid N2 and homogenized

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in 4 mL of ddH2O with a Polytron PT 3100 (Kinematica, Inc., Bohemia, NY 11716). The

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homogenate was centrifuged at 12,000 g for 5 min, the supernatant was collected and lyophilized

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under vacuum. The dried residue was stored at -20°C until further analysis. Each residue was

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resuspended in 150 µL of ddH2O and centrifuged at 16,000 g in a microfuge for 3 min. The


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supernatant was filtered through a 0.45 µm syringe filter, a 10 µL aliquot was analyzed by liquid

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scintillation counting (Packard Tri-Carb 1600TR) to calculate the total amount of radioactivity

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extracted, and the remaining sample was reserved for HPLC analysis.

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Glyphosate, AMPA and Shikimate Determination. In the greenhouse study of

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phytotoxicity, analysis of glyphosate, AMPA, and shikimate in leaves of NGR and GR plants at

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3, 7 and 14 DAT of AMPA or glyphosate was performed by gas chromatography-mass

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spectrometry following published methods.13, 15, 16

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In the metabolism study, a 50 µL sample was analyzed on a HPLC system (Water

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Corporation, Milford, MA 01757) consisting of a model 600E multi-solvent delivery system, a

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model 717plus autosampler, and a β-ram radioisotope detector (IN/US Systems, Tampa, FL

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33610). Separations were performed with an isocratic solvent system of 5 mM KH2PO4 at 0.5

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mL min-1 for typically 15 min on a Hamilton PRP-X400 column (250 × 4.1 mm) as described in

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Lorraine-Colwill et al.,27 except 0.01% NaN3 was not added to the mobile phase. To reduce

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background noise from the low pH of the mobile phase Ultima Gold Scintillation fluid was

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diluted 3:1 with 500 mM MOPS pH 7.0 before addition to the β-ram detector. Glyphosate and

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AMPA were detected at ca. 5.5 and 7 min, respectively, with this method. The dpm of each peak

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was calculated from the total sample dpm (as described above) and the fraction of the radioactive

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HPLC peak of the total radioactive peak areas.

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Statistical analysis. Graphs were generated with Sigma Plot 12 (Build 12.0.0.182, Systat

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Software, Inc., San Jose, CA). The data were subjected to analysis of variance, and the means

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were separated with the Fischer’s LSD at P = 0.05 or P = 0.1 using the Agricolae module28 on R

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platform (version 3.2.3, R Foundation for Statistical Computing, Vienna, Austria).29

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RESULTS AND DISCUSSION

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Effects of AMPA on Conventional and Glyphosate-Resistant Canola and

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Glyphosate on Glyphosate-Resistant Canola. AMPA at 0.25 and 1 kg ha-1 did not cause

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statistically significant effects on GR or NGR canola growth at 14 DAT as determined by fresh

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and dry weights (Fig. 1). A previous study with these cultivars found slight (ca. 10%)

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inhibition of shoot fresh weight 14 DAT of 1 kg ha-1 of AMPA with both GR and NGR

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plants.13 In the present study, AMPA stimulated height of GR plants (Fig. 2A). The difference

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in results with GR canola in the present study may be due different growing conditions (e.g.,

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pot size). Glyphosate at 1 and 3.33 kg ha-1 stimulated growth of GR canola (Fig. 1). Plant

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height (Fig. 2A) and chlorophyll (Fig. 2B) results were similar to those for fresh and dry weight

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(Fig. 1). The canola responses to AMPA differ from the responses of soybean. At the 1 kg/ha

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rate, AMPA reduced chlorophyll and fresh weight by 50 and 15%, respectively, of both GR and

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NGR soybean.9 In this study, canola plant height and chlorophyll levels of AMPA-treated

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plants were similar to or higher than the untreated control. This difference is not due to

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differential uptake, as the levels of AMPA found in tissues of GR and NGR canola 14 DAT

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(Fig. 3) were similar to or higher than those reported 14 days after GR and NGR soybean were

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treated with the same dose.9 The AMPA toxicity to GR soybean may explain why the GOX

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transgene is not used in GR soybean.

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Stimulation of growth in GR canola by high glyphosate doses was unexpected.

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Stimulation by a sub-toxic dose of a toxicant is termed hormesis.30 Low levels of glyphosate

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stimulate growth of virtually all plant species tested,31, 32 but this effect is not found at ultra

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doses in GR crops.32 We found 135 ± 5 SE and 140 ± 16 SE µg g-1 dry weight of shikimate in

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GR plants treated with 1 and 3.33 kg of glyphosate per hectare, respectively, versus no


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detectable shikimate in untreated plants. This is the level of shikimate elevation found at low,

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hormetic doses of glyphosate in glyphosate-sensitive plants31-33 and in GR plants at glyphosate

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doses that kill NGR plants.34 These results support the view that the concentrations of

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glyphosate needed to cause the same growth stimulation effects are higher in GR plants than

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NGR plants. So, stimulation of growth of GR plants by normally toxic concentrations might be

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expected if the biphasic, hormetic dose-response relationships of glyphosate effects on plant

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growth are shifted to higher doses in GR plants. Williams et al.35 recently reported a

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recommended field rate (1.68 kg ha-1) of glyphosate significantly enhanced yield of GR maize

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versus untreated GR maize.

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The difference in effect of AMPA on NGR and GR canola (Fig. 2) may have been due

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to the generally higher AMPA concentration in the NGR than the GR plants (Fig. 3). Whether,

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the higher AMPA concentration in NGR than GR plants is due to less growth (thus, more

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AMPA per unit of tissue) or whether less growth is due to higher AMPA cannot be determined

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from this experiment. The AMPA concentrations in the glyphosate-treated GR plants were

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comparable to those in AMPA-treated GR plants (Fig. 3). However, the glyphosate

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concentrations at 3, 7 and 14 DAT (Fig. 4) were always higher than AMPA concentrations in

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the same tissues. These data show that although glyphosate is metabolized by transgenic GOX,

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there is still enough glyphosate to cause damage if it were not for the GR CP4 EPSPS imparted

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by the cp4 transgene. The elevated shikimate levels mentioned earlier are evidence that some

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native EPSPS that functions in the shikimate pathway is inhibited in these plants. Native and

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CP4 EPSP synthases are expected to work on the same shikimate-3-P pool, so the small level

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of elevated shikimate that we found could due to a lower expression of the cp4 gene in some

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cells.



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Determination of the metabolism and translocation of glyphosate in GR canola using

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labeled glyphosate was conducted to further understand the fate of glyphosate in the transgenic

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crop with two genes for glyphosate resistance versus conventional NGR canola, using such

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small amounts of glyphosate that phytotoxicity does not confound the results with NGR canola.

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Metabolism and Translocation of [14C]-Glyphosate in Young Canola Leaves. Although

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there was variability among the treatments, typically 30-50% of the 14C-glyphosate applied was

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absorbed by the plant as determined from the amount of radioactivity present in the various

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washings of the leaf’s surface. One day after the application of 4.18 kBq of 14C-glyphosate to

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the treated leaf, most of the radioactivity translocated as glyphosate toward the younger sink

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leaves of the NGR seedlings, with glyphosate accumulating to increasingly greater levels as the

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age of the sink leaves decrease (Fig. 5 A & B). There was some radioactivity that was not in the

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form of glyphosate or AMPA in the first leaf, located below all treated leaves. In NGR plants,

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AMPA was detected only in the second and fifth leaves. About 90% of the detected 14C

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remained as glyphosate in the plant. At this same time after treatment, the distribution of

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glyphosate and its metabolites was drastically different in GR canola (Fig. 5 C & D). About

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30% of the total applied radioactivity remained in leaf two as glyphosate, suggesting little

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metabolism. However, there was considerable accumulation of AMPA and other metabolites in

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treated leaves three and four, as well as in untreated leaf five. The untreated leaf five contained

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considerably more AMPA than glyphosate. Less than half of the 14C detected in the plant

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remained as glyphosate, and most of this was in the treated leaf two.

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At 7 DAT, there was still little metabolic degradation of glyphosate in the NGR plants,

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with most of the glyphosate being translocated from the treated leaves (leaves two, three and

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four) to leaves five and six (Fig. 6 A & B). Almost 50% of the radioactivity was found as


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glyphosate in leaf 5 at this time. In the GR plant, glyphosate was metabolized mostly to

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AMPA and a lesser amount to unidentified products (Fig.6 C & D). Glyphosate was found

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predominantly in the treated leaves, with only trace amounts in leaves five and six. About half

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of the radioactivity was in the untreated leaves, mostly as AMPA. The lack of substantial

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AMPA accumulation in leaves two and three of the GR plants could be due to three factors: 1)

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the GOX enzyme is not very active in older leaves; 2) glyphosate is translocated faster to

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younger leaves than it can be metabolized in the older, treated leaf and/or 3) AMPA produced

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in the older leaves is translocated rapidly to younger leaves. Results with NGR plants indicate

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that glyphosate is translocated to younger leaves, where it is rapidly converted to AMPA in GR

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plants. In summary, the metabolism of glyphosate is drastically different between NGR and GR

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canola, resulting in relatively little of the applied glyphosate being found in GR canola within a

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week after application, and most of this is in older leaves. The rate of conversion of AMPA to

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other products in GR canola is apparently much slower than glyphosate conversion to AMPA.

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Thus, AMPA accumulates in mostly young leaves, apparently without significant

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phytotoxicity.

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The relatively high glyphosate to AMPA ratio in the first experiment (Figs. 3 and 4) at 7

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DAT compared to the much lower ratio in the second experiment at 7 DAT (Fig. 6) was

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probably due to the much higher dose of glyphosate used in the first experiment. The much

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smaller amount of glyphosate (radiolabeled) used in the second experiment was not enough to

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be phytotoxic to the NGR variety. So, the second experiment shows the relative GOX activity

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between NGR and GR varieties, but does not indicate the proportion of glyphosate metabolized

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at field application rates of glyphosate.



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These studies provide more insight into the activity of transgene-imparted GOX activity

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in GR canola. Our results indicate that the GOX enzyme is highly active, especially in young

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tissues. We also found that AMPA up to 1 kg ha-1 is not toxic to NGR or GR canola and does

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not accumulate to phytotoxic levels in GR plants treated with glyphosate doses up to 3.33 kg

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ha-1. Furthermore, higher field rates (1-2 kg ha-1) of glyphosate may be hormetic in GR canola.

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AUTHOR INFORMATION

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Corresponding Author

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*Tel: +1 662 915 1036. Fax: +1 662 915 1035. E-mail: [email protected].

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Funding

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The sabbatical of E. Correa at the USDA, ARS, Natural Product Utilization Research Unit was

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funded by the São Paulo Research Foundation – FAPESP.

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Notes

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The authors declare no competing financial interests.

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ACKNOWLEDGMENTS

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We are grateful to Robert Johnson, Gloria Hervey, and Amber Reichley for their excellent

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technical assistance. We thank Dr. Krishna Reddy for the generous provision of the canola

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seed.

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ABBREVIATIONS USED

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AMPA, aminomethylphosphonic acid; EPSPS, 5-enolpyruvylshikimic acid-3-phosphate

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synthase; GOX, glyphosate oxidase; GR, glyphosate resistant; NGR, non-glyphosate resistant


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357 358

359 360

Plant FW (% change)

200

NGR

GR

**

150

100

50

0

-50

Plant DW (% change)

200

** **

150

100

50

0

-50 0.25

1

0.25

AMPA (kg/ha)

1

1

3.33

Glyphosate (kg/ha)

361 362

Figure 1. Effects of AMPA on NGR and GR canola and of glyphosate on GR canola shoot

363

fresh and dry weight 14 days after application. Error bars are ± 1 SE. Means with

364

** are different from the control at α = 0.1.

365

366


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A 35

NGR

GR ***

Height (% change)

30

*** 25 20

**

15

**

10 5 0 -5

Chlorophyll (% change)

B 70

**

60 50 40 30 20 10 0 -10 -20 -30 0.25

1

0.25

AMPA (kg/ha)

1

1 3.33 Glyphosate (kg/ha)

367 368

Figure 2. Effects of AMPA on NGR and GR canola and of glyphosate on GR canola A) shoot

369

height and B) chlorophyll concentration 14 days after application. Error bars are ±

370

1 SE. Means with *** are statistically different from the control at α = 0.05 and **

371

are different from the control at α = 0.1.

372

373



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Corrêa et al. - 20

-1

AMPA (µg g DW)

120

NGR

GR

***

100 80 60 40

**

** 20

AMPA (µg g-1 DW)

0 120 100 80 60

**

40

**

**

**

**

20

AMPA (µg g-1 DW)

0 120 100

***

80

**

60

**

40 20

**

**

0 0.25

1

0.25

1

1

AMPA (kg/ha)

3.33

Glyphosate (kg/ha)

374 375

Figure 3. AMPA in NGR and GR canola plants at 3 (top), 7 (middle) and 14 (bottom) days

376

after application of AMPA or glyphosate. Error bars are ± 1 SE. Means with ***

377

are statistically different from the control at α = 0.05 and ** are different from the

378

control at α = 0.1.

379


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-1

Glyphosate (µg g DW)

140

Day 3

Day 7

Day 14 ***

***

120 100 80

***

***

60

*** 40

*** 20 0 1

3.33

1

3.33

1

3.33

Glyphosate (kg/ha)

380 381

382

Figure 4. Glyphosate in GR canola plants at 3, 7, and 14 days after application of glyphosate.

383

Error bars are ± 1 SE. Means with *** are statistically different from the control at

384

α = 0.05.

385

386

387

388



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Corrêa et al. - 22

50

50

B 40

30

30

20

20

10

10

0

0

D

-1

C 40

40

30

30

20

20

10

10

0

0

L1

389 390 391 392 393

dpm g DW (x1,000)

Radioactivity (% distribution)

A 40

L2

L3

L4

L5

L1

L2

L3

L4

L5

Plant tissue Plant tissue 14 Figure 5. Metabolism of C-glyphosate 1 DAT in conventional (A and B) and GR canola (C and D). The data is expressed as percent distribution of total radioactivity in each leaf on the left side and as actual dpm g-1 DW of the leaves on the right. Glyphosate = , AMPA = , and unknown metabolite(s) = . Error bars are ± 1 SE. dpm = disintegrations per minute

394


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395

60 50

100

B

A

80

40 60

40

20 20

10 0 50

0

-1

D

C

dpm g DW (x1,000)

Radioactivity (% distribution)

30

80

40 60

30 40

20 20

10 0

0

L1

396 397 398 399 400

L2

L3

L4

Plant tissue

L5

L6

L1

L2

L3

L4

L5

L6

Plant tissue

Figure 6. Metabolism of 14C-glyphosate 7 DAT in conventional (A and B) and GR canola (C and D). The data is expressed as percent distribution of total radioactivity in each leaf on the left side and as actual dpm g-1 DW of the leaves on the right. Glyphosate = , AMPA = , and unknown metabolite(s) = . Error bars are ± 1 SE. dpm = disintegrations per minute

401



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402

TOC Graphic

403 404

405


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532x182mm (96 x 96 DPI)


Glyphosate-Resistant and Conventional Canola (Brassica napus L

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