Role of Glutamine Synthetase Isogenes and Herbicide Metabolism in

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Agricultural and Environmental Chemistry

Role of glutamine synthetase isogenes and herbicide metabolism in the mechanism of resistance to glufosinate in Lolium perenne L. spp. multiflorum biotypes from Oregon Caio Augusto Brunharo, Hudson K Takano, Carol A. Mallory-Smith, Franck E. Dayan, and B. D. Hanson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01392 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Agricultural and Food Chemistry

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Role of glutamine synthetase isogenes and herbicide metabolism in the mechanism of

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resistance to glufosinate in Lolium perenne L. spp. multiflorum biotypes from Oregon

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Caio A. C. G. Brunharo1*

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1Department

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ORCID: 0000-0001-9735-1648

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Hudson K. Takano2

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2Department

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Colorado State University, Fort Collins, CO 80523, USA

of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

of Bioagricultural Sciences and Pest Management, 1777 Campus Delivery,

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ORCID: 0000-0002-8018-3868

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Carol A. Mallory-Smith1

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1Department

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Franck E. Dayan2

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2Department

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Colorado State University, Fort Collins, CO 80523, USA

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ORCID: 0000-0001-6964-2499

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Bradley D. Hanson3

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3One

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95616, USA

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ORCID: 0000-0003-4462-5339

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*Corresponding author: 3050 SW Campus Way, Crop Sciences Building 331B, Department of

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Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

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[email protected]

of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA

of Bioagricultural Sciences and Pest Management, 1777 Campus Delivery,

Shields Avenue, Department of Plant Science, MS-4, University of California, Davis, CA

ACS Paragon Plus Environment


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ABSTRACT

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Glufosinate-resistant Lolium perenne L. spp. multiflorum biotypes from Oregon exhibited

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resistance levels up to 2.8-fold the field rate. One resistant biotype (MG) had an amino acid

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substitution in the glutamine synthetase 2 (GS2), whereas the other (OR) exhibited the wild-type

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genotype. We hypothesized that the amino acid substitution in GS2 is involved in the resistance

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mechanism in MG, and that non-target site resistance mechanisms are present in OR. OR

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metabolized glufosinate faster than the other two biotypes, with >75% of the herbicide

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metabolized, compared to 50% in MG and the susceptible biotype. A mutation in the GS2 co-

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segregating with resistance in MG did not reduce the enzyme activity, results further supported

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by our enzyme homology models. This research supports the conclusion that a metabolism

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mechanism of glufosinate resistance is present in OR, and that glufosinate resistance in MG is

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not due to an altered target-site.

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Keywords: Enzyme modelling, gene expression, herbicide absorption, herbicide degradation,

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herbicide translocation, non-target-site resistance, phosphinothricin,

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INTRODUCTION

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Lolium perenne L. spp. multiflorum (L. multiflorum) is a winter annual grass species native to

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Europe and initially introduced to the United States as a forage crop.1 Lolium multiflorum is also

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a major weed in agricultural areas in many parts of the world, primarily in regions of temperate

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climate. In Oregon, L. multiflorum interferes with a wide range of crops.2 Modern agriculture

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relies heavily on herbicides because of their efficacy and economic returns, whereas other

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management practices may be more costly or less selective.3 Overreliance on herbicides for weed

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management has, however, selected for herbicide-resistant L. multiflorum populations in at least

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12 countries.4 The obligate-outcrossing, self-incompatible mating system of this diploid (2n = 2

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= 14) species facilitates the dispersal of herbicide resistance genes within and among

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populations.5-6

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Glufosinate (2-amino-4-(hydroxymethylphosphinyl) butyric acid) is a commercially

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available nonselective herbicide composed of a racemic mixture (L/D-phosphinothricin) used for

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postemergence control of weeds in many agricultural and non-agricultural systems. The L-

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phosphinothricin form has herbicidal activity, irreversibly inhibiting glutamine synthetases (EC

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6.3.1.2; GS), a family of enzymes responsible for the ATP-dependent assimilation of ammonia

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as an amide moiety of glutamine, central to the assimilation of nitrogen in plants.7 A number of

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GS isoforms have been identified in Arabidopsis thaliana, Zea mays and L. multiflorum, and are

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commonly classified as cytosolic (GS1) and plastidic (GS2), depending on their cellular

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localization and specific role in plants.8-9 The GS1a isoform in Triticum aestivum, for example, is

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involved in nitrogen remobilization during leaf senescence,10 while GS1b is associated with

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nitrogen assimilation in roots of Oryza sativa.11 Inhibition of GS causes a dramatic decrease in

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glutamate and glutamine, leading to the accumulation of downstream metabolites in



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photorespiration (glyoxylate, phosphoglycolate and glycolate).12-13 The accumulation of high

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levels of glyoxylate leads to the inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase

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activase, a key enzyme in the Calvin-Benson cycle, resulting in the accumulation of reactive

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oxygen species, lipid peroxidation of membranes and cell death.14

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Although glufosinate has been used for over 40 years, the first glufosinate-resistant weed

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species were confirmed in 2009 in a vegetable farm in Malaysia15 and in a Corylus avellana L.

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orchard in Oregon.2 Despite recent efforts to understand the biological processes involved in

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glufosinate resistance, little information has been obtained about the underlying mechanisms

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involved in these phenotypes.16 Among the glufosinate-resistant L. multiflorum biotypes

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identified in Oregon by Avila-Garcia et al.,2, 17 one biotype (MG) exhibited an amino acid

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substitution in the target site, whereas the other biotype (OR) presented the wild-type genotype.

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The single nucleotide polymorphism (SNP) found in the GS2 gene encoded for a GS isozyme

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with an amino acid substitution at position 173 (D173N) (equivalent to position 171 on the T.

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aestivum GS2 sequence). If GS2 is altered only in MG (compared to the GS2 sequence of the

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susceptible L. multiflorum biotype; SFS), then it is possible that a conformational alteration in

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GS2 leads to an altered binding affinity between the target enzyme and glufosinate. For instance,

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a single amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS;

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EC. 2.5.1.19) at position 102 and/or 106, located in the enzyme active site, reduced the binding

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affinity with the inhibitor glyphosate, conferring resistance to this herbicide in several weed

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species.18-21 Conversely, because alterations in the glufosinate target site were not found in OR, it

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is possible that non-target site resistance (NTSR) mechanisms were selected in this population.

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Glufosinate metabolism, for example, has been found in Daucus carota L. and Nicotiana

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tabacum L.,22 as well as in a number of weed species.23 Although enhanced metabolism has not


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been established as a glufosinate resistance mechanism, up-regulation of certain groups of

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xenobiotic-detoxifying enzymes may confer resistance to other herbicides.24

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Elucidating the mechanisms of resistance to herbicides in weeds is important for

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understanding the underlying basic biological processes in plants, and for allowing practitioners

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to develop better weed management decisions. In this context, the objective of this research was

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to characterize the putative target-site and NTSR mechanisms of glufosinate resistance in L.

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multiflorum biotypes from Oregon.

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MATERIALS AND METHODS

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Plant material

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Previously characterized2, 17 L. multiflorum biotypes were used in this research: (i) the

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glufosinate-resistant biotype MG that has an amino acid substitution in the plastidic GS2

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(D173N); (ii) the glufosinate-resistant biotype OR that does not exhibit mutations in GS2; and

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(iii) the susceptible biotype SFS. The reported GR50 values (herbicide rate required to reduce

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plant growth by 50%) for MG, OR and SFS were 0.45, 0.49 and 0.15 kg ai ha-1, respectively.2, 17

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In vivo glutamine synthetase activity - dose-response assay

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A dose-response experiment was conducted to compare the response of MG, OR and SFS

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to glufosinate. Plants were treated at the BBCH-23 growth stage25 in a spray cabinet calibrated to

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deliver 187 L ha-1, and kept at 30/20 C and 13/11 h (day/night) in a greenhouse throughout the

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experiment. Eight glufosinate rates (Liberty 280 SL, Bayer CropScience, Research Triangle PK,

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NC) ranging from 0 to 2,240 g ai ha-1 were applied to single-plant experimental units. Enzyme

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activity, as percentage of the control, was evaluated using a modified procedure outlined in



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Dayan et al.,26 that relies on the formation of γ-glutamyl hydroxamate and further reaction with

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acidic ferric chloride. Twenty-four hours after herbicide treatment (HAT), crude leaf extracts

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were obtained from 200 mg of fully expanded leaves in the presence of 333 µL extraction buffer

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[50 mM tris(hydroxymethyl)methylamine (99%, Bio-Rad, Hercules, CA), 1 mM

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ethylenediaminetetraacetic acid (99%, Bio-Rad, Hercules, CA), 2 mM dithiothreitol (99%,

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Thermo Scientific, Waltham, MA), 10 mM MgCl2 (99%, Fisher Scientific, Waltham, MA), 5%

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polyvinylpyrrolidone (w/v) (99%, Sigma Aldrich, St Louis, MO)], filtered with

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Miracloth®(EMD Millipore, San Diego, CA), centrifuged at 12,000  g for 10 min at 4 C and

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kept on ice throughout the procedure. Twenty microliters of crude leaf extract were added to 180

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µL of assay buffer [25 mM imidazole-HCL (pH 7.5) (99%, Fisher Scientific), 50 mM L-

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glutamine (99%, Sigma Aldrich), 4 mM MnCl2 (99%, Fisher Scientific), 5 mM adenosine

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diphosphate (99%, Sigma Aldrich), 40 mM sodium arsenate (99%, Fisher Scientific), 25 mM

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hydroxylamine (99%, VWR, Radnor, PA)] and incubated for 30 min at 30 C. After the

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incubation period, 100 µL of ferric chloride reagent [250 mM trichloroacetic acid (100%, Sigma

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Aldrich), 200 mM anhydrous ferric chloride (99%, Fisher Scientific), 0.5 M HCl (100%, Sigma

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Aldrich)] were added to each sample to stop the reaction before spectrophotometric measurement

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at 540 nm. Each treatment was replicated three times and the experiment was repeated.

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In vitro glutamine synthetase activity – dose-response assay To evaluate the role of D173N in the mechanism of resistance to glufosinate in MG, a GS

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enzyme assay comparing MG, OR and SFS was performed in vitro to isolate other potential

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mechanisms that each biotype might exhibit to reduce glufosinate damage. Because glufosinate

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inhibits the GS isoforms with similar inhibition constant (Ki) values,27 an alteration in the


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binding kinetics between glufosinate and GS2 should be quantifiable with this assay. To test this

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hypothesis, GS inhibition by glufosinate was measured in vitro from plant leaf extracts.26 Briefly,

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200 mg of leaf tissue were ground with extraction buffer, filtered with Miracloth® and

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centrifuged. Twenty microliters of crude leaf extract were added to 180 µL of assay buffer in the

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absence of L-glutamine, along with 5 µL of glufosinate at different doses (to yield final

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concentrations of 0, 1, 3, 10, 30, 100 or 300 µM), and incubated for 30 min at 30 C to allow

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glufosinate binding to the GS isoforms. After the incubation period with the inhibitor, 20 µL of

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500 mM L-glutamine was added and incubated for another 30 min at 30 C. Absorbance was read

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at 540 nm after the addition of 100 µL of ferric chloride reagent. Three replications of each

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glufosinate treatment were performed and the experiment was conducted twice.

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In vitro time-dependent GS inhibition assay GS isoforms exhibit time-dependent inhibition dependent on the Ki and on the interaction

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between the enzyme active site and the inhibitor. If an amino acid substitution was involved in

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the mechanism of resistance to glufosinate in MG, then a differential time-dependent loss of

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enzyme activity would be observed when MG was compared to SFS. To test this hypothesis, GS

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activity was quantified over time in MG, OR and SFS. A 34 factorial design (three biotypes and

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four incubation periods) with three replications. Plant extracts were obtained by grinding plant

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tissue in the presence of the previously described GS extraction buffer, filtered with Miracloth®

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and centrifuged. Twenty microliters of crude leaf extract were added to 180 µL of assay buffer

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along with 5 µL of glufosinate (to yield a final concentration of 100 µM of glufosinate). Samples

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were incubated at 30 C for 0, 5, 15 and 30 min after addition of glufosinate and the reaction was



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stopped by adding 100 µL of ferric chloride reagent. Twenty microliters of 500 mM L-glutamine

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were added and incubated for another 30 min at 30 C. Absorbance was read at 540 nm.

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Effects of glufosinate on glutamate and glutamine levels Because glufosinate inhibits GS isoforms that are crucial to the photorespiratory nitrogen

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cycle, we expected that glutamate and glutamine concentrations in glufosinate-affected tissues

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should be altered. Furthermore, a differential alteration of these amino acids between

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glufosinate-resistant and -susceptible biotypes may help to elucidate the mechanisms of

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resistance. Therefore, glutamate and glutamine levels were quantified in MG, OR and SFS 24

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HAT, using a method described previously.14 Plants were treated with 560 g ai ha-1 glufosinate

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as described in the in vivo glutamine synthetase activity - dose-response assay section, in a 32

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factorial (three biotypes, two herbicide treatments) for each amino acid with three replications.

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Plants were frozen in liquid nitrogen, ground to a fine powder with mortar and pestle, and 200

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mg of plant tissue was weighed and transferred to 2-mL Eppendorf microtubes. Five milliliters

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of a 75:25 (v/v) methanol:water solution were added to each sample, sonicated for 30 min at

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(400 kHz), and centrifuged at 16000  g for 10 min. Supernatant was filtered through a 0.2 µm

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nylon filter and transferred to a 2-mL injection vial before mass spectrometry analysis.

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A triple quadrupole liquid chromatography mass spectrometer (LC-MS/MS; LCMS-

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8040, Shimadzu, Columbia, MD, USA) equipped with an electro spray ionization source was

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used to analyze glutamate, glutamine and the glufosinate parent compound in the plant samples.

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The LC–MS/MS system consisted of a Nexera X2 UPLC with 2 LC-30 AD pumps, an SIL-30

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AC MP autosampler, a DGU-20A5 Prominence degasser, a CTO-30A column oven, and SPD-

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M30A diode array detector coupled to an 8040-quadrupole mass-spectrometer. Separation


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occurred in a hydrophilic interaction column (iHilic-Fusion 100 × 2.1 mm, 3.5 µm; Hilicon,

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Umeå, Sweden) at a flow rate of 0.2 mL min-1 using a linear gradient of acetonitrile (B) and 25

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mM ammonium acetate (A): 2 min, 80% B; 8 min, 30% B; 12 min, 30% B; 12.1 min, 80% B.

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The MRMs were optimized to 181.95 → 136.05, 147.95 → 130.10, 147.10 → 130.00 for

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glufosinate, glutamate, and glutamine, respectively.

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GS1a, GS1b and GS2 expression Glufosinate, as previously noted, inhibits not only the plastid form of GS (GS2), but also

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its cytosolic counterparts. Therefore, an enhanced expression in one of the GS isogenes would

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indicate that more enzyme is present in the cellular pool and, consequently, more glufosinate

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must be absorbed to be effective, as is observed in other plant species with copy number

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variation of an herbicide target-site gene.28 First, primers were designed to amplify fragments of

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GS1a, GS1b and GS2 followed by a primer efficiency experiment. Sequence information was

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obtained from the International Weed Genomics Consortium29-30 of L. multiflorum GS annotated

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sequences, as well as the gene encoding the enzyme acetolactate synthase (ALS) which was used

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as internal control gene31 (for primer information, see Table S1). Approximately 50 mg of plant

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tissue from the youngest fully expanded leaves were collected, immediately frozen in liquid

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nitrogen for RNA extraction (RNeasy Plant Mini Kit, Qiagen, Germantown, MD) and cDNA

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synthesis (iSCRIPT, BIO-RAD, Hercules, CA) following manufacturer recommendations. PCR

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reactions contained 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 mM forward primer, 0.2 mM reverse

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primer, 10 ng DNA template, 2 U Platinum™ Taq DNA polymerase (Platinum™ Taq DNA

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polymerase kit, Life Technologies, Carlsbad, CA). The following PCR program was used: 10

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min at 94 C, 35 cycles of 30 s at 94 C, 30 s at 55 C and three min at 72 C, and a final extension



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step of 10 min at 72 C. PCR products sequenced resulted in mixed sequences, suggesting that

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primers were annealing in conserved regions of multiple GS isogenes. Therefore, a cloning step

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was performed to obtain individual sequences from GS1a, GS1b and GS2. Amplicons were

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ligated into a vector, transformed into Escherichia coli, and further selected with blue-white

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screen with X-Gal following the manufacturer’s protocol (TOPO TA Cloning Kit One Shot

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TOP10, Life Technologies, Carlsbad, CA) before sequencing (BigDye Terminator version 3.1

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Cycle Sequencing Kit, Applied Biosystems, California, USA) with the universal primer M13F(-

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21). The sequencing data were used to design q-RT-PCR primers that amplified GS isogene

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fragments (Table S2). The relevant sequences have been deposited to NCBI (accession number

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MK572811-813).

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To evaluate GS isogene expression, the plants were treated with 35 g ai ha-1 of

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commercial formulation of glufosinate as previously described. Tissue from three individual

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plants from each biotype was collected from nontreated and treated plants 24 HAT and the

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experiment was conducted twice. The q-RT-PCR reactions were performed in a 7500/7500 Fast

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Real-Time PCR System (Applied Biosystems, Foster City, California, USA) with 10 µL

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SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, Hercules, California, USA), 1 µL

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of forward primer, 1 µL of reverse primer (10 µM), and 10 ng of DNA in a total reaction volume

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of 20 µL. Primer efficiency experiments were performed and all primer combinations were

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within 100-110% efficiency (Table S2). Specificity was checked with a melt curve step in the q-

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RT-PCR program and no sign of primer dimers was observed.

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Absorption and translocation of 14C-glufosinate


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MG, OR and SFS seeds were germinated and seedlings grown in a growth chamber

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throughout the duration of the experiment with an 11/13 h and 16/10 C (day/night) regime, and

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light intensity of 800 µmol m-2 s-1. At the 13-BBCH stage,12 seedlings were transferred from soil

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to a hydroponic system comprised of 40 mL vials with PTFE/silicon septa containing a dilute

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nutrient solution and returned to the growth chamber.32 A completely randomized 35 factorial

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(biotype  time point) design with four replications was used and the experiment was repeated. Approximately 1.4 kBq of 14C-glufosinate (specific activity 6.35 MBq mg-1) were applied

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to the youngest fully expanded leaf in a 1-µL droplet 2 cm from the ligule towards the leaf apex.

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At 0, 6, 24, 48 and 72 HAT, plants were sectioned into treated leaf, aboveground tissue, and

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roots. The treated leaf was further sectioned into the upper portion of the treated leaf, the treated

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middle section, and the lower portion of the treated leaf. Treated leaves were individually

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washed with 10 mL of deionized water (diH2O) to remove non-absorbed herbicide and the rinsate

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samples were concentrated to approximately 1 mL in an evaporator (RapidVap, Labconco,

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Kansas City, MO) and 10 mL of scintillation cocktail was added for 14C quantification using

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liquid scintillation spectrometry (LSS) techniques. Glufosinate exudation was quantified by

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collecting a 10-mL aliquot from the growth solution, evaporating this aliquot to dryness and

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adding 10 mL of scintillation cocktail for LSS analysis. Plant parts were oven-dried and oxidized

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in a sample oxidizer (307 Sample Oxidizer, Perkin Elmer, Waltham, MA) for quantification of

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14C

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found in all plant parts compared to the amount applied).

using LSS techniques. The overall mass balance of 14C was 95.3 ± 2.0% (proportion of 14C

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Metabolism of glufosinate



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Plants were grown as described in the 14C-glufosinate absorption and translocation

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section. Approximately 16.5 kBq of radiolabeled glufosinate in a total of 10 µL of mixture were

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applied to the youngest fully expanded leaves. A completely randomized 33 factorial (biotype 

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time point) design with four replications was used and the experiment was repeated. At 24, 48

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and 72 HAT, treated leaves were sectioned and washed with 10 mL of diH2O to quantify

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unabsorbed glufosinate and whole-plant tissue was frozen and kept in a -80 C freezer until

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further analysis. Plant tissue was ground in liquid nitrogen using a mortar and a pestle, and

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ground samples were transferred to 50-mL Falcon tubes. Samples received 4 mL of 2%

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ammonium hydroxide (v/v, 28-30% NH3 basis, Sigma-Aldrich, St. Louis, MO) extraction

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solution, and were sonicated for 45 min at 65 C. After sonication, 10 mL of dichloromethane

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(>99.8%, Sigma-Aldrich, St. Louis, MO) were added to each sample for a liquid-liquid

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extraction and centrifuged for 1 h at 3,800  g. Three milliliters of the upper aqueous layer

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containing the majority (>99.9%) of the extracted 14C-glufosinate were subjected to a solid-phase

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extraction (SPE) step in a vacuum manifold (Visiprep SPE vacuum manifold, Supelco,

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Bellefonte, PA) set at -20 kPa. An additional 0.5 mL aliquot was collected from the aqueous

266

layer after centrifugation for mass balance quantification. SPE cartridges (Bond Elut Plexa PAX

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500 mg, Agilent Technologies, Folsom, CA) were conditioned with 3 mL of methanol (>99.9%,

268

Sigma-Aldrich, St. Louis, MO) and equilibrated with 3 mL of diH2O before sample loading.

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Wash steps were performed with 2 mL of diH2O and 2 mL of methanol. Finally, 14C-glufosinate

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elution was performed with four 1-mL 10% formic acid (>95%, Sigma-Aldrich, St. Louis, MO)

271

washes in methanol. The four elution steps were combined into a 4-mL sample and were

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subjected to a solvent exchange step, where eluted samples were dried and resuspended with

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0.48 mL of 0.025 M sodium tetraborate (99%, Sigma-Aldrich, St. Louis, MO) adjusted to pH 10


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with sodium hydroxide (>97%, Fisher Scientific, Fair Lawn, NJ). A derivatization step (Figure

275

S1) was performed by adding 0.5 mL of 20 mM 9-fluorenylmethoxycarbonyl chloride in excess

276

(FMOC, >99%, Sigma-Aldrich, St. Louis, MO) in acetonitrile (>99.9%, Sigma-Aldrich, St.

277

Louis, MO) to each sample and incubated on a reciprocating shaker for 2 h. The pre-column

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derivatization step with FMOC-Cl reduces the polar characteristics of glufosinate, facilitating

279

chromatographic retention and reducing the lower limit of detection. Because FMOC-Cl reacts

280

with amine groups in alkaline medium, the derivatization reaction was restricted to glufosinate,

281

as its known metabolites do not have the amine groups present on the parent compound.

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Furthermore, by manipulating the 14C-glufosinate-FMOC retention time, it was possible to

283

reduce co-elution of peaks related to the matrix and metabolites which increased method

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precision and accuracy. After the 2-h incubation period, reactions were stopped by the addition

285

of 20 µL of formic acid and samples injected in a HPLC system (1200 Infinity LC, Agilent,

286

Santa Clara, CA, USA) with an inline radioactivity detector (FlowStar LB 513, Berthold

287

Technologies, Bad Wildbad, Germany).

288

Analyte separation occurred in a Zorbax Eclipse Plus (C18, 250 mm  4.6 mm  5 µm)

289

with a gradient elution composed of mobile phase A (MP A) as 10% acetonitrile in 1% formic

290

acid (v/v) and mobile phase B (MP B) as 1% formic acid in acetonitrile (v/v), as follows: 100%

291

MP B from 0 to 3 min, 100% to 0% in 1 min and held for 6 min, with a total run time of 10 min

292

at 1.5 mL min-1. 14C-glufosinate-FMOC calibration curves were produced in the sample matrix

293

and subjected to the sample extraction procedure to reduce enhancement/suppression effects and

294

assess 14C-glufosinate degradation.

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Data analysis



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In vivo GS activity dose-response assay and in vitro GS activity dose-response assay data were

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fit to three-parameter log-logistic models and outliers were identified and removed based on the

299

internally studentized residuals method with α = 0.05.33 In vivo dose-response data did not meet

300

the nonlinear regression assumptions (i.e. normality of residuals and homoscedasticity of

301

variance), and were transformed based on a Box-Cox transformation with λ = 0.747.34 The

302

resistance indexes (RI) were calculated for MG and OR by comparing the amount of glufosinate

303

required to inhibit the GS activity by 50% compared to SFS (I50R/I50S) using a t-test. Data from

304

the in vitro time-dependent GS inhibition assay were analyzed as a factorial 34 (three biotypes

305

by four time points) using ANOVA and comparisons were made with Tukey’s HSD test.

306

Glutamate, glutamine, and glufosinate concentrations after glufosinate treatment were analyzed

307

as a 32 factorial for each compound, where glutamate and glufosinate data required a Box-Cox

308

transformation with λ = 0.1 and 0.2, respectively, to meet the ANOVA assumptions. Gene

309

expression data were analyzed as a 32 factorial (three biotypes and two glufosinate treatments)

310

for each gene, followed by Tukey’s HSD test.

311

Absorption data were subjected to nonlinear regression comparison and regression

312

parameters including Amax (the maximum absorption of glufosinate) and t90 (time required to

313

reach 90% of maximum absorption) were used to compare the biotypes.35 Translocation data

314

were analyzed as a 35 factorial with eight replications. An ANOVA was conducted and means

315

were separated using Tukey’s HSD test. Metabolism data were subjected to ANOVA and means

316

were separated using Tukey’s HSD test. All multiple comparisons performed had α = 0.05

317

adjusted with the Bonferroni correction to control for family-wise type I errors. Data from

318

different runs were pooled in accordance with the Levene’s homogeneity of variance test with α

319

= 0.05.


Page 15 of 38


15 320 321

Homology modeling of GS2 from SFS and MG

322

Sequences of GS2 from the SFS and MG L. multiflorum biotypes were aligned to the

323

sequence of Zea mays GS1 crystal structure (2d3c.pdb)36 using the Clustal Omega multiple

324

alignment tool.37 Lolium multiflorum sequences were longer than the GS1 sequence 2d3c for which

325

a crystal structure was available, so residue 58-414 from SFS and MG L. multiflorum sequences

326

were selected for homology modeling. Pairwise alignment of these segments was obtained using

327

EMBOSS Needle37 and calculated a 74% sequence identity and 87% sequence homology between

328

these sequences (Supplemental Information 1).

329

The 3D structure models of a GS2 from SFS and MG L. multiflorum proteins were

330

developed by aligning the primary structure and building a preliminary tertiary structure of the

331

proteins using MODELLER (version 9.21)38-39 based on the crystal structure of Z. mays GS1

332

(2d3c.pdb) serving as a template.36 Models for SFS and MG L. multiflorum with the lowest

333

DOPE scores were selected for further refinement using GROMACS (version 2018.3)40-41 (see

334

Supplemental Information 2 for codes) on Dell Precision T7500 workstation equipped with

335

100GB of DDR3 ECC RAM, 24-Core Intel® Xeon® 5600 series processors and a GeForce GTX

336

1060 6GB GDDR5 DirectX 12 graphics card. Briefly, the .pdb output from Modeller was

337

converted to .gro format with accompanied topology, restraint file, and post-processed structure,

338

and submitted to molecular dynamic simulation. A virtual box extending 1 nm around the protein

339

was built, solvated using the spc216 water model, and the charges on the protein were

340

neutralized by including a 0.15 M solution of NaCl.

341 342

The structure was relaxed to ensure that no steric clashes or inappropriate geometry were present prior to molecular dynamics simulation. The system was then subjected to two two-step



Page 16 of 38

16 343

equilibrations to optimize temperature and pressure. Finally, the protein was subjected to full

344

molecular dynamics for 1-ns without restraints. GS2 MG and SFS homology models were

345

evaluated using MolProbity42-43 to identify the conformation of residues with potential to reduce

346

glufosinate binding. Proteins and ligand interactions were visualized using PyMOL (The PyMOL

347

Molecular Graphics System, Version 2.0 Schrödinger, LLC).44

348 349

RESULTS

350

Glutamine synthetase activity

351

MG and OR exhibited RI’s of 2.3 and 3.3, respectively (Table 1). Little variation was

352

observed within each treatment or between experimental runs, as observed by the 95%

353

confidence intervals around the regressions (Figure S2) and the residual standard deviation of

354

1.93. GS activity reached values close to zero with the highest glufosinate dose.

355

On the other hand, there was no difference in GS inhibition by glufosinate in MG, OR

356

and SFS when tested in vitro, with little variation within treatments as observed by the 95%

357

confidence intervals around the regressions (Figure S3) (Table 2), with residual standard error of

358

9.4. No differences were observed among the biotypes when the time-dependency of GS

359

inhibition was evaluated with 100 µM of glufosinate (Figure 1).

360 361 362

Effects of glufosinate on glutamate and glutamine levels Overall, glutamate levels were greater than glutamine levels in nontreated samples.

363

Levels of free glutamate and glutamine decreased 24 h after glufosinate application in MG, OR

364

and SFS (Figure 2). Glutamate concentrations in SFS plants treated with glufosinate were four

365

times less than in OR, but similar to the concentrations in MG. There were no differences among


Page 17 of 38


17 366

the biotypes for glutamine, with or without glufosinate treatment. Glufosinate parent compound

367

concentration in OR was two less than in SFS and similar between MG and SFS (Figure 2).

368 369

GS1a, GS1b and GS2 expression

370

The overall expression of GS1a and GS1b was less than that of GS2 in all L. multiflorum

371

biotypes, with mean expression of GS2 of 0.44, GS1a of 0.14, and GS1b of 0.07 compared to the

372

normalization gene. GS1a and GS1b expression were similar among the biotypes with or without

373

glufosinate treatments (Figure 3). GS2 expression was greater in MG compared to OR and SFS

374

before glufosinate application, but was similar to the other biotypes 24 HAT. Upon glufosinate

375

treatment, GS2 expression in OR increased.

376 377

Absorption and translocation of 14C-glufosinate

378

Herbicide absorption dynamics were similar among MG, OR and SFS (Figure S4). The

379

regression estimates Amax and t90 from MG and OR were not different from those of SFS (Table

380

3). Most of the 14C-glufosinate was absorbed within 24 HAT and the maximum absorption of

381

glufosinate observed was greater than 85% for all biotypes within 72 HAT.

382

Herbicide translocation out of the treated leaf was similar among the biotypes up to 24

383

HAT (Figure 4). At 48 HAT, the resistant biotypes MG and OR had less translocation and by 72

384

HAT biotype OR translocated the least amount of 14C of the three biotypes (16.5% for OR,

385

19.2% for MG, and 20.7% for SFS). The majority of the 14C-glufosinate retained in the treated

386

leaf was translocated toward the leaf apex in all biotypes (data not shown). The maximum

387

differences in translocation were less than 5% when OR and SFS were compared at 48 and 72



Page 18 of 38

18 388

HAT, with up to 20% of the radioactivity moving out of the treated leaf. Glufosinate exudation

389

from roots was negligible (data not shown).

390 391 392

Metabolism of glufosinate A high coefficient of determination was observed when 14C-glufosinate-FMOC was

393

analyzed using HPLC in line with a radioactivity detector (>99%), with a wide range of linearity

394

(43 to 813 dpm µL-1) in the L. multiflorum matrix (data not shown). Biotypes MG, OR and SFS

395

had 14C-glufosinate metabolites at all sampling times (Figure 5). At 24 HAT, no differences in

396

the amount of metabolite were observed among biotypes, whereas at 48 and 72 HAT biotype OR

397

had greater metabolite amounts compared to MG and SFS, in some instances up to 30% more.

398

The method used in this research resulted in a mass balance >90% which was a significant

399

improvement compared to previously reported analytical methods that analyzed glufosinate

400

metabolism in weed species.16

401 402 403

Homology modeling of GS2 from glufosinate SFS and MG L. multiflorum Sequences of GS2 from the SFS and MG L. multiflorum biotypes had 74% sequence

404

identity and 87% sequence homology to the Z. mays GS1 sequence, making it possible to build

405

homology models that had 1.23 root mean square deviation of the α-C atoms from the crystal

406

structure (those having the most influence on the secondary and tertiary conformation) (Figure

407

S5). After GROMACS, 98.9% (350/354) of all GS2 residues from the SFS had torsion and bond

408

angles within allowable ranges. The four outliers were residues 2 Arg, 18 Lys, 171 Ile, and 242

409

Asp (Supplemental Information 3). Similarly, 98.69% of all GS2 residues from MG had torsion


Page 19 of 38


19 410

and bond angles within allowable ranges. The 5 outliers were residues 64 Pro, 138 Asp, 142 Pro,

411

204 Pro, and 336 Ser. In both models, all of the Asn, Gln, and His were oriented correctly.

412 413 414

DISCUSSION Weed resistance to herbicides threatens food security and the sustainability of cropping

415

systems, as weed competition reduces the yield potential and aesthetic value of crops. Non-

416

target-site resistance (NTSR) is of major concern to practitioners, as it is a more general adaptive

417

trait and may confer resistance to different classes of herbicides and herbicides that have not yet

418

been discovered. Here, we report a glufosinate-resistant L. multiflorum biotype with enhanced

419

herbicide metabolism. Furthermore, our initial hypothesis that the D173N amino acid

420

substitution in GS2 was involved in the glufosinate resistance phenotype in MG was not

421

supported by our data.

422

In vivo GS activity measured in whole-plants sprayed with different rates of glufosinate

423

was consistent with resistance indices from Avila-Garcia et al. (Table 1), even though these

424

authors adopted an ammonia accumulation bioassay to quantify the resistance levels.2, 17

425

Conversely, the resistance indices in our research were considerably different from Avila-Garcia

426

et al.17 when the GS was quantified, likely induced by the large standard errors obtained by the

427

authors. No differences in GS activity from the in vitro experiment were observed among the

428

biotypes, suggesting the the D173N amino acid substitution is not involved in the mechanism of

429

glufosinate resistance in MG. Alterations in target-site enzymes are known to confer resistance to

430

other herbicides such as the protoporphyrinogen oxidase (E.C. 1.3.3.4) and EPSPS inhibitors,45-46

431

primarily due to conformational changes in the enzyme active site. As demonstrated in Z. mays,

432

GS2 active sites involve interactions of amino acid residues at positions 131, 192, 249, 291, 311



Page 20 of 38

20 433

and 332 with glufosinate, whereas positions 127, 187, 251, 253, 328 and 316 interact with an

434

ADP molecule.36 Because the position 173 in L. multiflorum is equivalent to amino acid residue

435

167 in Z. mays, this amino acid substitution does not seem to interact directly with the catalitic

436

site of GS2. No crystal structure comparison has been conducted to evaluate the effect of D173N

437

in the L. multiflorum GS2, nor have studies been conducted to evaluate the activity of GS2 in

438

isolation of the other GS isozymes in MG. Our data suggest, however, that the overall combined

439

catalytic efficiency of the GS isozymes was not significantly reduced in biotype MG with

440

D173N.

441

Sequences of GS2 from the SFS and MG L. multiflorum biotypes have strong sequence

442

identity and homology to the Z. mays GS1 sequence, resulting in the production of highly

443

reliable homology models (Figure S5) suitable for examining the potential role of the D173N

444

mutation on resistance to glufosinate.17 The homology models of the GS2 from L. multiflorum

445

had binding domains that could hold both ADP and glufosinate based on the coordinate from the

446

crystal structure of Z. mays GS1 (2d3c.pdb), illustrating the highly conserved residues involved

447

in the activity of all GS (Figure 6A).36 A closer examination of the catalytic domain confirmed

448

that the interactions between ADP, glufosinate, and the three Mn atoms were maintained and that

449

the complex was stabilized via interactions to several amino acid side chains (Figure 6B).

450

While a D173N mutation in the MG biotype has been associated with resistance to

451

glufosinate,17 the biochemical characterization of GS activity from SFS and MG did not support

452

that this mutation imparted resistance to the enzyme in vitro (Table 2 and Figure S3). Analysis

453

of the tertiary structure of L. multiflorum GS2 revealed that this mutation was positioned

454

between 20.5 and 32.5 Å from either of the ligands and separated by a β-sheet and an α-helix

455

(Figure 7), which makes it very unlikely to affect GS activity or the binding of glufosinate. This


Page 21 of 38


21 456

observation is consistent with our biochemical characterization of the GS activity indicating no

457

difference in sensitivity to glufosinate between the SFS and MG biotypes. Consequently, more

458

work needs to be conducted to elucidate the potential function of the D173N mutation (if any) in

459

the MG biotype.

460

Differential expression of GS isogenes was not associated with resistance, as no

461

differences among L. multiflorum biotypes were observed after glufosinate treatment. These data

462

agree with the overall results from the enzyme activity assays, and further suggest that there is a

463

close relationship between GS transcripts and enzyme translated. Plastid GS is likely encoded by

464

a single gene in L. multiflorum,9 whereas cytoplasmic GS is encoded by a family of genes

465

depending on the plant tissue, enzyme function and developmental stage.9, 10, 47 Nord-Larsen et

466

al.9 observed that nitrogen starvation (a symptom similar to glufosinate action in plants) in L.

467

perenne increased the expression of GS1a and a decreased GS1b expression, likely because

468

GS1a is associated with nitrogen remobilization in tissues.48 In our experiments, however, GS1a

469

expression did not change after glufosinate treatment.

470

As expected, concentrations of glutamine and glutamate decreased after glufosinate

471

application. Because OR exhibited the highest resistance level, differences in the glutamate

472

concentration compared to SFS were more evident after glufosinate treatment (Figure 2). The

473

inhibition of GS activity by glufosinate inherently reduces glutamine and glutamate

474

concentration in plants,14 and a differential reduction among the biotypes would help elucidate

475

the mechanism of resistance to glufosinate. Furthermore, our experiments suggest that GS

476

enzyme activity assays are more reliable biomarkers for glufosinate resistance diagnostics than

477

glutamate and glutamine quantification.



Page 22 of 38

22 478

Although the physicochemical properties of glufosinate would suggest it is a phloem-

479

mobile herbicide (i.e. low pKa, low Kow),49 its ability to be translocated to other parts of the plant

480

is in fact very limited,50 explaining the observations from this research that the majority of the

481

applied herbicide was found in the treated leaf. The maximum differences in translocation were

482

less than 5% when OR and SFS were compared at 48 and 72 HAT, and these differences seem to

483

only partially explain the glufosinate resistance phenotype observed at the whole-plant level.

484

Enhanced glufosinate metabolism was observed in OR compared to SFS, and is the major

485

mechanism of resistance in OR. Furthermore, mass spectrometry data of the glufosinate parent ion

486

corroborate the metabolism experiment with radiolabeled glufosinate, as the lowest concentration

487

of the glufosinate parent compound was found in OR and the greatest concentration in SFS. L-

488

Phosphinothricin metabolites are not toxic and D-phosphinothricin seems to be stable in plants,

489

supporting the hypothesis that glufosinate metabolism could confer resistance.22, 51 Considering

490

that L-phosphinothricin has herbicidal activity and is subjected to biological degradation while D-

491

phosphinothricin is not metabolized in plants,22 a comparative analysis between biotypes seems

492

reasonable as D-phosphinothricin had similar behavior in all L. multiflorum biotypes studied and

493

the differences observed likely originated from L-phosphinothricin metabolism (Figure 5).

494

In summary, the D173N amino acid substitution does not account for the glufosinate

495

resistance in MG and the mechanism(s) of resistance in this biotype remains unclear. Our results

496

strongly suggest that the differences in glufosinate metabolism in OR are involved in the

497

mechanism of resistance to glufosinate. These results support our initial hypothesis that NTSR

498

mechanisms are involved in glufosinate resistance in OR. Ongoing research will elucidate the

499

mechanisms of resistance to glufosinate in MG, as well as the inheritance mechanisms, and the

500

genetic mechanisms contributing to the observed phenotypes in MG and OR.


Page 23 of 38


23 501 502

SUPPORTING AND SUPPLEMENTAL INFORMATION

503

Supporting information contains primer sequences for amplification of both initial GS

504

isogenes (Table S1) and q-RT-PCR (Table S2), the molecular structure of glufosinate, FMOC

505

and the derivatization product (Figure S1), in vivo and in vitro dose-response curves (Figure S2

506

and S3), glufosinate absorption (Figure S4), evaluation of the homology models using

507

MolProbity (Figure S5). Supplemental information contains the sequence analysis of SFS, MG

508

and Z. mays GS1 (Supplemental Information 1), GROMACS code used to refine the structures of

509

GS2 from SFS and MG (Supplemental information 2), and overlay of homology model of L.

510

multiflorum GS2 on crystal structure of Z. mays GS1 (Supplemental information 3).

511 512 513 514 515 516 517 518 519 520 521 522 523



Page 24 of 38

24 524

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Page 28 of 38

28 676

Table 1. In vivo glutamine synthetase dose-response assay with MG, OR and SFS L. multiflorum

677

biotypes. Log-logistic regression estimates/a Biotype/b

b

d

I50

RI/c

MG

1.21 ± 0.06

95.01 ± 2.09

51.17 ± 3.79

2.27 ± 0.19***

OR

1.73 ± 0.11

91.74 ± 1.76

72.71 ± 4.19

3.30 ± 0.26***

SFS

1.03 ± 0.04

101.01 ± 2.16

21.07 ± 1.44

-

678

/aEquation:

679

the upper limit, and e is the I50 (amount of glufosinate required to inhibit glutamine synthetase

680

activity by 50% in g a.i. ha-1). /bValues are means ± SE of the detransformed data. /cRI =

681

Resistance Index [e(MG or OR)/e(SFS)]. /ns not statistically significant (α = 0.05). /*** P-value

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