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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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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 A. C. G. Brunharo,*,† Hudson K. Takano,‡ Carol A. Mallory-Smith,† Franck E. Dayan,‡ and Bradley D. Hanson§
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†
Department of Crop and Soil Science, Oregon State University, 3050 Southwest Campus Way, Crop Sciences Building Corvallis, Oregon 97331, United States ‡ Department of Bioagricultural Sciences and Pest Management, Colorado State University, 1777 Campus Delivery, Fort Collins, Colorado 80523, United States § Department of Plant Science, University of California, Davis, One Shields Avenue, MS-4, Davis, California 95616, United States S Supporting Information *
ABSTRACT: Glufosinate-resistant Lolium perenne L. spp. multiflorum biotypes from Oregon exhibited resistance levels up to 2.8-fold the field rate. One resistant biotype (MG) had an amino acid substitution in glutamine synthetase 2 (GS2), whereas the other (OR) exhibited the wild-type genotype. We hypothesized that the amino acid substitution in GS2 is involved in the resistance mechanism in MG and that non-target site resistance mechanisms are present in OR. OR metabolized glufosinate faster than the other two biotypes, with >75% of the herbicide metabolized in comparison to 50% in MG and the susceptible biotype. A mutation in GS2 co-segregating with resistance in MG did not reduce the enzyme activity, with results further supported by our enzyme homology models. This research supports the conclusion that a metabolism mechanism of glufosinate resistance is present in OR and that glufosinate resistance in MG is not due to an altered target site. KEYWORDS: enzyme modeling, gene expression, herbicide absorption, herbicide degradation, herbicide translocation, non-target site resistance, phosphinothricin
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INTRODUCTION Lolium perenne L. spp. multiflorum (Lolium multiflorum) is a winter annual grass species native to Europe and initially introduced to the United States as a forage crop.1 L. multiflorum is also a major weed in agricultural areas in many parts of the world, primarily in regions of temperate climate. In Oregon, L. multiflorum interferes with a wide range of crops.2 Modern agriculture relies heavily on herbicides because of their efficacy and economic returns, whereas other management practices may be more costly or less selective.3 Overreliance on herbicides for weed management has, however, selected for herbicide-resistant L. multiflorum populations in at least 12 countries.4 The obligate-outcrossing, self-incompatible mating system of this diploid (2n = 2x = 14) species facilitates the dispersal of herbicide resistance genes within and among populations.5,6 2-Amino-4-[hydroxy(methyl)phosphoryl]butanoic acid (glufosinate) is a commercially available non-selective herbicide composed of a racemic mixture (L/D-phosphinothricin) used for post-emergence control of weeds in many agricultural and non-agricultural systems. The L-phosphinothricin form has herbicidal activity, irreversibly inhibiting glutamine synthetases (GS; EC 6.3.1.2), a family of enzymes responsible for the adenosine triphosphate (ATP)-dependent assimilation of ammonia as an amide moiety of glutamine, central to the assimilation of nitrogen in plants.7 A number of GS isoforms have been identified in Arabidopsis thaliana, Zea © XXXX American Chemical Society
mays, and L. multiflorum and are commonly classified as cytosolic (GS1) and plastidic (GS2), depending upon their cellular localization and specific role in plants.8,9 The GS1a isoform in Triticum aestivum, for example, is involved in nitrogen remobilization during leaf senescence,10 while GS1b is associated with nitrogen assimilation in roots of Oryza sativa.11 Inhibition of GS causes a dramatic decrease in glutamate and glutamine, leading to the accumulation of downstream metabolites in photorespiration (glyoxylate, phosphoglycolate, and glycolate).12,13 The accumulation of high levels of glyoxylate leads to the inhibition of ribulose-1,5bisphosphate carboxylase/oxygenase activase, a key enzyme in the Calvin−Benson cycle, resulting in the accumulation of reactive oxygen species, lipid peroxidation of membranes, and cell death.14 Although glufosinate has been used for over 40 years, the first glufosinate-resistant weed species were confirmed in 2009 in a vegetable farm in Malaysia15 and in a Corylus avellana L. orchard in Oregon.2 Despite recent efforts to understand the biological processes involved in glufosinate resistance, little information has been obtained about the underlying mechanisms involved in this phenotype.16 Among the Received: Revised: Accepted: Published: A
March 1, 2019 May 3, 2019 May 8, 2019 May 8, 2019 DOI: 10.1021/acs.jafc.9b01392 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
buffer [25 mM imidazole−HCl at pH 7.5 (99%, Fisher Scientific), 50 mM L-glutamine (99%, Sigma-Aldrich), 4 mM MnCl2 (99%, Fisher Scientific), 5 mM adenosine diphosphate (99%, Sigma-Aldrich), 40 mM sodium arsenate (99%, Fisher Scientific), and 25 mM hydroxylamine (99%, VWR, Radnor, PA, U.S.A.)] and incubated for 30 min at 30 °C. After the incubation period, 100 μL of ferric chloride reagent [250 mM trichloroacetic acid (100%, SigmaAldrich), 200 mM anhydrous ferric chloride (99%, Fisher Scientific), and 0.5 M HCl (100%, Sigma-Aldrich)] was added to each sample to stop the reaction before spectrophotometric measurement at 540 nm. Each treatment was replicated 3 times, and the experiment was repeated. 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 enzyme assay comparing MG, OR, and SFS was performed in vitro to isolate other potential mechanisms that each biotype might exhibit to reduce glufosinate damage. Because glufosinate inhibits the GS isoforms with similar inhibition constant (Ki) values,28 an alteration in the binding kinetics between glufosinate and GS2 should be quantifiable with this assay. To test this hypothesis, GS inhibition by glufosinate was measured in vitro from plant leaf extracts.27 Briefly, 200 mg of leaf tissue was ground with extraction buffer, filtered with Miracloth, and centrifuged. A total of 20 μL of crude leaf extract was added to 180 μL of assay buffer in the absence of L-glutamine, along with 5 μL of glufosinate at different doses (to yield final concentrations of 0, 1, 3, 10, 30, 100, or 300 μM), and incubated for 30 min at 30 °C to allow for glufosinate binding to the GS isoforms. After the incubation period with the inhibitor, 20 μL of 500 mM L-glutamine was added and incubated for another 30 min at 30 °C. Absorbance was read at 540 nm after the addition of 100 μL of ferric chloride reagent. Three replications of each glufosinate treatment were performed, and the experiment was conducted twice. In Vitro Time-Dependent GS Inhibition Assay. GS isoforms exhibit time-dependent inhibition dependent upon Ki and the interaction between the enzyme active site and the inhibitor. If an amino acid substitution was involved in the mechanism of resistance to glufosinate in MG, then a differential time-dependent loss of enzyme activity would be observed when MG was compared to SFS. To test this hypothesis, GS activity was quantified over time in MG, OR, and SFS. A 3 × 4 factorial design (three biotypes and four incubation periods) was used with three replications. Plant extracts were obtained by grinding plant tissue in the presence of the previously described GS extraction buffer, filtered with Miracloth, and centrifuged. A total of 20 μL of crude leaf extract was added to 180 μL of assay buffer along with 5 μL of glufosinate (to yield a final concentration of 100 μM glufosinate). Samples were incubated at 30 °C for 0, 5, 15, and 30 min after the addition of glufosinate, and the reaction was stopped by adding 100 μL of ferric chloride reagent. A total of 20 μL of 500 mM L-glutamine were added and incubated for another 30 min at 30 °C. Absorbance was read at 540 nm. Effects of Glufosinate on Glutamate and Glutamine Levels. Because glufosinate inhibits GS isoforms that are crucial to the photorespiratory nitrogen cycle, we expected that glutamate and glutamine concentrations in glufosinate-affected tissues should be altered. Furthermore, a differential alteration of these amino acids between glufosinate-resistant and -susceptible biotypes may help to elucidate the mechanisms of resistance. Therefore, glutamate and glutamine levels were quantified in MG, OR, and SFS 24 HAT, using a method described previously.14 Plants were treated with 560 g of ai ha−1 glufosinate as described in the In Vivo Glutamine Synthetase Activity: Dose−Response Assay section, in a 3 × 2 factorial (three biotypes and two herbicide treatments) for each amino acid with three replications. Plants were frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle, and 200 mg of plant tissue was weighed and transferred to 2 mL Eppendorf microtubes. A total of 5 mL of a 75:25 (v/v) methanol/water solution was added to each sample, sonicated for 30 min at (400 kHz), and centrifuged at 16000g for 10 min. The supernatant was filtered through a 0.2 μm nylon filter and transferred to a 2 mL injection vial before mass spectrometry analysis.
glufosinate-resistant L. multiflorum biotypes identified in Oregon by Avila-Garcia et al.,2,17 one biotype (MG) exhibited an amino acid substitution in the target site, whereas the other biotype (OR) presented the wild-type genotype. The single nucleotide polymorphism (SNP) found in the GS2 gene encoded for a GS isozyme with an amino acid substitution at position 173 (D173N) (equivalent to position 171 on the T. aestivum GS2 sequence). If GS2 is altered only in MG (compared to the GS2 sequence of the susceptible L. multiflorum biotype; SFS), then it is possible that a conformational alteration in GS2 leads to an altered binding affinity between the target enzyme and glufosinate. For instance, a single amino acid substitution in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) at position 102 and/or 106, located in the enzyme active site, reduced the binding affinity with the inhibitor glyphosate, conferring resistance to this herbicide in several weed species.18−21 Conversely, because alterations in the glufosinate target site were not found in OR, it is possible that non-target site resistance (NTSR) mechanisms were selected in this population. Glufosinate metabolism, for example, has been found in Daucus carota L. and Nicotiana tabacum L.22 as well as in a number of weed species.23 Although enhanced metabolism has not been established as a glufosinate resistance mechanism, upregulation of certain groups of xenobiotic-detoxifying enzymes may confer resistance to other herbicides.24,25 Elucidating the mechanisms of resistance to herbicides in weeds is important for understanding the underlying basic biological processes in plants and allowing practitioners to develop better weed management decisions. In this context, the objective of this research was to characterize the putative target site and NTSR mechanisms of glufosinate resistance in L. multiflorum biotypes from Oregon.
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MATERIALS AND METHODS
Plant Material. Previously characterized2,17 L. multiflorum biotypes were used in this research: (i) the glufosinate-resistant biotype MG that has an amino acid substitution in the plastidic GS2 (D173N), (ii) the glufosinate-resistant biotype OR that does not exhibit mutations in GS2, and (iii) the susceptible biotype SFS. The reported herbicide rate required to reduce plant growth by 50% (GR50) for MG, OR, and SFS were 0.45, 0.49, and 0.15 kg of active ingredient (ai) ha−1, respectively.2,17 In Vivo Glutamine Synthetase Activity: Dose−Response Assay. A dose−response experiment was conducted to compare the response of MG, OR, and SFS to glufosinate. Plants were treated at the BBCH-23 growth stage26 in a spray cabinet calibrated to deliver 187 L ha−1 and kept at 30/20 °C and 13/11 h (day/night) in a greenhouse throughout the experiment. Eight glufosinate rates (Liberty 280 SL, Bayer CropScience, Research Triangle Park, NC, U.S.A.) ranging from 0 to 2240 g of ai ha−1 were applied to singleplant experimental units. Enzyme activity, as a percentage of the control, was evaluated using a modified procedure outlined by Dayan et al.27 that relies on the formation of γ-glutamyl hydroxamate and further reaction with acidic ferric chloride reagent. At 24 h after herbicide treatment (HAT), crude leaf extracts were obtained from 200 mg of fully expanded leaves in the presence of 333 μL of extraction buffer [50 mM tris(hydroxymethyl)methylamine (99%, Bio-Rad, Hercules, CA, U.S.A.), 1 mM ethylenediaminetetraacetic acid (99%, Bio-Rad, Hercules, CA, U.S.A.), 2 mM dithiothreitol (99%, Thermo Scientific, Waltham, MA, U.S.A.), 10 mM MgCl2 (99%, Fisher Scientific, Waltham, MA), and 5% (w/v) polyvinylpyrrolidone (99%, Sigma-Aldrich, St. Louis, MO, U.S.A.)], filtered with Miracloth (EMD Millipore, San Diego, CA, U.S.A.), centrifuged at 12000g for 10 min at 4 °C, and kept on ice throughout the procedure. A total of 20 μL of crude leaf extract was added to 180 μL of assay B
DOI: 10.1021/acs.jafc.9b01392 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Absorption and Translocation of 14C-Glufosinate. MG, OR, and SFS seeds were germinated, and seedlings were grown in a growth chamber throughout the duration of the experiment with an 11/13 h and 16/10 °C (day/night) regime and light intensity of 800 μmol m−2 s−1. At the 13th BBCH stage,12 seedlings were transferred from soil to a hydroponic system comprised of 40 mL vials with polytetrafluoroethylene (PTFE)/silicon septa containing a dilute nutrient solution and returned to the growth chamber.33 A completely randomized 3 × 5 factorial (biotype × time point) design with four replications was used, and the experiment was repeated. Approximately 1.4 kBq of 14C-glufosinate (specific activity of 6.35 MBq mg−1) was applied to the youngest fully expanded leaf in a 1 μL droplet 2 cm from the ligule toward the leaf apex. At 0, 6, 24, 48, and 72 HAT, plants were sectioned into treated leaf, aboveground tissue, and roots. The treated leaf was further sectioned into the upper portion of the treated leaf, the treated middle section, and the lower portion of the treated leaf. Treated leaves were individually washed with 10 mL of deionized water (diH2O) to remove non-absorbed herbicide; the rinsate samples were concentrated to approximately 1 mL in an evaporator (RapidVap, Labconco, Kansas City, MO, U.S.A.); and 10 mL of scintillation cocktail was added for 14C quantification using liquid scintillation spectrometry (LSS) techniques. Glufosinate exudation was quantified by collecting a 10 mL aliquot from the growth solution, evaporating this aliquot to dryness, and adding 10 mL of scintillation cocktail for LSS analysis. Plant parts were oven-dried and oxidized in a sample oxidizer (307 Sample Oxidizer, PerkinElmer, Waltham, MA, U.S.A.) for quantification of 14 C using LSS techniques. The overall mass balance of 14C was 95.3 ± 2.0% (proportion of 14C found in all plant parts compared to the amount applied). Metabolism of Glufosinate. Plants were grown as described in the 14C-glufosinate absorption and translocation section. Approximately 16.5 kBq of radiolabeled glufosinate in a total of 10 μL of mixture was applied to the youngest fully expanded leaves. A completely randomized 3 × 3 factorial (biotype × time point) design with four replications was used, and the experiment was repeated. At 24, 48, and 72 HAT, treated leaves were sectioned and washed with 10 mL of diH2O to quantify unabsorbed glufosinate and whole plant tissue was frozen and kept in a −80 °C freezer until further analysis. Plant tissue was ground in liquid nitrogen using a mortar and pestle, and ground samples were transferred to 50 mL Falcon tubes. Samples received 4 mL of 2% (v/v) ammonium hydroxide (28−30% NH3 basis, Sigma-Aldrich, St. Louis, MO, U.S.A.) extraction solution and were sonicated for 45 min at 65 °C. After sonication, 10 mL of dichloromethane (>99.8%, Sigma-Aldrich, St. Louis, MO, U.S.A.) was added to each sample for a liquid−liquid extraction and centrifuged for 1 h at 3800g. A total of 3 mL of the upper aqueous layer containing the majority (>99.9%) of extracted 14C-glufosinate were subjected to a solid-phase extraction (SPE) step in a vacuum manifold (Visiprep SPE vacuum manifold, Supelco, Bellefonte, PA, U.S.A.) set at −20 kPa. An additional 0.5 mL aliquot was collected from the aqueous layer after centrifugation for mass balance quantification. SPE cartridges (Bond Elut Plexa PAX 500 mg, Agilent Technologies, Folsom, CA, U.S.A.) were conditioned with 3 mL of methanol (>99.9%, Sigma-Aldrich, St. Louis, MO, U.S.A.) and equilibrated with 3 mL of diH2O before sample loading. Wash steps were performed with 2 mL of diH2O and 2 mL of methanol. Finally, 14C-glufosinate elution was performed with four 1 mL of 10% formic acid (>95%, Sigma-Aldrich, St. Louis, MO, U.S.A.) washes in methanol. The four elution steps were combined into a 4 mL sample and were subjected to a solvent exchange step, where eluted samples were dried and resuspended with 0.48 mL of 0.025 M sodium tetraborate (99%, Sigma-Aldrich, St. Louis, MO, U.S.A.) adjusted to pH 10 with sodium hydroxide (>97%, Fisher Scientific, Fair Lawn, NJ, U.S.A.). A derivatization step (Figure S1 of the Supporting Information) was performed by adding 0.5 mL of 20 mM 9-fluorenylmethoxycarbonyl chloride (FMOC, >99%, Sigma-Aldrich, St. Louis, MO, U.S.A.) in excess in acetonitrile (>99.9%, Sigma-Aldrich, St. Louis, MO, U.S.A.) to each sample and incubated on a reciprocating shaker for 2 h. The pre-column derivatization step with FMOC−Cl reduces the polar
A triple quadrupole liquid chromatography mass spectrometer (LC−MS/MS, LCMS-8040, Shimadzu, Columbia, MD, U.S.A.) equipped with an electrospray ionization source was used to analyze glutamate, glutamine, and the glufosinate parent compound in the plant samples. The LC−MS/MS system consisted of a Nexera X2 UPLC with two LC-30AD pumps, a SIL-30 AC MP autosampler, a DGU-20A5 prominence degasser, a CTO-30A column oven, and SPD-M30A diode array detector coupled to an 8040 quadrupole mass spectrometer. Separation occurred in a hydrophilic interaction column (iHilic-Fusion, 100 × 2.1 mm, 3.5 μm, Hilicon, Umeå, Sweden) at a flow rate of 0.2 mL min−1 using a linear gradient of acetonitrile (B) and 25 mM ammonium acetate (A): 2 min, 80% B; 8 min, 30% B; 12 min, 30% B; and 12.1 min, 80% B. The multiple reaction monitoring (MRM) was optimized to 181.95 → 136.05, 147.95 → 130.10, and 147.10 → 130.00 for glufosinate, glutamate, and glutamine, respectively. GS1a, GS1b, and GS2 Expression. Glufosinate, as previously noted, inhibits not only the plastid form of GS (GS2) but also its cytosolic counterparts. Therefore, an enhanced expression in one of the GS isogenes would indicate that more enzyme is present in the cellular pool and, consequently, more glufosinate must be absorbed to be effective, as observed in other plant species with copy number variation of an herbicide target site gene.29 First, primers were designed to amplify fragments of GS1a, GS1b, and GS2. Sequence information was obtained from the International Weed Genomics Consortium30,31 of L. multiflorum GS annotated sequences as well as the gene encoding the enzyme acetolactate synthase (ALS), which was used as an internal control gene32 (for primer information, see Table S1 of the Supporting Information). Approximately 50 mg of plant tissue from the youngest fully expanded leaves were collected and immediately frozen in liquid nitrogen for RNA extraction (RNeasy Plant Mini Kit, Qiagen, Germantown, MD, U.S.A.) and cDNA synthesis (iSCRIPT, Bio-Rad, Hercules, CA, U.S.A.) following manufacturer recommendations. Polymerase chain reaction (PCR) contained 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 mM forward primer, 0.2 mM reverse primer, 10 ng of DNA template, and 2 units of Platinum Taq DNA polymerase (Platinum Taq DNA polymerase kit, Life Technologies, Carlsbad, CA, U.S.A.). The following PCR program was used: 10 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 3 min at 72 °C, and a final extension step of 10 min at 72 °C. PCR products sequenced resulted in mixed sequences, suggesting that primers were annealing in conserved regions of multiple GS isogenes. Therefore, a cloning step was performed to obtain individual sequences from GS1a, GS1b, and GS2. Amplicons were ligated into a vector, transformed into Escherichia coli, and further selected with a blue−white screen with X-Gal following the protocol of the manufacturer (TOPO TA Cloning Kit One Shot TOP10, Life Technologies, Carlsbad, CA, U.S.A.) before sequencing (BigDye Terminator version 3.1 Cycle Sequencing Kit, Applied Biosystems, Foster City, CA, U.S.A.) with the universal primer M13F(-21). The sequencing data were used to design quantitative reverse transcription polymerase chain reaction (q-RT-PCR) primers that amplified GS isogene fragments (Table S2 of the Supporting Information). The relevant sequences have been deposited to the National Center for Biotechnology Information (NCBI, accession numbers MK572811− MK572813). To evaluate GS isogene expression, the plants were treated with 35 g of ai ha−1 of the commercial formulation of glufosinate as previously described. Tissue from three individual plants from each biotype was collected from non-treated and treated plants 24 HAT, and the experiment was conducted twice. The q-RT-PCR reactions were performed in a 7500/7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) with 10 μL of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, U.S.A.), 1 μL of forward primer, 1 μL of reverse primer (10 μM), and 10 ng of DNA in a total reaction volume of 20 μL. Primer efficiency experiments were performed, and all primer combinations were within 100−110% efficiency (Table S2 of the Supporting Information). Specificity was checked with a melt curve step in the q-RT-PCR program, and no sign of primer dimers was observed. C
DOI: 10.1021/acs.jafc.9b01392 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. In Vivo Glutamine Synthetase Dose−Response Assay with MG, OR, and SFS L. multiflorum Biotypes log−logistic regression estimatesa biotype
b
MG OR SFS
b
d
I50
RIc
1.21 ± 0.06 1.73 ± 0.11 1.03 ± 0.04
95.01 ± 2.09 91.74 ± 1.76 101.01 ± 2.16
51.17 ± 3.79 72.71 ± 4.19 21.07 ± 1.44
2.27 ± 0.19d 3.30 ± 0.26d
Equation: Y = d/1 + exp[b(log x − log e)], where b denotes the relative slope around e, d is the upper limit, and e is the amount of glufosinate required to inhibit glutamine synthetase activity by 50% (I50) in grams of ai per hectare. bValues are means ± standard error (SE) of the detransformed data. cRI = resistance index [e(MG or OR)/e(SFS)]. dp value of 99%), with a wide range of linearity (43−813 dpm μL−1) in the L. multiflorum matrix (data not shown). Biotypes MG, OR, and SFS had 14C-glufosinate metabolites at all sampling times (Figure 5). At 24 HAT, no differences in the amount of
before glufosinate application but was similar to the other biotypes 24 HAT. Upon glufosinate treatment, GS2 expression in OR increased. Absorption and Translocation of 14C-Glufosinate. Herbicide absorption dynamics were similar among MG, OR, and SFS (Figure S4 of the Supporting Information). The regression estimates Amax and t90 from MG and OR were not different from those of SFS (Table 3). Most 14C-glufosinate was absorbed within 24 HAT, and the maximum absorption of glufosinate observed was greater than 85% for all biotypes within 72 HAT. Table 3. Asymptotic Regression Parameters of 14CGlufosinate Absorption in L. multiflorum Biotypes regression parametera biotype
Amax (%)
t90 (h)
MG OR SFS
86.1 ± 1.5b 88.8 ± 1.5b 88.8 ± 1.5
21.9 ± 1.5b 25.2 ± 1.9b 22.1 ± 1.6
a
Equation: Y = (Amaxt)/[(10/θ)t90 + t], where Y is the absorption (as a percentage of applied), Amax is the maximum percentage of absorption at large values of t, t is time, and t90 is the time required to reach 90% of maximum absorption. bNot statistically different based on a t test comparing means to the susceptible biotype SFS.
Herbicide translocation out of the treated leaf was similar among the biotypes up to 24 HAT (Figure 4). At 48 HAT, the resistant biotypes MG and OR had less translocation, and by 72 HAT, biotype OR translocated the least amount of 14C of the three biotypes (16.5% for OR, 19.2% for MG, and 20.7% for SFS). The majority of 14C-glufosinate retained in the treated leaf was translocated toward the leaf apex in all biotypes (data not shown). The maximum differences in Figure 5. Metabolism of 14C-glufosinate in biotypes MG, OR, and SFS of L. multiflorum. Circles represent biotype MG; triangles represent OR; and squares represent SFS. Data points represent mean responses, and error bars represent standard errors.
metabolite were observed among biotypes, whereas at 48 and 72 HAT, biotype OR had greater metabolite amounts compared to MG and SFS, in some instances up to 30% more. The method used in this research resulted in a mass balance of >90%, which was a significant improvement compared to previously reported analytical methods that analyzed glufosinate metabolism in weed species.16 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 identity and 87% sequence homology to the Z. mays GS1 sequence, making it possible to build homology models that had 1.23 root-meansquare deviation of the α-C atoms from the crystal structure (those having the most influence on the secondary and tertiary conformations) (Figure S5 of the Supporting Information). After GROMACS, 98.9% (350/354) of all GS2 residues from the SFS had torsion and bond angles within allowable ranges. The four outliers were residues 2Arg, 18Lys, 171Ile, and 242Asp (Supporting Information 3). Similarly, 98.69% of all GS2 residues from MG had torsion and bond angles within allowable ranges. The five outliers were residues 64Pro, 138Asp, 142Pro, 204Pro, and 336Ser. In both models, all Asn, Gln, and His were oriented correctly.
Figure 4. Translocation of 14C-glufosinate out of the treated leaf as a percentage of absorption in biotypes MG, OR, and SFS of L. multiflorum. Data points represent mean responses, and error bars represent standard errors. ns, not significant; ∗∗, statistically significant, after Bonferroni correction (α = 0.017). F
DOI: 10.1021/acs.jafc.9b01392 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 6. (A) Surface topology of the L. multiflorum GS2 homology model showing the location of L-glufosinate and ADP bound within the catalytic domain. Coordinates of the ligands were transferred from the crystal structure of corn GS1 (2d3c.pdb). (B) Catalytic domain of L. multiflorum GS2 after molecular dynamics simulation using GROMACS, showing interactions between ADP, L-glufosinate, and the three Mn atoms. Note how the complex is stabilized via interaction to a number of amino acid side chains.
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DISCUSSION Weed resistance to herbicides threatens food security and the sustainability of cropping systems, because weed competition reduces the yield potential and aesthetic value of crops. Nontarget site resistance (NTSR) is of major concern to practitioners, because it is a more general adaptive trait and may confer resistance to different classes of herbicides and herbicides that have not yet been discovered. Here, we report a glufosinate-resistant L. multiflorum biotype with enhanced herbicide metabolism. Furthermore, our initial hypothesis that the D173N amino acid substitution in GS2 was involved in the glufosinate resistance phenotype in MG was not supported by our data. In vivo GS activity measured in whole plants sprayed with different rates of glufosinate was consistent with resistance indices from Avila-Garcia et al. (Table 1), even though these authors adopted an ammonia accumulation bioassay to quantify the resistance levels.2,17 Conversely, the resistance indices in our research were considerably different from AvilaGarcia et al.17 when GS was quantified, likely induced by the large standard errors obtained by the authors. No differences in GS activity from the in vitro experiment were observed among the biotypes, suggesting that the D173N amino acid substitution is not involved in the mechanism of glufosinate resistance in MG. Alterations in target site enzymes are known to confer resistance to other herbicides, such as the protoporphyrinogen oxidase (E.C. 1.3.3.4) and EPSPS inhibitors,46,47 primarily as a result of conformational changes in the enzyme active site. As demonstrated in Z. mays, GS2 active sites involve interactions of amino acid residues at positions 131, 192, 249, 291, 311, and 332 with glufosinate, whereas positions 127, 187, 251, 253, 328, and 316 interact with an adenosine diphosphate (ADP) molecule.37 Because position 173 in L. multiflorum is equivalent to amino acid residue 167 in Z. mays, this amino acid substitution does not seem to interact directly with the catalitic site of GS2. No crystal structure comparison has been conducted to evaluate the effect of D173N in the L. multiflorum GS2 nor have studies been conducted to evaluate the activity of GS2 in isolation of the other GS isozymes in MG. Our data suggest, however, that the overall combined catalytic efficiency of the GS isozymes was not significantly reduced in biotype MG with D173N.
Sequences of GS2 from the SFS and MG L. multiflorum biotypes have strong sequence identity and homology to the Z. mays GS1 sequence, resulting in the production of highly reliable homology models (Figure S5 of the Supporting Information) suitable for examining the potential role of the D173N mutation on resistance to glufosinate.17 The homology models of GS2 from L. multiflorum had binding domains that could hold both ADP and glufosinate based on the coordinate from the crystal structure of Z. mays GS1 (2d3c.pdb), illustrating the highly conserved residues involved in the activity of all GS (Figure 6A).37 A closer examination of the catalytic domain confirmed that the interactions between ADP, glufosinate, and the three Mn atoms were maintained and that the complex was stabilized via interactions to several amino acid side chains (Figure 6B). While a D173N mutation in the MG biotype has been associated with resistance to glufosinate,17 the biochemical characterization of GS activity from SFS and MG did not support that this mutation imparted resistance to the enzyme in vitro (Table 2 and Figure S3 of the Supporting Information). Analysis of the tertiary structure of L. multiflorum GS2 revealed that this mutation was positioned between 20.5 and 32.5 Å from either of the ligands and separated by a β-sheet and an αhelix (Figure 7), which makes it very unlikely to affect GS activity or the binding of glufosinate. This observation is consistent with our biochemical characterization of the GS activity, indicating no difference in sensitivity to glufosinate between the SFS and MG biotypes. Consequently, more work needs to be conducted to elucidate the potential function of the D173N mutation (if any) in the MG biotype. Differential expression of GS isogenes was not associated with resistance, because no differences among L. multiflorum biotypes were observed after glufosinate treatment. These data agree with the overall results from the enzyme activity assays and further suggest that there is a close relationship between GS transcripts and enzyme translated. Plastid GS is likely encoded by a single gene in L. multiflorum,9 whereas cytoplasmic GS is encoded by a family of genes depending upon the plant tissue, enzyme function, and developmental stage.9,10,48 Nord-Larsen et al.9 observed that nitrogen starvation (a symptom similar to glufosinate action in plants) in L. perenne increased the expression of GS1a and decreased GS1b expression, likely because GS1a is associated with G
DOI: 10.1021/acs.jafc.9b01392 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
In summary, the D173N amino acid substitution does not account for the glufosinate resistance in MG and the mechanism(s) of resistance in this biotype remain unclear. Our results strongly suggest that the differences in glufosinate metabolism in OR are involved in the mechanism of resistance. Ongoing research will elucidate the mechanisms of resistance to glufosinate in MG, the inheritance mechanisms, and the genetic mechanisms contributing to the observed phenotypes in MG and OR.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01392. Sequence analysis of SFS, MG, and Z. mays GS1 (Supporting Information 1), GROMACS code used to refine the structures of GS2 from SFS and MG (Supporting Information 2), and overlay of the homology model of L. multiflorum GS2 on the crystal structure of Z. mays GS1 (Supporting Information 3) (PDF) Molecular structure of glufosinate, FMOC, and the derivatization product (Figure S1), in vivo and in vitro dose−response curves (Figures S2 and S3), glufosinate absorption (Figure S4), and evaluation of the homology models using MolProbity (Figure S5) (PDF) Primer sequences for amplification of both initial GS isogenes (Table S1) and q-RT-PCR (Table S2) (PDF)
Figure 7. Position of the D to N mutation on residue 173 (red spheres) of L. multiflorum GS2 relative to the catalytic domain holding ADP and L-glufosinate. The mutation is between 20.5 and 32.5 Å from either of the ligands and separated by a β-sheet and an α-helix.
nitrogen remobilization in tissues.49 In our experiments, however, GS1a expression did not change after glufosinate treatment. As expected, concentrations of glutamine and glutamate decreased after glufosinate application. Because OR exhibited the highest resistance level, differences in the glutamate concentration compared to SFS were more evident after glufosinate treatment (Figure 2). The inhibition of GS activity by glufosinate inherently reduces glutamine and glutamate concentrations in plants,14 and a differential reduction among the biotypes would help elucidate the mechanism of resistance to glufosinate. Furthermore, our experiments suggest that GS enzyme activity assays are more reliable biomarkers for glufosinate resistance diagnostics than glutamate and glutamine quantification. Although the physicochemical properties of glufosinate would suggest that it is a phloem-mobile herbicide (i.e., low pKa and low Kow),50 its ability to be translocated to other parts of the plant is in fact very limited,51 explaining the observations from this research that the majority of the applied herbicide was found in the treated leaf. The maximum differences in translocation were less than 5% when OR and SFS were compared at 48 and 72 HAT, and these differences seem to only partially explain the glufosinate resistance phenotype observed at the whole plant level. Enhanced glufosinate metabolism was observed in OR compared to SFS and is the major mechanism of resistance in OR. Furthermore, mass spectrometry data of the glufosinate parent ion corroborate the metabolism experiment with radiolabeled glufosinate, because the lowest concentration of the glufosinate parent compound was found in OR and the greatest concentration was found in SFS. L-Phosphinothricin metabolites are not toxic, and D-phosphinothricin seems to be stable in plants, supporting the hypothesis that glufosinate metabolism could confer resistance.22,52 Considering that Lphosphinothricin has herbicidal activity and is subjected to biological degradation while D-phosphinothricin is not metabolized in plants,22 a comparative analysis between biotypes seems reasonable because D-phosphinothricin had similar behavior in all L. multiflorum biotypes studied and the differences observed likely originated from L-phosphinothricin metabolism (Figure 5).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Caio A. C. G. Brunharo: 0000-0001-9735-1648 Franck E. Dayan: 0000-0001-6964-2499 Notes
The authors declare no competing financial interest.
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REFERENCES
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