Glyphosate Can Decrease Germination of Glyphosate-Resistant

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Glyphoste can decrease germination of glyphosate-resistant soybeans Marcelo Pedrosa Gomes, Elisa Monteze Bicalho, Élise Smedbol, Fernanda Vieira da Silva Cruz, Marc Lucotte, and Queila Souza Garcia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05601 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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Journal of Agricultural and Food Chemistry

Glyphoste can decrease germination of glyphosate-resistant soybeans Marcelo Pedrosa Gomesa,*, Elisa Monteze Bicalhoa, Élise Smedbol a,b, Fernanda Vieira da Silva Cruza, Marc Lucotteb, Queila Souza Garciaa,* a

Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento

de Botânica, Avenida Antônio Carlos, 6627, Pampulha, Caixa Postal 486, 31270-970, Belo Horizonte, Minas Gerais, Brazil b

Université du Québec à Montréal, GEOTOP & Institut des Sciences de

l’environnement, C.P. 8888, Succ. Centre-Ville, H3C 3P8, Montréal, Québec, Canada. *

Corresponding authors

*(M.P.G.) E-mail: [email protected] Phone: +553134092690. Fax:

+553134092671 *(Q.S.G.) E-mail: [email protected] Phone: +553134092690. Fax: +553134092671

1 ACS Paragon Plus Environment


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ABSTRACT: We investigated the effects of different concentrations of glyphosate acid

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and one of its formulations (Roundup®) on seed germination of two glyphosate

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resistant (GR)- and one non-GR variety of soybean. As expected, the herbicide affected

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the shikimate pathway in non-GR seeds but not in GR seeds. We observed that

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glyphosate can disturb the mitochondrial electron transport chain leading to H2O2

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accumulation in soybean seeds, which was, in turn, related to lower seed germination.

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In addition, GR-seeds showed increased activity of antioxidant systems when compared

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to non-GR seeds, making them less vulnerable to oxidative-stress induced by

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glyphosate. The differences in the responses of GR varieties to glyphosate exposure

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corresponded to their differences in enzymatic activity related to H2O2-sacavenging and

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mitochondrial Complex III (the proposed site of ROS induction by glyphosate). Our

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results shown that glyphosate ought to be used carefully as a pre-emergence herbicide in

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soybean field crop systems since this practice may reduce seed germination.

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Keywords: antioxidant, mitochondria, reactive oxygen species, pesticide, shikimate,

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Roundup


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Graphical Abstract



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INTRODUCTION

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Soybean [Glycine max (L) Merrill] is one of the most important economic crop

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cultures worldwide and its production was greatly leveraged by the introduction of

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glyphosate-resistant (GR) plants1. Nowadays, about 60% of the worldwide cultivated area

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is planted with GR soybeans 2. GR-plants are provided with an exogenous gene, cp4 5-

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enolpyruvylshikimate-3-phosphate synthase (EPSPS), which encoded the bacterial

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version of the enzyme CP4EPSPS, conferring plants tolerance against glyphosate (N-

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phosphonomethyl glycine)3. Non-GR plants, in contrast, are susceptible to the effects of

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glyphosate linked to the inhibition of EPSPS - which prevents the biosynthesis of

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aromatic amino acids 4.

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The primary mode of action of glyphosate is the inhibition of EPSPS, however,

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the herbicide has several secondary effects on plant physiology 5, which have even been

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observed in GR-plants

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shown either deleterious

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mechanism by which the herbicide could affect the germination process is still unclear.

6–8

. When applied to seeds, glyphosate-based herbicides have 9,10

, little or no effect

11,12

on germination, but the exact

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Although recommended for use on post-emergence stages, glyphosate-based

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herbicides are often applied in combination with, or following, the application of pre-

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emergence herbicides in programs of weed control

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glyphosate just before sowing can result in the exposure of crop seeds to residual

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concentrations of the herbicide in soil. Little is known, however, about the effects of

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glyphosate on seed germination of GR plants. Recently, we have shown that glyphosate

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(glyphosate acid and its commercial formulation Roundup®) impairs the mitochondrial

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electron transport chain (ETC) in Dimorphandra wilsonii (a non-GR Brazilian legume)

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seeds, with Complex III as its precise target

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mitochondrial ETC Complex III appeared quite sensitive to glyphosate (glyphosate acid),

14

13

. In this scenario, the use of

. Similarly, in Lemna minor leaves,


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which resulted in the formation of reactive oxygen species (ROS) by the electron shuttling

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from semiquinone to oxygen

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related to the herbicide toxicity to L. minor

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species 16–20. Unlike what was expected, however, the impairment of respiratory ETC in

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D. wilsonii seeds did not induce oxidative stress and ROS accumulation was prevented

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by the activity of antioxidant enzymes such as catalase (CAT) and ascorbate peroxidase

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(APX). ROS provide important signals during seed germination

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plants to cope with excessive ROS production is directly linked to seed germinability 22.

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Therefore, we hypothesized that antioxidant systems have a central role in seeds

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germinating under the presence of glyphosate. Moreover, it is known that oxidative

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responses under stress conditions (such as the presence of herbicide) differ among plant

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species, organs, tissues and even among varieties of the same species 17,23.

15

. The oxidative stress due to ROS accumulation was 15

- as also observed in some other plant

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, and the ability of

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In this context, the present study evaluates the effects of the glyphosate acid and

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the glyphosate-based commercial formulation Roundup® on seed germination of three

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varieties of soybean. Since GR and non-GR soybean seeds differ in their antioxidant

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system activities 24, by evaluating the effects of the herbicides on GR and non-GR seeds,

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we determined if the effect of glyphosate on soybean seed germination is linked to

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oxidative metabolism. Moreover, two different varieties of GR-soybean seeds were tested

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aiming to understand if the herbicides could have differential effects on GR-varieties.

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More specifically, we aimed at (i) understanding the mechanisms of glyphosate action on

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germination and (ii) verifying whether pre-emergent usage of the herbicide on soybean

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crops could affect seed germination, possibly reducing yields.

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MATERIAL AND METHODS

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Plant material and germination assays. The non-GR soybean variety BRS-284

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(EMBRAPA) and the GR varieties L 8307 RR (Riber-KWS) and AS 3810 IPRO 5 ACS Paragon Plus Environment


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(Agroeste) collected in 2016 were used in this study. The seeds were surface sterilized

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in 2.5% sodium hypochlorite for 5 min and thoroughly rinsed with deionized water

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before sowing. Germination assays were carried out by placing four replicates of 25

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seeds in germination boxes lined with filter paper (Whatman No. 1) moistened with 20

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ml of deionized water or with treatment solutions. The germination boxes were arranged

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in a completely randomized order in a growth chamber at 30 ºC in the dark. Seeds were

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considered to have germinated when approximately 2 mm of the primary root had

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emerged. Seed viability was evaluated by the tetrazolium test before germination and in

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non-germinated seeds after germination experiments. Seeds were pre-imbibed in

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distilled water for 6 hours and then immersed in 0.5% tetrazolium (2,3,5-Triphenyl-

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tetrazolium chloride, Sigma) solution for 2 hours in the dark. The initial viability of the

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seed lot was 70% for non-GR and 100% for GR varieties. Germination percentages

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were corrected by initial viability.

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Analytical-grade glyphosate (Pestanal grade) obtained from Sigma-Aldrich

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(Oakville, Canada) and the commercial glyphosate Roundup® formulation (Monsanto,

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Brazil) were used in the experiments. Stock solutions (1000 mg l-1) of glyphosate and

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Roundup® were prepared in ultrapure water and used to obtain the solutions with

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desired concentrations of the chemicals used to moisten seeds (20 ml/germination box).

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Seeds were submitted to different concentrations of glyphosate and Roundup® (0, 5, 25

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and 50 mg active ingredient l-1), considering field application of 2.8 kg glyphosate ha-1

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(19 g l-1 of active equivalent (a.e.))16. To assure reproducibility of the obtained data, the

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germination experiment was repeated twice, with no statistically significant differences

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according to ANOVA (data not shown).

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Biochemical evaluations. Biochemical evaluations were performed in the whole seed

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after the extraction of the tegument, here considered as embryo (cotyledons + 6 ACS Paragon Plus Environment

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embryonic axe). The mean germination time (average length of time required for final

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germination of a seed lot) 25 for all the varieties studied was less than 48 hours.

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Therefore, we followed physiological changes of early seed germination after 24 hours

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of treatment induction. Biochemical analyses were performed on seeds treated in

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parallel experiments under the same conditions used in the germination tests. Embryos

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(embryonic axis + cotyledons) of six seeds from each germination box were manually

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extracted and ground in liquid nitrogen, constituting one replicate (with a total of 4

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replicates/treatment). Samples were stored at -80 ºC until analyzed. Shikimate concentrations were evaluated following the methods of Bijay and

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Dale 26 with modifications: 0.1 g of embryos was grown in liquid nitrogen and

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homogenized with 1 ml of 0.25 M hydrochloric acid. Then, the extract was centrifuged

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at 25,000 x g for 15 min and 100 µl of the centrifuged supernatant was reacted with 1

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ml of 1% periodic acid solution. After 1 h, 1 ml of 1 M sodium hydroxide and 0.6 ml of

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0.1 M glycine were added to the samples and the absorbance was measured at 380 nm.

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Hydrogen peroxide (H2O2) concentrations were measured following Velikova et

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al. 27, using 0.1 g of embryos. H2O2 was extracted in 2 ml of 0.1% tricloroacetic acid

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(TCA) and after centrifugation at 12,000 x g for 15 min, 300 µl of the centrifuged

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supernatant was reacted with 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and

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1 ml of 1 M KI. Samples were read at 390 nm and H2O2 concentrations were given on a

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standard curve.

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Antioxidant enzymes were extracted by macerating 0.1 g of embryos in 1 ml of

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an extraction buffer containing 100 mmol l−1 potassium phosphate buffer (pH 7.8), 100

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mmol l−1 EDTA, 1 mmol l−1 L-ascorbic acid, and 2% PVP (m/v). The protein contents

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of all the samples were determined using the Bradford method. Catalase (CAT; E.C.

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1.11.1.6) and ascorbate peroxidase (APX; E.C. 1.11.1.11) activities of the embryos were 7 ACS Paragon Plus Environment


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determined spectrophotometrically. Catalase assay was performed following 28 at 25 ºC

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in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 250 mM

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hydrogen peroxide and distilled water. Catalase activity was determined following the

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decomposition of H2O2, for 1.5 min at 10-second intervals, monitoring changes in

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absorbance at 240 nm, with a molar extinction coefficient of 0.0394 mM-1 cm-1.

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Ascorbate peroxidase activity was determined following 29 at 28 ºC in a reaction

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mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM L-ascorbic

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acid, 2 mM hydrogen peroxide and distilled water. The APX activity was estimated my

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monitoring ascorbate oxidation rate (ε = 2.8 mM-1 cm-1) at 290 nm for 3 min at 15-s

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intervals.

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The enzymatic activities of the mitochondrial ETC Complexes I to IV were

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determined by spectrophotometry on embryo homogenates (200 mM phosphate buffer

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pH 7.5) using 30 mg of protein in each assay from seeds exposed to 0 or 50 mg l-1 of the

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tested herbicides. Complex I (NADH:ubiquinone oxidoreductase) and Complex II

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(succinate dehydrogenase) assays were performed following 30, and their activities were

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calculated by monitoring the rates of NADH (ε = 5.5 mM-1 cm-1) and ubiquinone

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(ε = 9.6 mM-1 cm-1) decreases per µg of protein. Complex III (ubiquinol-cytochrome c

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reductase) activity was determined following Birch-Machin et al. 31, monitoring

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cytochrome cIII reduction rates (ε = 19 mM-1 cm-1). Complex IV (cytochrome c

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oxidase) activity was determined following 32, calculating the rate of increase in

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absorbency caused by the oxidation of cytochrome cII to cytochrome cIII (ε = 19 mM-1

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cm-1). All activities were expressed as per microgram of protein (which was determined

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by the Bradford method).

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To identify if mitochondria Complex III enzyme is the site of ROS (hydrogen peroxide) induction by glyphosate herbicides in seeds of soybean 14,15, seeds were 8 ACS Paragon Plus Environment

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exposed to 0 or 50 mg l-1 solutions (glyphosate acid or Roundup®) with 15 µM of

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rotenone (an inhibitor of mitochondrial ETR Complex I) or 15 µM of antimycin A (an

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inhibitor of mitochondrial ETR Complex III Qo site) 15,33. Seed germination was

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recorded and H2O2 concentrations in embryos (24 h after exposure) were assessed 27.

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Statistical Analyses. Statistical analyses were performed using JMP 7.0 software (SAS

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Institute Inc.). The results were expressed as the averages of four replicates. The data

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was tested for normality (Shapiro–Wilk) and homogeneity (Bartlett) and then

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statistically evaluated. Germination, shikimate, oxidative stress marker and assays

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involving Complex III inhibitors were evaluated using three-way analysis of variance

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(ANOVA). Interactions between herbicide (glyphosate acid and Roundup®),

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concentrations (0, 5, 25 and 50 mg a.i l-1)/inhibitor (rotenone and antimycin) and

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varieties (Non-GR, L 8307 RR and AS 3810 IPRO) were included in the model.

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Meanwhile, two-way ANOVA was used in evaluations of the activity of mitochondrial

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ETC complexes. In these evaluations, the interaction between herbicide and varieties

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was included in the model. When differences were detected by ANOVA, means were

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compared by a post hoc Student t test (significance at P 0.05). The herbicides, however, decreased

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seed germination of non-GR (70.2 and 66.8% for glyphosate and Roundup®,

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respectively) and GR-variety L 8307 (74.2 and 68.7% for glyphosate and Roundup®,

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respectively) (P < 0.05; Fig. 1). Glyphosate acid exposure resulted in a greater decrease

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(P < 0.05) in seed germination of non-GR (70.2%) than of GR- L 8307 (74.2%), while

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the effects of Roundup® were similar between these two varieties (68.7 and 66.8%,

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respectively, P > 0.05).

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In the affected varieties (non-GR and L 8307), glyphosate acid and Roundup®

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concentrations had different effects on germination percentage of seeds (P < 0.0001;

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Fig. 1). Detrimental effects of glyphosate acid on seed germination of both varieties

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were only observed at the highest concentration (50 mg l-1) (P < 0.05). In contrast, all

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the studied concentrations of Roundup® led to decreases on germination percentage of

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these seeds (P < 0.05). Moreover, comparing the effects of the same herbicide

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concentration on each variety, it was noted that Roundup® induced a greater decrease in

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germination than glyphosate acid (P < 0.05) – with the exception of seeds exposed to 50

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mg l-1 (P > 0.05). None of the concentrations of glyphosate acid or Roundup® induced

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decreases in the viability of the soybean seeds of the three varieties tested (P > 0.05).

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Shikimate was greater in embryos of non-GR seeds of (104.55 µg g-1 FW) than

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in GR seeds (74.70 and 68.61 µg g-1 FW in L 8307 RR and AS 3810 IPRO,

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respectively) (P < 0.0001). A significant interaction was observed between

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concentration and variety (P < 0.0001). In non-GR seeds treated with both glyphosate

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acid and Roundup, shikimate concentrations increased in embryos regardless of the

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herbicide concentration used (Fig. 1); in both GR varieties, however, shikimate

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concentrations did not differ among treatments (P < 0.05; Fig. 1). 10 ACS Paragon Plus Environment

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Oxidative stress markers. Hydrogen peroxide (H2O2) concentrations were greater in

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embryos of seeds treated with Roundup® (P < 0.0001) and in embryos of GR (2.53 and

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2.41 µg g-1 FW in L 8307 RR and AS 3810 IPRO, respectively) than in non-GR seeds

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(1.6 µg g-1 FW). Significant interactions were observed between herbicide,

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concentration and variety (P = 0.0006). In non-GR seeds and in the L 8307 RR variety,

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all Roundup® treatments greatly increased H2O2 concentrations in embryos, which was

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only observed in seeds treated with 50 mg glyphosate acid l-1 (Fig. 2).

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Ascorbate peroxidase (APX) and catalase (CAT) activity was lower in embryos

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of non-GR-seeds (13.75 mM ascorbate min-1 mg-1 protein and 3.02 nM H2O2 min-1 mg-1

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protein, respectively) (P < 0.0001; Fig. 2). Between GR-varieties, APX and CAT activity

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in embryos was greater in AS 3810 IPRO (21.01 mM ascorbate min-1 mg-1 protein and

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4.98 nM H2O2 min-1 mg-1 protein, respectively) than in L 8307 RR (19.56 mM ascorbate

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min-1 mg-1 protein and 4.59 nM H2O2 min-1 mg-1 protein, respectively) seeds (P < 0.0001;

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Fig. 2). Significant interactions were observed between concentration and variety (P

Glyphosate Can Decrease Germination of Glyphosate-Resistant

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