Glyphosate and aminomethylphosphonic acid content in glyphosate

Subscriber access provided by UNIV OF LOUISIANA

Agricultural and Environmental Chemistry

Glyphosate and aminomethylphosphonic acid content in glyphosateresistant soybean leaves, stems and roots and associated phytotoxicity following a single glyphosate-based herbicide application Élise Smedbol, Marc Lucotte, Sophie Maccario, Marcelo Pedrosa Gomes, Serge Paquet, Matthieu Moingt, Lila Lucero Celis Mercier, Millaray Rayen Perez Sobarzo, and Marc-André Blouin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00949 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

GLYPHOSATE AND AMINOMETHYLPHOSPHONIC ACID CONTENT IN

2

GLYPHOSATE-RESISTANT SOYBEAN LEAVES, STEMS AND ROOTS AND

3

ASSOCIATED PHYTOTOXICITY FOLLOWING A SINGLE GLYPHOSATE-BASED

4

HERBICIDE APPLICATION

5

*ÉLISE SMEDBOL1, MARC LUCOTTE1, SOPHIE MACCARIO1, MARCELO

6

PEDROSA GOMES2, SERGE PAQUET3, MATTHIEU MOINGT1, LILA LUCERO

7

CELIS MERCIER3, MILLARAY RAYEN PEREZ SOBARZO4 , MARC-ANDRÉ

8

BLOUIN4

1 Université

9

du Québec à Montréal, GEOTOP & Institut des Sciences de

10

l’environnement, 201, Avenue du Président-Kennedy, H2X 3Y7, Montréal, Québec,

11

Canada

12

2 Universidade

biológicas, 80050-540, Curitiba, Paraná, Brazil

13 14

3

17

Université du Québec à Montréal, Département des sciences biologiques, 141, Avenue du Président-Kennedy, H2X 1Y4, Montréal, Québec, Canada

15 16

Federal do Paraná, Departamento de Botânica, Setor de Ciências

4

Université du Québec à Montréal, Département de Chimie, 2101, rue JeanneMance, H2X 2J6, Montréal, Québec, Canada

18 19 20

*Correspondance: É. Smedbol, Université du Québec à Montréal, GEOTOP &

21

Institut des Sciences de l’environnement, 201, Avenue du Président-Kennedy, H2X 3Y7,

22

Montréal, Québec, Canada

23

E-mail: [email protected]

ACS Paragon Plus Environment


Page 2 of 38

25

Abstract

26

Glyphosate-based herbicides (GBH) applications were reported to induce physiological

27

damages to glyphosate-resistant (GR) soybean, which were mainly attributed to

28

aminomethylphosphonic acid (AMPA). In order to study glyphosate and AMPA dynamics

29

in plants and associated phytotoxic effects, a greenhouse experiment was set where GR

30

soybeans were exposed to GBH (0.7 to 4.5 kg glyphosate ha-1) and sampled over time (2,

31

7, 14 and 28 days after treatment (DAT)). Hydrogen peroxide content increased 2 DAT,

32

while a decrease was observed for the effective quantum yield (2, 7, 14 DAT), stomatal

33

conductance (2 DAT) and biomass (14 DAT). Glyphosate content was higher in leaves,

34

followed by stems, then roots. AMPA content tended to increase with time, especially in

35

roots, and the amount of AMPA in roots was negatively correlated to mostly all

36

phytotoxicity indicators. This finding is important since AMPA residues are measured in

37

agricultural soils several months after GBH applications, which could impact productivity

38

in GR crops.

39

Keywords: glyphosate and AMPA toxicokinetics, glyphosate-based herbicides,

40

glyphosate-resistant soybean, H2O2, stomatal conductance, biomass, effective quantum

41

yield

42

2 ACS Paragon Plus Environment

Page 3 of 38


44

1.

Introduction

45

Application of glyphosate-based herbicides (GBH) on glyphosate-resistant (GR) crops is

46

now the main weed control strategy worldwide in field crops and, for example,

47

approximately 78% of all soybean crops are GR

48

(phosphonomethyl)glycine) has previously been described as slighlty mobile in plants,

49

residues of this molecule as well as aminomethylphosphonic acid (AMPA), its main

50

metabolite, are measured in grains several weeks/months after GBH applications

51

Glyphosate and AMPA are also detected in GR soybean leaves and stems at all growth

52

stages, following GBH applications 5. Glyphosate fate in plants after foliar application is

53

relatively well documented, however, very little is known about that of AMPA.

54

Glyphosate, the active molecule of GBH, is a phosphonic acid, usually sprayed in a

55

watersoluble form (isopropylamine salt) and is best absorbed by plants with the addition

56

of surfactants contained in the commercial formulations 6. After foliar application, it is

57

proposed that glyphosate is absorbed through cuticular uptake and/or stomatal infiltration

58

at varying rates following the species tested, the applied concentration and the surfactant

59

concentration 7. Different degrees of glyphosate absorption are reported in the literature,

60

ranging from 4 to 70% of the applied dose 7-9. About 40-45% of the absorbed glyphosate

61

is then systematically translocated in the entire plant 7. After reaching the vascular tissues,

62

glyphosate is preferably distributed towards specific tissues following the photoassimilates

63

pathway

64

stems is estimated at 5.39 and 4.02 DAT in plants exposed to glyphosate doses of 0.8 and

65

1.6 kg ha-1 11. Glyphosate dissipation in plants can occur by its metabolization into AMPA,

66

particulary so in the second generation of GR plants expressing glyphosate oxidoreductase

10.

1.

While glyphosate (N-

2-4.

The half-time dissipation of glyphosate measured in GR maize leaves and



12.

Page 4 of 38

67

(GOX) enzyme, isolated from Ochrobactrum anthropi

68

resistant to GBH through its metabolization into AMPA, by the expression of GOX which

69

cleaves glyphosate C-N bond

70

residues are expected to be present. The second resistance pathway in GR crops is the

71

introduction of the CP4 gene isolated from Agrobacterium sp., coding for an insensitive 5-

72

enolpyruvolshikimate 3-phosphate synthase (EPSPS) enzyme

73

insentitive EPSPS, while some crops have both an insensitive EPSPS and express GOX 15.

74

However, AMPA residues are also measured in GR plants not expressing GOX but only

75

an insensitive EPSPS, as well as in non-GR plants

76

for glyphosate metabolization into AMPA in plants. Glyphosate dissipation in plants could

77

also occur via its excretion in root exudates, which is estimated to be in the order of 8-12

78

% of the applied glyphosate dose 16. Moreover, non-treated plants may absorb glyphosate

79

via the rhizosphere from treated plants17. Whether or not this phenomenon may also occur

80

for AMPA is actually unknown.

81

Since GR plants are not resistant to AMPA, it is suggested that glyphosate degradation into

82

AMPA in plant tissues could be responsible for the phytotoxity observed in GR crops, such

83

as reduced photosynthesis, stomatal conductance and transpiration

84

chlorophyll content and biomass 19, occuring after GBH applications. Similar phytotoxic

85

effects are also reported in non-GR plants following GBH applications, with for example,

86

reduced photosynthesis and chlorophyll content in phytoplankton

87

assimilation in non-GR soybean

88

oxygenase (Rubisco) activity

89

effects on photosynthesis and carbon metabolism can occur in both GR and non-GR

13.

23

Indeed, these GR crops are

Thus, in the case of GR plants expressing GOX, AMPA

22,

9, 13,

14.

All GR crops have an

which suggests another pathway

18

20, 21,

or reduced

reduced CO2

reduced ribulose 1,5-biphosphate carboxylase

or carbohydrates accumulation in leaves

24.

Since these


Page 5 of 38


90

species, the phytotoxicity observed after GBH applications could result either from the

91

effects of glyphosate, AMPA or both molecules simultaneously. In non-GR plants, it was

92

proposed that AMPA could reduce chlorophyll biosynthesis by competitioning with δ-

93

aminolevulinic acid (ALA) synthetase or glycine, while glyphosate seemed to induce

94

chlorophyll degradation through the enhanced formation of reactive oxygen species 25. This

95

suggests that glyphosate and AMPA may interfere with different mecanisms both resulting

96

in a decrease of the chlorophyll content. The main objective of this study is to investigate

97

glyphosate degradation into AMPA in GR soybean, as well as to relate the distribution of

98

both molecules among plant tissues with observed phytotoxic effects upon GBH

99

application. The effects of GBH applications on phytotoxicity indicators such as ΦPSII,

100

H2O2, biomass, chlorophyll content and stomatal conductance, have been evaluated over

101

the first month following a single GBH application along with the determination of

102

glyphosate and AMPA content.

103

2.

Material and Methods

104

2.1

Growth conditions and glyphosate applications

105

GR soybean seeds (5091RR2Y, ÉliteMD) were planted in two gallon (7.57 l) pots filled with

106

the surface soil (0-30 cm) of an eutric Brunisol

107

Canada). The Brunisol had a sandy loam texture with 3% of organic matter, pH of 7.59 ±

108

0.35 and mineral content of 4980 ± 172 µg g-1 for Ca; 124 ± 48 µg g-1 for Al; 118 ± 3 µg

109

g-1 for Fe; 90 ± 4. µg g-1 for Mg; 75 ± 3 µg g-1 for K; 40 ± 2 µg g-1 for Mn; 1.6 ± 0.1 µg g-

110

1

111

34-0-0), but not inoculated with diazotrophs, one week prior to the GBH applications. GBH

112

(Factor 540®, Interprovincial Cooperative Limited, Saskatoon, Canada) was applied

26

collected in Boisbriand (Québec,

for Cu; 1.4 ± 0.2 µg g-1 for Zn. Soybeans were fertilized with ammonium nitrate (N-P-K:



Page 6 of 38

113

manually to V3 stage soybeans (three trifoliate leaves) at doses of glyphosate of 0.7; 1.8;

114

2.3; 3.6 and 4.5 kg ha-1. For each treatment, three plants (pseudoreplicates) were grown

115

in three pots. Soybeans were harvested 2, 7, 14 and 28 DAT and plants from each pot were

116

pooled (n=3). A similar number of plants not treated with glyphosate were used as controls

117

(n=3). Pots were randomly placed (random number generator) in the greenhouse under

118

natural light conditions complemented with sodium vapor lamps to maintain a 12h:12h

119

light/dark cycle and mean photosynthetic active radiation (PAR) level of 850 µmol photons

120

m-2 s-1. Temperature was maintained between 20-25 °C. In vivo measurements were

121

performed on the third fully expanded leaf on at least two plants from each pot at four time

122

points (2; 7; 14 and 28 DAT). All plants were then harvested (within a 3-hour period) and

123

rinsed, and fresh weight (FW) measured. The third fully expanded leaves were frozen in

124

liquid nitrogen and stored at -80 °C while the rest of the plants was frozen at -20 °C for

125

further measurements. Dry weight (DW) measurement were performed later by drying

126

them to a constant weight at 60 °C.

2.2

Photosynthesis and pigment evaluations

127

Chlorophyll fluorescence measurements were performed using a portable chlorophyll

128

fluorometer PAM-2500 (Waltz, Effeltrich, Germany). Samples were dark acclimated for

129

15 minutes then exposed to ten actinic light intensities (11, 30, 47, 71, 113, 168, 257, 380,

130

570, 846 µmol photons m-2 s-1), for 20 seconds intervals, with a saturating pulse at the end

131

of each interval. The effective quantum yield was calculated as:

132 133

ΦPSII = (Fm’ - Ft) / Fm’

(1)


Page 7 of 38


134 135

where Fm’ = maximal fluorescence in light and Ft = fluorescence level before light

136

illumination 27.

137

Chlorophyll content was measured using an atLEAF PLUS chlorophyll meter (atLeaf,

138

Delaware, United States). Data were converted in SPAD units

139

determination (r2) of 0.9999, then in chlorophyll content (μg cm-2) with a r2 of 0.9883.

28

with a coefficient of

140 141

2.3

Gas exchange

142

Stomatal conductance (gs) measurements were performed using a SC-1 leaf porometer

143

(Decagon Devices Inc., Washington, United States) and expressed in mmol m-2 s-1. The

144

accuracy of the leaf porometer was of ± 10% of measurement. Three measurements were

145

performed on each leaf and the mean value was used as the stomatal conductance value.

146

The porometer was calibrated under the greenhouse conditions prior to measurements for

147

each timepoint.

148 149

2.4

Oxidative stress marker

150

Hydrogen peroxide (H2O2) content in soybeans was measured following Velikova et al.

151

(2000). Briefly, 200 mg of frozen leaves were grounded on ice with 2 ml of trichloroacetic

152

acid (0.1%, w/v), then centrifuged for 15 minutes (12 000 x g) at 4 °C. 0.5 ml of the

153

supernatant was added to 0.5 ml of 10 mM phosphate buffer (KH2PO4 + K2HPO4, pH 7)

154

and 1 ml of potassium iodide 1 M. Absorbance was read at 390 nm and values where

155

reported on a standard curve with H2O2 concentrations ranging from 0 to 9 µg ml-1.

156



Page 8 of 38

157

2.5

Glyphosate and AMPA content

158

Glyphosate and AMPA content were determined following the slighly modified method

159

described in Gomes et al. (2016). In short, 50 mg of fresh material (leaves, stems and roots)

160

were weighed and homogenized in a 50 ml Falcon tube containing 10 ml of deionized

161

water, 10 ml of HPLC grade methanol and 5 ml of GC grade dichloromethane for 1 minute

162

(25 000 rpm) using a T18 digital ULTRA-TURRAX® (IKA, North Carolina, USA). Falcon

163

tubes were then centrifuged 20 minutes at 3400 rpm. 50 µl (leaves and stems) or 100 µl

164

(roots) of supernatant were transferred and evaporated to dryness under nitrogen flow.

165

Samples were derivatized by the addition of 1 ml of trifluoroacetic anhydride (TFAA) and

166

500 µl of trifluoroethanol (TFE), prior to heating at 100 oC for one hour. After cooling to

167

room temperature for 15 minutes, samples were evaporated to dryness under nitrogen flow,

168

then resuspended with 1 ml of ethyl acetate, prior to injection (0.5 µl). A Varian CP 3800

169

gas chromatograph coupled with an electron capture detector and equipped with a Rxi®-

170

5Sil MS column (Restek, Pennsylvania, USA) (30 m x 0.25mm x 0.25 μm) was used for

171

glyphosate and AMPA quantification. Injector and detector were held at 280 °C and 300

172

°C, respectively. Hydrogen was used as carrier gas with a column flow of 1.8 ml min-1.

173

The oven temperature program was an initial temperature of 70 °C held for 0.8 min, a 5 °C

174

min-1 increase up to 130 °C held for 5 min, followed by a 60 °C min-1 increase up to 250

175

°C held for 12 min, for a total run time of 31.8 min.

176 177

2.6

Statistical analyzes

178

Analyzes of variance were performed followed by multiple mean comparisons for ΦPSII,

179

H2O2 content, stomatal conductance, chlorophyll content and biomass (FW and DW), using


Page 9 of 38


180

Dunnett’s parametric test (DW, H2O2) or Steel’s non-parametric test when data did not

181

fulfill the conditions of residuals normality (FW, ΦPSII, chlorophyll content, stomatal

182

conductance), for comparison with the control treatment (0 kg ha-1).

183 184

Two-factor analyzes of variance (ANOVA) were also performed (DAT, Plant tissue

185

[leaves, stems, roots], DAT*Plant tissue), followed by Tukey’s post hoc tests with

186

glyphosate and AMPA content in soybean leaves, stems and roots, for all GBH doses. Data

187

were previously logarithmically (ln) converted in order to fulfill the conditions of residuals

188

normality. When the interaction between DAT and plant compartment was non-significant,

189

the two-factor ANOVA was repeated without the interaction variable (DAT*Plant tissue).

190 191

Spearman’s rank correlations were performed with parameters measured in leaves (ΦPSII,

192

H2O2 content, chlorophyll content, stomatal conductance and glyphosate and AMPA

193

content), glyphosate and AMPA content in roots, as well as biomass (DW). The Bonferroni

194

correction was performed and p-value was adjusted to 0.01 (0.05 / 5 variables), the tests

195

being significant when p-value < 0.01. All other statistical analyzes were significant when

196

p-value < 0.05 and all analyzes were performed using the JMP 14 software from SAS.

197 198

3

Results

199

3.1

Physiological indicators

200

The effective quantum yield was significantly reduced 2 DAT, for GBH applications with

201

doses of 1.8 to 4.5 kg glyphosate ha-1 (Figure 1A). Φ PSII was also significantly reduced 7

202

DAT and 14 DAT at the higher dose of 4.5 kg glyphosate ha-1 (Figure 1B, 1C). No 9 ACS Paragon Plus Environment


Page 10 of 38

203

significant difference was observed 28 DAT (Figure 1D). Plant biomass was significantly

204

reduced 14 DAT, when compared to the control treatment, at the dose of 4.5 kg glyphosate

205

ha-1 for fresh weight and dry weight (Figure 1G, 1K). Plants fresh weight increased 7 and

206

28 DAT at doses of 3.6 and 0.7 kg glyphosate ha-1, respectively (Figure 1F, 1H). There

207

was no other significant difference regarding biomass (FW and DW) for all other

208

timepoints (Figure 1E, 1I, 1J, 1L). GBH applications significantly reduced stomatal

209

conductance 2 DAT at the highest dose (4.5 kg glyphosate ha-1) (Figure 2I). This was

210

followed by a significant increase of the stomatal conductance 14 DAT, occurring at doses

211

of 0.7, 1.8 and 4.5 kg glyphosate ha-1 (Figure 2K), as well as an increase 28 DAT at the

212

two highest doses of 3.6 and 4.5 kg glyphosate ha-1 (Figure 2L). There was no significant

213

differences 7 DAT (Figure 2J). H2O2 content was significantly increased 2 DAT for the

214

two highest doses (3.6 and 4.5 kg ha-1) (Figure 2E), but there was no signifiant differences

215

further in the experiment (7, 14 and 28 DAT) (Figure 2F, 2G, 2H). Finally, regardless of

216

the time of exposure and glyphosate doses, chlorophyll concentrations were not affected

217

by treatments (Figure 2A, 2B, 2C, 2D).

218 219

3.2

Glyphosate and AMPA content

220

Glyphosate and AMPA content in plant tissues (leaves, stems, roots)

221

Differences regarding glyphosate content in plant tissues was the most significant variable

222

of the two-way ANOVA model (Table 1). Glyphosate content in GR soybean was

223

significantly higher in leaves than in stems and roots for all applied doses (0.7 to 4.5 kg ha-

224

1)

225

measured in roots was significantly lower at 0.7, 3.6 and 4.5 kg glyphosate ha-1 doses

(Figure 3A, 3B, 3C, 3D, 3E). When compared to glyphosate content in stems, the amount


Page 11 of 38


226

(Figure 3A, 3D, 3E). A significant difference was observed regarding AMPA content

227

measured in the different plant tissues (leaves, stems and roots) but only at the two highest

228

3.6 and 4.5 kg glyphosate ha-1 doses (Table 1), the content in roots being significantly lower

229

than in leaves and stems (Figure 3I, 3J). For the three lowest GBH doses (0.7, 1.8 and 2.3

230

kg glyphosate ha-1), the AMPA content measured in leaves, stems and roots was not

231

significantly different (Figure 3F, 3G, 3H).

232 233

Glyphosate and AMPA content over time (DAT)

234

For the lowest GBH application (0.7 kg glyphosate ha-1), the glyphosate content measured

235

in plants (leaves + stems + roots) was significantly lower 14 and 28 DAT in comparison to

236

7 DAT (Figure 3A). With increasing glyphosate doses (2.3, 3.6 and 4.5 kg glyphosate ha-

237

1),

238

following time after application in comparison to 2 DAT (Figure 3C, 3D, 3E). AMPA

239

content was significantly higher 28 DAT when comparing to 2 DAT (Figure 3G-3J) at

240

doses ranging from 1.8 to 4.5 kg glyphosate ha-1.

the opposite effect was observed, glyphosate content being significantly higher

241 242

Glyphosate and AMPA content in plant tissues over time (DAT*Plant tissue)

243

The interaction of glyphosate content in plant tissues over time was significant only at the

244

lowest and highest doses (0.7 and 4.5 kg glyphosate ha-1) (Table 1). At the lowest dose (0.7

245

kg glyphosate ha-1), glyphosate content in leaves decreased over time, the amount

246

measured 28 DAT being significantly lower than 2 DAT (Figure 3A). Moreover,

247

glyphosate content in stems, for all timepoints (2, 7, 14 and 28 DAT), was not different,

248

while glyphosate content in roots was lower 2 DAT when comparing to 7 DAT (Figure



Page 12 of 38

249

3A). Finally, the glyphosate content measured in roots 2 DAT was lower than in leaves at

250

all time points (2-28 DAT) and in stems 2 and 7 DAT (Figure 3A). At the highest dose (4.5

251

kg glyphosate ha-1), there was only one significant difference regarding glyphosate content

252

in leaves, which was significantly higher 28 DAT, when compared to 14 DAT (Figure 3E)

253

In stems, the amount of glyphosate was significantly higher 28 DAT when comparing to 2

254

DAT, while there was no differences regarding roots (Figure 3E). Similarly to the lowest

255

GBH application, the amount of glyphosate measured in stems and roots was lower 2 DAT,

256

when comparing to the amount in leaves (Figure 3E). For AMPA, the interaction of AMPA

257

content in plant tissues over time was significant for all GBH applications (Table 1). From

258

1.8 to 4.5 kg glyphosate ha-1, the AMPA content measured in roots 28 DAT was almost

259

always significantly higher than the amount measured at 2 DAT (Figure 3G-3J), which was

260

not the case for the lowest dose of 0.7 kg glyphosate ha-1 (Figure 3F). The AMPA content

261

in leaves and stems were not really different over time for the different GBH applications.

262

Similarly to glyphosate, AMPA content in roots 2 DAT was significantly lower from

263

AMPA content in leaves and stems for the same timepoint, but only for the two highest

264

doses of 3.6 and 4.5 kg glyphosate ha-1 (Figure 3I-3J).

265 266

3.3

Correlations between glyphosate and AMPA content and physiological

267

indicators of GBH exposure

268

Glyphosate content in leaves was significantly correlated with ΦPSII (-), while AMPA

269

content in leaves was also correlated with ΦPSII (-), as well as with chlorophyll content (-)

270

(Table 2). Glyphosate content in roots was solely correlated with H2O2 content (+)

271

measured in leaves. On the other hand, AMPA content in roots was significantly correlated 12 ACS Paragon Plus Environment

Page 13 of 38


272

with all indicators, except for ΦPSII (Table 2). Solely DW was used as the biomass proxy

273

in the correlation matrix, since FW and DW values are not independent.

274 275

4.

Discussion and conclusions

276

A reduction of ΦPSII was observed 2 DAT, 7 and 14 DAT. The lowest dose being

277

significantly different from the control treatment or lowest observed concentration effect

278

(LOEC), was compared for each DAT, giving 0.7; 4.5 and 4.5 kg glyphosate ha-1

279

respectively at 2, 7 and 14 DAT (Figure 1A, 1B, 1C). There was no LOEC value at 28

280

DAT suggesting that soybeans were able to recover from GBH applications over time.

281

Reduced ΦPSII in GR soybeans exposed to GBH was previously reported 30, as well as the

282

potential of recovery 30, 31. It was proposed that these effects on photosynthesis were linked

283

to AMPA, produced from the degradation of glyphosate in plants. Indeed, following the

284

application of AMPA alone, its content measured in plants was correleted to a reduction

285

of the chlorophyll content of GR soybeans and the associated decreased FW 13. In our case,

286

ΦPSII reduction was correlated with both AMPA and glyphosate content in leaves (Table

287

2).

288 289

A reduction in photosynthesis efficiency can lead to the production of reactive oxygen

290

species (ROS) and H2O2 accumulation which were already observed in non-GR plant

291

species such as phytoplankton 21, willow shrubs 25 and duckweed 32. In GR soybeans, H2O2

292

accumulation was observed in germinating seeds exposed to GBH 33. In our study, it seems

293

that ΦPSII reduction could have resulted in an increase of H2O2 content in leaves. Indeed,

294

H2O2 content was significantly higher 2 DAT in comparison with the control treatment for 13 ACS Paragon Plus Environment


Page 14 of 38

295

GBH applications at the highest 3.6 and 4.5 kg glyphosate ha-1 doses (Figure 2E). However,

296

as for ΦPSII, it seems that plants could recover from early stress following GBH

297

application, since there were no significant differences for H2O2 content later in the

298

experiment (7, 14 and 28 DAT) (Figure 2F-2H). Moreover, it seems that ΦPSII reduction

299

and the H2O2 increase were unsufficient to affect the chlorophyll content (Figure 2A-2D).

300

An effective antioxidant response could have prevented damages to chlorophyll and could

301

also explain the recovery observed in this experiment, regarding ΦPSII and H2O2 content

302

(Figure 1D and Figure 2F-2H). For example, increased superoxide dismutase, catalase and

303

ascorbate peroxidase activities were observed following GBH applications in a

304

phytoplankton community 21. In GR and non-GR soybeans, there was a slight increase of

305

oxidative stress and the antioxidant response was modulated by GBH applications.

306

However, lipid peroxidation did not occur, and it was suggested that the pool of amino

307

acids implicated in stress response (proline and asparagine), unaffected in GR soybeans

308

leaves and roots exposed to GBH, could have prevent damages 34. While the chlorophyll

309

content was not significantly different from the control treatment at all glyphosate doses

310

and time points, there was still a significant negative correlation between AMPA content

311

measured in leaves and roots, and the chlorophyll content. The absence of a similar

312

correlation between glyphosate and chlorophyll content (Table 2) seems to support the

313

hypothesis that the effects of GBH application on GR soybeans chlorophyll content are

314

mostly due to AMPA effects.

315 316

A significant biomass reduction was observed 14 DAT for both FW and DW. Bernal et al.

317

(2012) related a similar decrease in FW of GR maize 14 DAT to the effects of AMPA.


Page 15 of 38


318

Indeed, they observed that the AMPA to glyphosate ratio was higher over the first 14 DAT,

319

indicating that glyphosate degradation into AMPA could occur more rapidly during the

320

first two weeks after GBH applications, due to an increased GOX activity

321

soybeans, previous studies attempted to link the biomass reduction with AMPA foliar

322

applications. However, contradictory results were reported, i.e. decreased FW when

323

exposed to AMPA 14 DAT

324

present study, neither glyphosate content measured in leaves or roots was correlated with

325

biomass (Table 2). A positive correlation was solely observed between AMPA content

326

measured in roots and DW. Otherwise, biomass did not appear negatively correlated with

327

glyphosate nor AMPA content measured in plants. On the other hand, DW was highly

328

correlated with all other parameters i.e. stomatal conductance (ρ= -0.7644; p-value=

329

Glyphosate and aminomethylphosphonic acid content in glyphosate

Recommend Documents