Foliar Desiccators Glyphosate, Carfentrazone, and Paraquat Affect the

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The foliar desiccators glyphosate, carfentrazone and paraquat affect the technological and chemical properties of cowpea grains Igor da Silva Lindemann, Gustavo Heinrich Lang, Jessica Fernanda Hoffmann, Cesar Valmor Rombaldi, Mauricio de Oliveira, Moacir Cardoso Elias, and Nathan Levien Vanier J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01912 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Journal of Agricultural and Food Chemistry

1

The foliar desiccators glyphosate, carfentrazone and paraquat affect the

2

technological and chemical properties of cowpea grains

3 4

Igor da Silva Lindemann1, Gustavo Heinrich Lang1, Jessica Fernanda Hoffmann1, Cesar

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Valmor Rombaldi1, Maurício de Oliveira1, Moacir Cardoso Elias1, Nathan Levien

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Vanier1*

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1

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96010-900, Pelotas, RS, Brazil

Department of Agroindustrial Science and Technology, Federal University of Pelotas,

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Igor da Silva Lindemann ([email protected])

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Gustavo Heinrich Lang ([email protected])

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Jessica Fernanda Hoffmann ([email protected])

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Cesar Valmor Rombaldi ([email protected])

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Maurício de Oliveira ([email protected])

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Moacir Cardoso Elias ([email protected])

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* Corresponding author: Nathan Levien Vanier ([email protected])

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Tel/Fax: +005553981175570

ACS Paragon Plus Environment


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Abstract

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The effects of the use of glyphosate (GLY), glyphosate plus carfentrazone (GLY/CAR),

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and paraquat (PAR) as plant desiccators on the technological and chemical properties of

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cowpea grains were investigated. All studied desiccants provided lower cooking time to

24

freshly harvested cowpea. However, the coat color of PAR- and GLY/CAR-treated

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cowpea was reddish than control treatment. Principal component analysis (PCA) from

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liquid chromatography-mass spectrometry (LC-MS) data sets showed a clear distinction

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among cowpea from the different treatments. Catechin-3-glucoside and epicatechin

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significantly contributed for discriminating GLY-treated cowpea, while citric acid was

29

responsible for discriminating GLY/CAR-treated cowpea. Quercetin derivative and

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gluconic acid were responsible for discriminating control treatment. Residual

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glyphosate and paraquat content was higher than the maximum limits allowed by Codex

32

Alimentarius and European Union Commission. Improvements in the technological and

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chemical properties of cowpea may not be overlapped by the risks that those desiccants

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exhibit when exceeding the maximum limits of tolerance in food.

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Keywords: Vigna unguiculata, coat color, cooking time, phenolics, storage of cowpea.


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1. Introduction

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Cowpea (Vigna unguiculata L.) is considered a good source of carbohydrates,

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proteins, fibers, vitamins, as well as iron and zinc.1 Moreover, cowpea exhibits phenolic

39

compounds

40

antihypertensive properties, which are distributed mainly in the grain coat, but are also

41

present in cotyledon. Those compounds include flavonols, phenolic acids, flavan-3-ols

42

and anthocyanins.2

with

putative

antioxidant,

antimutagenic,

anti-inflammatory

and

43

Once the plants from cowpea are resistant to dry- and warm-weather conditions,

44

they are mainly cultivated in semi-arid regions all over the world.3 In Brazil, cowpea

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was firstly cultivated in small farms located in the North and Northeast of the country;4

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however, nowadays, cowpea has been cultivated in huge farms located in the Center-

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East of the country, where the manual harvesting gave space to the mechanical

48

harvesting, since the harvested area in each farm may vary from few hundreds to more

49

than 3,000 hectares.

50

Cowpea naturally exhibit indeterminate growth type of plant. This means that

51

the plants may retain green leaves during grain ripening, which impairs or delays

52

mechanical harvesting.5 Thus, desiccation treatment is required in order to allow the

53

mechanical harvesting of cowpea. The strategy is largely employed in major crops,

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especially in species and cultivars expressing stay-green phenotype.6 Between the most

55

commonly herbicides used by Brazilian farmers and in other countries are glyphosate,

56

protox inhibitors and paraquat, as well as the combination of glyphosate and protox

57

inhibitors in the same spraying treatments. It is important to note that the cited

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herbicides have the post-harvest interval varying from 2 days (glyphosate) to 7 days

59

(paraquat). The farmers utilize these treatments in order to mechanically harvest the

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crop with lower losses, lower fuel consumption and better production yield.



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Glyphosate acts by inhibition of the 5-enolpiruvil-shikimato-3-fosfato synthase

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(EPSPS) enzyme, which participate in the shikimate pathway. Once this molecule is

63

applied as herbicide, the synthesis of aromatic amino acids is stopped, which, in turn,

64

impair the synthesis of proteins and some specialized metabolites.7,8 Intermediate toxic

65

compounds could be also synthesized when glyphosate is metabolized, but it is still not

66

fully understood.9

67

In

order

to

improve

glyphosate

efficiency,

some

herbicides

from

68

protoporphyrinogen oxidase (Protox) group are used in combination. Protox inhibitors

69

are powerful inhibitors of the Protox enzyme, responsible for the chlorophyll and heme

70

biosynthesis. In sum, this enzyme catalyzes the oxidation of protoporphyrinogen IX to

71

protoporphyrin IX, and it will end in the formation of singlet and triplet oxygen radicals

72

followed by cell and cell organelles disintegration.10, 11

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Paraquat is also used to desiccate crops.6 It acts inhibiting photosystem I.

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Paraquat produces radical oxygen species (ROS), such as hydrogen peroxide (H2O2)

75

and free hydroxyl (OH-).12 ROS rapidly interact with lipids, promoting irreversible lipid

76

peroxidation with subsequent cell organelles, proteins and nucleic acids destruction.13

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The effects of herbicides in the desiccation of crops are well established but few

78

literature reports deal with the impact of desiccation on grain quality. According to

79

Unver et al.14 plants may exhibit different responses to biotic and abiotic stresses, which

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severally impacts the plant bioactive compounds’ content. Komives and Schröder15

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published a review article regarding glyphosate mechanism of action and its effects on

82

secondary metabolites of plants and stated that more studies are necessary to elucidate

83

the synthesis of specialized metabolites as a function of desiccation treatments.

84

Once the desiccation is necessary to improve the mechanical harvesting, we

85

evaluate the effects of the use of glyphosate, glyphosate plus carfentrazone, and


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paraquat as plant desiccators on the technological and chemical properties of cowpea

87

grains. The residual herbicide in the grains was also assessed in the present study.

88 89

2. Materials and Methods

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2.1. Materials and sample preparation

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Cowpea from “Bico de ouro” variety was produced in the 2015/2016 growing

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season in the countryside of Primavera do Leste, State of Mato Grosso, Brazil. Four

93

field plots of 25 hectares each were used. In each 25-hectare plot, three areas of 10 m x

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20 m were considered as replicates for sample analyses. Seedling and agronomical

95

practices of insects and fungal control were exactly the same in all the four field plots.

96

Moreover, soil fertility was similar between the field plots.

97

Three treatments were tested: 1) treatment A (GLY) consisted of 1824 g a.i. of

98

glyphosate (Glizmax® Prime, Monsanto do Brasil, São Paulo, Brazil) per hectare; 2)

99

treatment B (GLY/CAR) consisted of 1824 g a.i. of glyphosate + 0.032 g a.i. of

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carfentrazone (Aurora®, FMC Corporation, Philadelphia, USA) per hectare; and 3)

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treatment C (PAR) consisted of 552 g a.i. of paraquat (Tocha®, HuBei XianLong

102

Chemical Industry Co., China) per hectare. One field crop (25 hectare) was kept without

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herbicide application before harvesting, being used as control. Figure 1 presents images

104

from the three different field crops taken after 24 h of desiccation and control treatment.

105

GLY, GLY/CAR and PAR were applied 70 days after seeding by using a self-

106

propelled sprayer. GLY, GLY/CAR and PAR were mechanically harvested after 4, 3

107

and 2 days of herbicide treatment, respectively, when plants exhibited total leaf

108

abscission and the grains exhibited around 13% moisture content. Untreated cowpea

109

plants from control treatment were manually harvested after 70 days and placed under



110

the ground. Grains were separated from these plants and submitted to drying in the field

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until 13% moisture content be achieved.

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Cowpea grains were cleaned, packaged into raffia bags and immediately

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transported to Laboratório de Pós-Colheita, Industrialização e Qualidade de Grãos

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(LABGRÃOS) from Universidade Federal de Pelotas (UFPEL), where the storage and

115

analyses were carried out. The evaluations of coat color, cooking time, total phenolics

116

and proanthocyanidins content, and individual phenolics were carried out at the harvest.

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Coat color and cooking time was also evaluated in cowpea stored after 8 months at 25

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°C under dark.

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2.2. Coat color and cooking time of cowpea

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Color attributes of bean samples were determined by using a Minolta

122

colorimeter (CR-410, Konica Minolta, Japan). The colorimetric parameter a* was

123

obtained and used for sample comparison. The a* value expresses red (+) or green (-),

124

being set with a white background plate which was directly obtained from the

125

apparatus.

126

In order to determine the cooking time, grains were soaked for 14 hours in

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distilled water and then submitted to the cooking procedure at the Mattson Bean Cooker

128

(MBC), as described by Wang and Daun 16. Cooking time was defined as the time, in

129

minutes, that 50% of the cowpea grains were fully cooked, which was easily perceived

130

by plungers dropping.

131

The changes in a* value (∆a*) and cooking time (∆cooking time) of cowpea

132

during 8 months of storage were also determined. Cowpea was stored at 25 ºC in

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polyethylene bags of 0.16 mm of film thickness and capacity for 1.5 kg of cowpea.

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Grains were put in a new polyethylene package every 30 days in order to avoid the


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absence of oxygen. ∆a* and ∆cooking time were calculated by subtracting the values

136

obtained at the 8th storage month from those at the beginning of storage.

137 138

2.3. Total phenolics and proanthocyanidins

139

2.3.1. Free phenolics extraction

140

The extraction of free phenolics was performed according to the method

141

described by Qiu, Liu, and Beta,17 with some modifications. The flour from whole

142

grains (2 g) was extracted twice with Acetone/Water solvent (70:30 v/v). For each

143

extraction, the mixture was kept on a mechanical shaker (Certomat Biotech

144

International, Germany) for 1 h at 150 rpm at room temperature. After centrifuging it

145

(5430-R, Eppendorf AG, Germany) at 1430 x g for 5 min, the supernatants obtained

146

from each extraction were combined and concentrated until dryness by using a rotary

147

evaporator at 35 °C. The dried extracts were redissolved in 20 mL of Acetone/Water

148

solvent (70:30 v/v) and used as crude extracts for total quantification of the free

149

phenolics.

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2.3.2. Bound phenolics extraction

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The bound phenolics extraction was performed according to the optimized

153

method described by Alves et al.18 with minor modifications. The residue obtained after

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two steps of the extraction of free phenolics using Acetone/Water (70:30 v/v) was

155

washed with distilled water, filtrated, and left to dry in the hood, under dark. Initially,

156

5.0 mL of distilled water was added to the residue from the extraction of free phenolics.

157

Afterwards, the residue was hydrolyzed with 5 µL of α-amylase (50 units/µL) from

158

Bacillus licheniformis (Sigma–Aldrich, United States) at 37 °C during 15 min.

159

Enzymatic reaction was then stopped by heating the material to 90 °C during 5 min. The



160

material was subject to alkaline hydrolysis with 40 mL of 4 M NaOH in a shaker for 4

161

h. After digestion, the solution was adjusted to a pH of 1.5–2.0 with 6 M HCl and then

162

extracted three times with 70 mL aliquots of ethyl acetate. The combined ethyl acetate

163

fractions were evaporated until dry and reconstituted in 5 mL of Acetone/Water solvent

164

(70:30 v/v), constituting the bound phenolics extract.

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2.3.3. Phenolics quantification

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Free and bound phenolics content was determined by using the Folin–Ciocalteu

168

method, with some modifications.19 Briefly, 100 µL of the properly diluted extracts

169

were mixed with 400 µL of distilled water, 0.25 mL of 1 N Folin–Ciocalteu reagent,

170

and then 1.25 mL of 7.5 g/100 mL sodium carbonate were added. After reacting for 120

171

min, the absorbance of the mixture was measured at 725 nm (UV 17000

172

spectrophotometer, Shimadzu, Japan). The quantification was performed using a

173

calibration curve made with gallic acid dissolved in Acetone/Water solvent (70:30 v/v).

174

Results were expressed as mg of gallic acid equivalents (GAE) per 100 g of cowpea on

175

a dry weight basis.

176 177

2.4. Proanthocyanidins

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The proanthocyanidin content was determined based on the method described by

179

Diaz et al.20 0.5 mL of the extract was pipetted (same extract used for free phenolics

180

quantitation) in glass tubes. Then, 3.0 mL of acidified butanol (butanol:HCl, 950 mL:50

181

mL) and 100 µL of ferric reagent were added. The mixture was boiled (97-100 °C) for

182

30 minutes. The absorbance was measured at 550 nm and the results were expressed in

183

mg of leucocyanidins per 100 g of dry sample.

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2.5. LC-ESI-qTOF-MS analysis

186

The same extract used for total phenolics determination was used for LC-ESI-

187

qToF-MS analysis. Samples were filtered through a 0.45 µm nylon membrane filter

188

(Merck Millipore Corporation, Darmstadt, Hesse, Germany). The LC-ESI-qTof-MS

189

analysis was performed on a Prominence UFLC system (Shimadzu, Japan) coupled to a

190

quadrupole time-of-flight mass spectrometer (Impact HD, Bruker Daltonics, Bremen,

191

Germany). Metabolites were separated using a Bidentate C18 column (100 × 2.1 mm,

192

MicroSolv Technology Corp., Leland, NC, USA). Mobile phases were 0.1% aqueous

193

formic acid (pH 2.8; solvent A) and acetonitrile (solvent B). The gradient program was

194

set as follows: started at 5% B, increased linearly to 90% B at 15 min, and maintained

195

for 3 min at 90% B; returned to 5% B in 2 min and maintained at 5% B for an additional

196

6 min at a flow rate of 0.2 mL min−1. The injection volume was 10 µL. All samples

197

were injected in duplicate.

198

Parameters for MS analysis were set using negative ionization mode with spectra

199

acquired over a mass range from m/z 50 to 1200. The parameters were: capillary

200

voltage, +4.0 kV; drying gas temperature, 180 °C; drying gas flow, 8.0 L/min;

201

nebulizing gas pressure, 2 bar; collision RF, 150 Vpp; transfer time 70 µs, and pre-pulse

202

storage, 5 µs. Moreover, automatic MS/MS experiments were performed adjusting the

203

collision energy values as follows: m/z 100, 15 eV; m/z 500, 35 eV; m/z 1000, 50 eV,

204

and using nitrogen as collision gas.

205

The MS data were analyzed using Data Analysis 4.0 software (Bruker Daltonics,

206

Bremen, Germany). Data mining and alignment (m/z, retention time and intensity) of

207

LC-MS/MS records was performed on ProfileAnalysisTM software (version 2.0, Bruker

208

Daltonics, Bermen, Germany) and submit to principal component analysis (item 2.7).



209

Tentative metabolite identification of significant peaks on PCA was performed

210

by matching the accurate m/z values and MSn fragmentation patterns with data from

211

databases (METLIN, KEGG compounds, PubChem, Mass bank, Maven, FooDB, and

212

ReSpect) and reference literature with a mass accuracy window of 5 ppm. The identities

213

of citric acid and epicatechin were confirmed with external standards (Sigma-Aldrich).

214 215

2.6. Glyphosate, carfentrazone and paraquat determination

216

Residual glyphosate and paraquat content were determined in accordance to the

217

method described by the European Union Reference Laboratory for Residues of

218

Pesticides.21 Residual carfentrazone content was determined using the AOAC 2007.01

219

method of AOAC International.22 Results were expressed as mg per kilograms of dry

220

weight.

221 222

2.7. Statistical analysis

223

Analytical determinations for the samples were performed in triplicate, and

224

standard deviations were reported. A comparison of the means was ascertained with

225

Tukey’s test to a 5% level of significance using an analysis of the variance (ANOVA).

226

The LC–ESI-qTOF-MS data (aligned peak intensities, mass and retention times)

227

of four treatments was analyzed by principal component analysis (PCA) to identify

228

potential discriminate metabolites. PCA was performed using MetaboAnalyst 3.0.

229 230

3. Results and discussion

231

3.1. Color and cooking time are affected by desiccation

232

Grains harvested from plants treated with PAR exhibited the highest a* value

233

(Fig. 2). It is well-known that PAR treatment generates ROS, which improves the


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activity of polyphenol oxidase (PPO) enzyme in plants.23,24 The activation of PPO may

235

increase quinone production, darkening the seed coat color.25 Cowpea harvested from

236

GLY-treated plants exhibited higher a* value than those from GLY/CAR and control

237

treatments. Although GLY acts inhibiting shikimate pathway being apparently not so

238

stressful to the plants compared to herbicides that directly generate high amount of

239

ROS, GLY may have up-regulated plant phenolics synthesis as well as their

240

polymerization, giving a reddish color to cowpea seed coat. This hypothesis remains to

241

be tested.

242

Interestingly, the highest cooking time of 15.9 minutes was determined for

243

cowpea untreated with desiccants (Fig. 2B). Lignification of the cell wall and

244

polymerization of phenolic constituents mainly from the seed coat have been described

245

as the main phenomena contributing for the increase in the cooking time of stored

246

beans.26 The softening of cowpea during cooking depends on starch gelatinization,

247

which, in turn, depends on the amount and ability of water intake through the cotyledon.

248

The lower cooking time of cowpea subjected to desiccation treatments with GLY,

249

GLY/CAR, and PAR may be a result of the destruction of membranes from organelles

250

and cells, that increased the ability of water intake through cotyledon and heat transfer

251

during cooking. This has likely favored starch gelatinization and cotyledon softening

252

when analyzing freshly harvested cowpea. Another factor that may have contributed for

253

the highest cooking time of untreated cowpea is the longer time that grains from

254

untreated plants (control treatment) took to achieve the optimum moisture content prior

255

threshing.

256 257

3.2. Total phenolics and proanthocyanidins content change as a function of desiccation

258

treatment



259

Free and bound phenolics were firstly quantitated by Folin-Ciocalteu method.

260

According to Zhang et al.27 phytochemicals, such as phenolics, mainly exist as

261

glycosides linked to various sugar moieties or as other complexes linked to organic

262

acids, amines, lipids, carbohydrates, and other phenols. Paiva et al.28 stated that

263

phenolic compounds are commonly present in the bound form and are typically

264

components of complex structures, such as lignins and hydrolysable tannins, and linked

265

to the cell wall structural components, such as cellulose, lignin, and proteins through

266

ester bonds. In the present study, free phenolics corresponded to around 88% of total

267

phenolics in cowpea.

268

The highest free and bound phenolics content was determined in grains

269

harvested from GLY-treated plants (Figs. 3A and 3B). GLY treatment have also

270

provided the greatest proanthocyanidins content in cowpea, as presented in Fig. 3C. To

271

our knowledge, GLY treatment up-regulated the activity of enzymes linked to phenolics

272

synthesis as a natural mechanism of specialized metabolites production. GLY is not so

273

abrupt in promoting plant senescence compared to GLY/CAR and PAR, and while the

274

plants took 1- or 2-days more to be ready for mechanical harvesting they continue to

275

produce those specialized metabolites.

276

Proanthocyanidins results (Fig. 3C) followed a similar trend to free and bound

277

phenolics results (Fig. 3A and 3B). Proanthocyanidins are also known as condensed

278

tannins and are considered polymers of flavan-3-ol units, such as catechin and

279

epicatechin, which are described as end-products of flavonoids biosynthesis.29

280

Proanthocyanidins exhibits 15- to 30-times greater antioxidant capacity than phenolic

281

acids.30 The low proanthocyanidins and free and bound phenolics of grains from control

282

treatment suggests that there was little collaboration of phenolics polymerization to the

283

initial cooking time of freshly harvested cowpea, leading to the conclusion that the


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integrity of the cotyledon cells was the main contributor to the cooking time differences

285

observed between grains from control and herbicide-treated plants.

286 287

3.3. Principal component analysis

288

Principal component analysis was applied to verify the influence of herbicide

289

application on secondary metabolism of cowpea. Figure 4A and 4B shows the PCA

290

score and loading plot for components 1 and 2. These components explained 66% of

291

total variance.

292

The identities of metabolites responsible for separations were presented in Table

293

1. Catechin-3-glucoside ([M-H]- m/z 451.1258) and epicatechin ([M-H]- m/z 289.0723)

294

significantly contributed for discrimination of GLY-treated cowpea from the other

295

treatments (Fig. 4B, Table 1). Extracted ion chromatograms of catechin-3-glucoside and

296

epicatechin are presented in Fig. 5A. Both catechin and epicatechin are described as

297

end-route compounds from the flavonoid biosynthesis pathway, being found as

298

monomers, dimmers, trimmers, tetramers, as well as in more complex polymeric forms.

299

According to Zabalza et al.31, glyphosate treatment induces a non-regulated carbon

300

entrance through 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS),

301

which is the first enzyme of the shikimate pathway, and accumulation of metabolites

302

upstream EPSPS. In sum, the findings from Zabalza et al.31 clarified that phenylalanine

303

ammonia-lyase (PAL) activity is up-regulated due to glyphosate treatment,

304

accompanied by the synthesis of flavonoid precursor and flavonoids.

305

Citric acid ([M-H]- m/z 191.0205) was responsible for discrimination of

306

GLY/CAR-treated cowpea (Fig. 5B). A previous work conducted by Wells and

307

Appleby32 provided evidence that another protox inhibitor molecule called “Lactofen”

308

associated to glyphosate potentiated the glyphosate transport throw the symplast, as



309

well as glyphosate penetration in plant cells. Thus, it is hypothesized that similar

310

behavior occurred in the present work, with carfentrazone favoring glyphosate

311

assessment in the grain cells, which, in turn, favored citric acid accumulation. Zhu et

312

al.33 observed an increase in the expression of five cDNAs codifying for enolases, when

313

soybean susceptible to glyphosate was treated with glyphosate. Enolase is the ninth

314

enzyme from the glycolysis pathway, acting in the conversion of 2-phosphoglycolate to

315

phosphoenolpyruvate. The authors concluded that greater amounts of intermediates of

316

the tricarboxylic acid cycle are accumulated in such situation. The statements of Zhu et

317

al.33 support our findings.

318

Quercetin derivative ([M-H]- m/z 625.1426) and gluconic acid ([M-H]- m/z

319

195.0515) were responsible for discrimination of control treatment (Figs. 5C and 5D;

320

Table 1). According to the studies performed by Ojwang et al.34, quercetin is the main

321

flavonol found in white, black, red, and brown cowpea. De Abreu et al.35 reported that

322

quercetin is a potential antioxidant of ROS. Although levels of ROS have not been

323

determined in the present study, the applied desiccants are known to increase ROS

324

levels. Thus, it is assumed that greater quercetin derivatives content was determined in

325

samples from control treatment because less ROS were formed at the absence of

326

desiccant treatment. Baxter et al.36 reported increases in gluconic acid content in

327

Arabidopsis cells when subjected to oxidative stress, justifying this behavior by the re-

328

routing of glycolytic carbon flow to the oxidative pentose phosphate pathway. The

329

authors also reported a decrease in the activity of the enzymes related to tricarboxylic

330

acid cycle. This fact supports the low citric acid content as well as the higher gluconic

331

acid content determined in samples from control treatment.

332

Intermediate gluconic acid level was determined in PAR treatment (Fig. 5C),

333

that may be a result of the re-routing of carbon flow to the oxidative pentose phosphate


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pathway associated with ROS generation and an abrupt plant desiccation. In this case,

335

gluconic acid accumulation was stimulated but for a fewer time than control treatment.

336 337

3.4. Residual glyphosate, carfentrazone and paraquat content

338

Flour from uncooked GLY-, GLY/CAR- and PAR-treated cowpea was prepared

339

from grains stored during 30 months at 15 °C. Residual glyphosate content of 13 and 14

340

mg per kilogram of dry weight were determined in GLY- and GLY/CAR-treated

341

cowpea, respectively. Carfentrazone was below the limit of quantification of 0.01 mg

342

per kilogram of dry weight in cowpea from GLY/CAR-treated cowpea. Glyphosate

343

values from both treatments are higher than the maximum limit of 2 and 0.1 mg per

344

kilogram stablished by Codex Alimentarius37 and European Union38. Paraquat content

345

was 0.84 mg per kilogram of dry weight in PAR-treated cowpea. Paraquat value

346

determined in the present study is higher than the maximum limits of 0.5 and 0.02 mg

347

per kilogram of grains allowed by Codex Alimentarius37 and European Union38,

348

respectively. Effects of glyphosate exposure on human’s health have been studied for

349

years, being a controversial issue in research. Scientific evidences suggest that

350

incidence of Parkinson’s disease is associated to exposure to toxicants such as

351

paraquat.39,40

352 353

3.5. Susceptibility to changes in coat color and cooking time during storage

354

Once cowpea is subjected to desiccation, different metabolic responses occur

355

depending on the herbicide used, as seen previously in sections 3.1, 3.2, and 3.3. One

356

problem that arises is the susceptibility of the grains obtained from the different

357

desiccation treatments to the postharvest color and cooking time changes. Thus, cowpea

358

from the GLY, GLY/CAR, PAR and control treatments were stored at 25 °C during 8



359

months, in order to evaluate differences in coat color (∆a*) and cooking time (∆cooking

360

time). Results are presented in Figs. 6 and 7.

361

From this issue, we observed that grains from control treatment exhibited an

362

increase in coat color and cooking time during storage (Figs. 6 and 7), which are

363

phenomena involved in the appearance of hard to cook defect in stored beans.25 GLY

364

and GLY/CAR treatment increased coat color to a similar level than control treatment.

365

Interestingly, PAR treatment provided the lowest ∆a*, which may be a reflect of a

366

lower grain metabolism. Cowpea from PAR treatment exhibited just 58% of

367

germination while cowpea from other treatments exhibited between 86 and 94% of

368

germination (data not shown). In Fig. 7, some extremely darken cowpea are perceived

369

mixed to the samples at the eighth month of storage, mainly in PAR and control

370

treatments. This may be a result of the variations in metabolic responses of the grains as

371

a function of maturity; when the desiccant is applied, some grains are fully mature while

372

others are still green. This issue needs to be addressed in future studies.

373

The GLY and PAR treatments provided the lowest ∆cooking time (Fig. 6B). The

374

lower grain metabolism supported by the germination levels in PAR-treated cowpea has

375

probably maintained the cotyledon cells with a mild lignification degree. Interestingly,

376

GLY-treated cowpea was the less susceptible to increases in cooking time during 8

377

months of storage at 25 °C. This fact supports that GLY treatment impacts mainly the

378

metabolism of grain coat flavonoids than in lignification process of cell walls from

379

cowpea cotyledon.

380

To the best of our knowledge, this is the first time that the effects of the most

381

common desiccants used in Brazil on the quality of cowpea from “Bico de Ouro”

382

variety were determined. All studied desiccants provided lower cooking time compared

383

to freshly harvested cowpea. However, the coat color of PAR- and GLY/PAR-treated


Page 16 of 33

Page 17 of 33


384

cowpea was reddish than control treatment. This is not desired by farmers, traders,

385

industry nor consumers. During storage, GLY- and PAR-treated cowpea exhibited

386

better resistance to changes in coat color and cooking time than control and GLY/CAR

387

treatments. It is better because cowpea without reddish color is marketed at higher

388

values. Any possibility of improvements in the technological and chemical properties of

389

cowpea may be off-set by the risks of those desiccants exhibit when exceeding the

390

maximum limits of tolerance in food. This is why Federal inspection may be urgently

391

intensified.

392

Transcriptome, proteome and metabolome studies may help to deeply

393

understand the metabolic responses of cowpea to the different commercial desiccants.

394

Moreover, studies dealing with other types of herbicides, desiccant doses, period of

395

desiccant application and conditions will help farmers and bean industries to find

396

alternatives for a safe cowpea harvesting in large areas.

397 398

Abbreviations used

399

cDNA – complementary deoxyribonucleic acid

400

EPSPS - 5-enolpiruvil-shikimato-3-fosfato synthase

401

ESI – Electrospray ionization source

402

GLY – Glyphosate

403

GLY/CAR – Glyphosate plus carfentrazone

404

LC-MS – liquid chromatography-mass spectrometry

405

PAR – Paraquat

406

PPO – Polyphenol oxidase

407

PROTOX - protoporphyrinogen oxidase

408

qTOF – quadrupole-time of flight mass analyzer



409

ROS – Reactive oxygen species

410 411

Acknowledgments

412

We would like to thank Dr. Galileu Rupollo for the technical and financial support for

413

this research, as well as Conselho Nacional de Desenvolvimento Científico e

414

Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

415

Fundacão de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS),

416

Secretaria do Desenvolvimento Econômico, Ciência e Tecnologia do Estado do Rio

417

Grande do Sul (SDECT-RS), and Polo de Inovação Tecnológica em Alimentos da

418

Região Sul (Polo de Alimentos).

419 420

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421

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533

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Toxicology. 2017, 380, 1–10.

carbosulfan,

cypermethrin,

through

NADPH

fluopicolide,

hexythiazox,

oxidase-mediated

microglial

indoxacarb,

activation.

542 543

Figure captions

544

Figure 1. Images taken after 24 hours of plant desiccation with glyphosate (A),

545

glyphosate plus carfentrazone (B) and paraquat (C), and after 24 hours of manual

546

harvesting in case of control treatment (D).

547

Figure 2. a* value (A) and cooking time (B) of cowpea harvested from plants

548

desiccated with different herbicides.

549

Figure 3. Free phenolics (A), bound phenolics (B) and proanthocyanidins (C) content

550

of cowpea grains obtained from plants desiccated with different herbicides.

551

Figure 4. Principal component analysis of score plot (A) and loading ions (B) derived

552

from LC-MS data using negative electrospray ionization of cowpea extracts.



553

Figure 5. Extracted ion chromatogram of catechin-3-glucoside and epicatechin (A),

554

citric acid (B), gluconic acid (C), and quercetin glucoside derivative (D) from cowpea

555

grains harvested from plants desiccated with different herbicides.

556

Figure 6. ∆a* (A) and ∆cooking time (B) of cowpea stored during 8 months at 25 °C as

557

a function of the plant desiccant applied prior harvesting.

558

Figure 7. Visual appearance of cowpea grains at the beginning and at the 4th and 8th

559

months of storage at 25 °C under dark.

560 561

Table captions

562

Table 1. Mainly compounds differentiating cowpea grains treated with different

563

herbicides derived from LC-MS data using negative electrospray ionization.

564


Page 24 of 33

Page 25 of 33

565 566


Table 1. Mainly compounds differentiating cowpea grains treated with different herbicides derived from LC-MS data using negative electrospray ionization Retention time (min)

Experimental m/z

Theoretical m/z

Error (ppm)

mSigma

Fragmentation m/z

Identification

6.86

289.0723

289.0718

-0.60

45

-

Epicatechin

Quantification (mg . 100g-1) GLY 0.92±0.0

GLY/CAR 0.66±0.0

PAR 0.89±0.1

CONTROL 1.12±0.1

1

567 568

1.69

195.0515

195.0510

-0.4

8.8

129.0216 75.0088

Gluconic acid2

2.65±0.0

2.78±0.0

3.12±0.0

3.77±0.0

9.52

625.1426

625.1410

-2.5

46.2

300.0266

1.69±0.0

1.62±0.0

1.65±0.0

1.87±0.0

1.98

191.0205

191.0197

-4.0

15.6

111.0088 85.0299

Quercetin glucoside derivative2 Citric acid1

33.79±0.1

34.22±0.0

30.95±0.1

30.19±0.2

5.72

451.1258

451.1246

-2.7

59.8

289.0722 Catechin-3- 1.95±0.0 1.58±0.0 1.64±0.0 1.65±0.0 245.0821 glucoside2 137.0239 1 Confirmed by comparison to the external standard compound; 2 Confirmed by MS/MS; mSigma = fit between measured and theoretical isotopic pattern. The smaller the mSigma value the better the isotopic fit.

569



570

Fig. 1

571


Page 26 of 33

Page 27 of 33


Fig. 2

7

A a

6 b

a* Value

5

c

c

4 3 2 1 0 GLY

GLY/CAR

PAR

CONTROL

18

a

B 16 14 Cooking time (min)

572

b b

b

GLY

GLY/CAR

12 10 8 6 4 2 0 PAR

CONTROL



573

Fig. 3

1600

A -1

Free phenolics content (mg.100 g )

1400

a

1200

b

bc

GLY/CAR

PAR

CONTROL

b

b

b

GLY/CAR

PAR

CONTROL

b

b

c

1000 800 600 400 200 0 GLY

-1

Bound phenolics content (mg.100 g )

200 180

B

a

160 140 120 100 80 60 40 20 0 GLY

C

-1

Proanthocyanidins content (mg.100 g )

400

a 300

c 200

100

Free

0

GLY

GLY/CAR

PAR

CONTROL

574


Page 28 of 33

Page 29 of 33


575

Fig. 4

576



577

Fig. 5

578


Page 30 of 33

Page 31 of 33


579

Fig. 6

10

A

a

a

∆Cooking time (min)

8

b 6

c 4

2

0 GLY

GLY/CAR

PAR

CONTROL

10

B a

8

ab ∆a* Value

b 6

c 4

2

0 GLY

GLY/CAR

PAR

CONTROL

580



581

Fig. 7

582


Page 32 of 33

Page 33 of 33


583

TOC Graphic

584


Foliar Desiccators Glyphosate, Carfentrazone, and Paraquat Affect the

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