GlycinergicâFipronil Uptake Is Mediated by an Amino Acid Carrier

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

Glycinergic-fipronil uptake is mediated by an amino acid carrier system and induces the expression of amino acid transporter genes in Ricinus communis seedlings Yun Xie, Jun-Long Zhao, Chuan-Wei Wang, Ai-Xin Yu, Niu Liu, Li Chen, Fei Lin, and Hanhong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06042 • Publication Date (Web): 19 Apr 2016 Downloaded from http://pubs.acs.org on April 22, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 34

Journal of Agricultural and Food Chemistry

1

Glycinergic-fipronil uptake is mediated by an amino

2

acid carrier system and induces the expression of

3

amino acid transporter genes in Ricinus communis

4

seedlings

5

Yun Xie,†§Jun-Long Zhao,†§Chuan-Wei Wang,† Ai-XinYu†, Niu Liu † Li Chen† ,

6

Fei Lin*† and Han-Hong Xu*†

7

†

8

bioresources, South China Agricultural University, Guangzhou, Guangdong 510642;

9

People’s Republic of China and Key Laboratory of Natural Pesticide and Chemical

10

Biology, Ministry of Education, South China Agricultural University, Guangzhou,

11

Guangdong 510642, People’s Republic of China

State Key Laboratory for Conservation and Utilization of Subtropical Agro-

12 13 14 15

Corresponding Authors

16

*Han-Hong Xu and Fei Lin Tel: +86-20-85285127. E-mail: [email protected].

17

Author Contributions

18

§

Yun Xie and Jun-Long Zhao contributed equally to this work.

19 20 21 22 23

ACS Paragon Plus Environment


24

Abstract: Phloem-mobile insecticides are efficient for piercing and sucking insects

25

control. Introduction of sugar or amino acid groups to the parent compound can

26

improve the phloem mobility of insecticides, so a glycinergic -fipronil conjugate

27

(GlyF),

28

((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and

29

synthesized. Although the “Kleier model” predicted that this conjugate is not phloem

30

mobile, GlyF can be continually detected during five-hour collecting of Ricinus

31

communis phloem sap. Further, an R. communis seedling cotyledon disc uptake

32

experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide

33

m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic

34

acid (pCMBS), and is likely mediated by amino acid carrier system. In order to

35

explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AATs

36

genes were identified from the R. communis genome in silico. Phylogenetic analysis

37

revealed that AATs in R. communis were organized into the ATF (amino acid

38

transporter) and APC (amino acid, polyamine and choline transporter) superfamilies,

39

with 5 subfamilies in ATF and 2 in APC. Furthermore, the expression profiles of 20

40

abundantly expressed AATs (Cycle threshold (Ct) values lower than 27) were

41

analyzed at 1 h, 3 h, and 6 h after GlyF treatment by RT-qPCR. The results

42

demonstrated that expression level of four AATs genes, RcLHT6, RcANT15, RcProT2,

43

and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. Based on

44

the observation that the expression profile of the four candidate genes is similar to the

45

time course observation for GlyF foliar disc uptake, we suggest that those four genes

46

are possible candidates involved in the uptake of GlyF. Our results contribute to a

47

better understanding of the mechanism of GlyF uptake as well as phloem loading in

48

molecular biology perspective and facilitate functional characterization of candidate

2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-


Page 2 of 34

Page 3 of 34


49

AATs genes in the future studies.

50

Keywords: glycinergic, fipronil, amino acid transporter, uptake, Ricinus communis

51 52

INTRODUCTION

53

It is known that combining an endogenous nutrient such as monosaccharide or

54

amino acid to a pesticide compound enhances the plasma membrane permeation of

55

the conjugate. For example, our previous work synthesized a glycosyl-fipronil

56

conjugate, GTF, in which we demonstrated the involvement of active transport

57

system1. In 1997, an amino acid herbicide conjugate 2,4D-Lys was synthesized2,

58

which showed a distinctive distribution and the uptake of this compound is mediated

59

by an amino acid carrier system3. Therefore, we decide to design and synthesis an

60

amino acid-insecticide conjugate which possibly has similar uptake mechanism as

61

2,4D-Lys. However, there are few reports of the mechanisms of insecticide conjugate

62

uptake and little knowledge of the carriers involved, especially in molecular biology

63

perspective.

64

Following the identification of first amino acid transporter AAP1/NAT2 of

65

Arabidopsis4,5, more than 60 putative amino acid transporters have been identified

66

(see http://aramemnon.botanik.uni-koeln.de/). Many studies focused on the substrate

67

specificity, localization, and biological functions of these transporters6,7,8. These

68

transporters are divided into two superfamilies, the amino acid, polyamine and

69

choline transporters superfamily (APC) and the amino acid transporter family

70

(ATF)9,10,11. ATF proteins have a 9 to 11 transmembrane (TM) domain topology12 and

71

contain at least five subfamilies including the amino acid permease (AtAAPs)13, the

72

lysine/histidine transporters (AtLHTs)14, the proline transporters (AtProTs)15, the

73

aromatic and neutral amino acid transporters (AtANTs)16 and the putative auxin



Page 4 of 34

74

transporters (AtAUXs)17. APC proteins have a 12 or 14 TM topology18 and are

75

organized into two subfamilies, the cationic amino acid transporters (AtCATs)

76

L-type amino acid transporters (AtLATs)11.

19

and

77

R. communis seedling is an ideal model to study the mobility of test compounds

78

due to easy collection of phloem sap by surface incision. The full genome of R.

79

communis was not sequenced until 2010 and only a very small number of its amino

80

acid transporters have been investigated20. RcAAP1 and RcAAP2 were found to be

81

expressed abundantly in the cotyledon and root tissues of developing seedlings21.

82

RcAAP3 was observed to be expressed in source and sink tissues in all plants22. Thus,

83

there is large gap in our knowledge about the function of amino acid transporters in

84

the uptake of amino acid-pesticide conjugates in R. communis.

85

The uptake mechanism of exogenous compounds is very complicated and involves

86

multiple genes. Monitoring the gene expression profiles of amino acid carrier in

87

responding to the drug treatments provided supplement of physiological evidence that

88

certain carrier system involving the amino-acid-pesticide conjugates uptake process.

89

RT-qPCR (real-time quantitative polymerase chain reaction) is a convenient and

90

efficient method to measure the expression profile of certain genes compared with the

91

traditional Northern blot hybridization method23. The purpose of the present work was

92

(1) to design and synthesize a glycinergic - fipronil conjugates 2-(3-(3-cyano-1-(2,6-

93

dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl) sulfinyl )-1H-pyrazole-5-yl)

94

ureido ) acetic acid (GlyF) (Figure 1); (2) to assess its phloem mobility and uptake

95

mechanism in R. communis seedlings; and (3) to identify genes encoding putative

96

amino acid transporters in silico and monitor their expression profiles in response to

97

GlyF treatment. After demonstrating the possibility of amino acid carrier-mediated

98

uptake of GlyF, four amino acid carrier genes were found to be up-regulated by GlyF


Page 5 of 34


99

treatment, and thus likely serve as the transporters are the primary candidate involving

100

in the uptake and phloem loading of GlyF. This work represents the first step to

101

recognizing the role of abundant amino acid transporters in the uptake mechanism of

102

endogenous nutrient-pesticide conjugates. And this system could be exploited to

103

achieve optimal pesticide distribution in plants.

104

MATERIALS AND METHODS

105

General Information for Synthesis. All reagents and solvents were purchased

106

from Energy Chemical Company. Melting points were determined on a X-6 micro

107

melting point detector (BEIJING ZHONGYIBOTENG-TECH CO.,Ltd., China)

108

without calibration. 1H NMR and 13C NMR spectra were obtained with a Bruker AV-

109

600 instrument. Deuterated solvents were obtained from Cambridge Isotope

110

Laboratories (Andover, MA). DMSO and CDCl3 solvent peaks (2.56 and 7.26 ppm

111

for 1H and 39.6 and 77.0 ppm for

112

shift references. The mass spectrographic analysis was recorded on a Waters ZQ4000

113

(Waters,

114

chromatography (TLC) was carried out on pre-coated plates (silica gel GF254) and

115

spots were visualized with a ZF-20D ultraviolet (UV) analyzer. Silica gel (200−300

116

mesh) was used for column chromatography.

117

Synthesis Procedure. The synthesis of target compound glycinyl fipronil (GlyF) is

118

illustrated in Scheme 1.

119

tert-butyl2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)4((trifluoromethyl)

120

sulfinyl)-1H-pyrazole-5-yl)ureido)acetate（Scheme 1, b）

121

In an ice bath, potassium hydroxide (KOH, 1.68 g, 30 mmol) was added to the

122

solution of fipronil (4.37 g, 10 mmol) in dry acetone (40 mL), followed by dropwise

123

addition of phenyl chloroformate (1.88 g, 12 mmol). The reaction was stirred for 1 h

MA,

USA)

with

13

C, respectively) were used as internal chemical

electrospray

ionization.


Analytical

thin-layer


124

at 0 °C and overnight at room temperature (RT). The solvent was evaporated to give

125

the

126

((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)carbamate (compound 1). The residues

127

were subjected to the next step without further purification. To the solution of crude

128

compound 1 (0.56 g, 1 mmol) in anhydrous tetrahydrofuran (THF 30 mL), 1,8-

129

diazabicyclo [5.4.0] undec-7-ene (DBU, 0.015 g, 0.1 mmol), triethylamine (1.39 mL,

130

10 mmol) and tert-Butyl glycine hydrochloride (0.74 g, 5 mmol) were added. The

131

reaction was stirred at 65°C for 12 h and then cooled to room temperature. The

132

aqueous phase was extracted with ethyl acetate (3×30 mL). The combined ethyl

133

acetate layer was washed with saturated sodium chloride solution (3 ×20 mL), dried

134

by magnesium sulfate. The residues were purified by column chromatography

135

(hexane/EtOAc 2:1) to provide 2 (0.3 g). White solid; yield, 54%; mp, 208.4-208.6°

136

C. 1H NMR (DMSO-d6) δ 9.97 (s, 1H), 8.45 (s, 1H), 8.41 (s, 1H), 6.88 (s, 1H), 3.80 –

137

3.70 (m,2H), 1.39 (s,9H); 13C NMR (DMSO-d6) δ 168.7, 152.2, 141.2, 135.6, 135.2,

138

134.2, 133.9, 127.1, 127.0, 125.9, 124.7, 122.1, 111.1, 106.1, 81.0, 42.3, 27.3-27.1

139

(3C). EI-MS, m/z: 595.3,[M +H]+.

140

2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)

141

sulfinyl )-1H-pyrazole-5-yl) ureido ) acetic acid （scheme 1,c）

142

Trifluoroaceticacid (TFA, 20 mL) was added to a solution of 2b (0.59 g, 1 mmol)

143

anhydrous dichloromethane (CH2Cl2, in anhydrous dichloromethane (20 mL)). The

144

reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated

145

and the residue was purified by column chromatography (hexane/EtOAc 2:1) to give

146

the desired product 3 (0.53g). White amorphous solid; yield, 90%; mp, 199.0-

147

199.9°C .1H NMR (DMSO-d6) δ 12.80 (s, 1H), 9.94 (s, 1H), 8.46 (s, 1H), 8.42 (s,

148

1H), 6.89 (t, J = 5.4 Hz, 1H), 3.81 (dd, J = 8.4, 5.6Hz,2H); 13C NMR (DMSO-d6) δ

intermediate

1

phenyl(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-


Page 6 of 34

Page 7 of 34


149

170.9, 152.1, 141.2, 135.6, 135.3, 134.1, 133.9, 127.1, 127.0, 126.0, 124.8, 122.1,

150

111.2, 105.8, 41.6. EI-MS, m/z: 539.2, [M +H]+

151

Physicochemical Properties. The physicochemical properties [ionization constant

152

in aqueous solution (pKa), octanol/water partitioning coefficient (log Kow)] of GlyF

153

were predicted by the ACD LogD software suite version 14.0.

154

Plant Materials. R. communis No. 9, purchased from the Agricultural Science

155

Academy of Zibo, Shandong, China, were nurtured as previously described1. After six

156

days growing, seedlings (hypocotyl of almost 20 mm in length) were selected for

157

experimentation.

158

Membrane Potential Measurements. Plasma membrane potential of the

159

protoplasts of R. communis cotyledons was measured by flow cytometry using a

160

fluorescent membrane potential indicator dye bis (1,3-dibutylbarbituric acid)-

161

trimethine oxonol [DiBAC4(3)]24 . The protoplasts were incubated with buffer

162

solution without (control) or with the GlyF at 100 µM for 6 h. The treated protoplast

163

suspension was then co-incubated with 3 µM DiBAC4(3) for 30 min at 28 °C prior to

164

analysis.

165

Phloem Sap Collection. Phloem sap was collected as previously described, with

166

modifications25. The cotyledons of gathered seedlings with removed endosperm were

167

put in the cavern of 12 -hole cell culture plate and merged with buffer solution

168

containing GlyF at a 100 µM concentration. The roots of the seedlings were immersed

169

in 0.5 mM CaCl2, 0.25 mM MgCl2, 1 mM MES sodium salt solution. After 1 h of

170

incubation, the hypocotyl was severed in the hook region for phloem exudation. The

171

interval of phloem sap collection was 1 h, and the duration was 5 h. The collected

172

phloem sap was diluted with pure water (phloem sap:pure water, 1:4, v/v) and

173

quantified by HPLC.



174

Uptake by R. communis Foliar Disks. As previously described1, the disks (1.13

175

cm2 surface) were obtained using a 12 mm diameter cork and treated with incubation

176

medium containing 20 mM MES (pH 5.6), 250 mM mannitol, 0.25 mM MgCl2, and

177

0.5 mM CaCl2. 12 disks were used for a single set. The disks were transferred into

178

medium supplemented with GlyF to perform the time-course uptake experiment and

179

investigate the concentration-dependence of GlyF uptake. Disks were incubated in the

180

medium, pH ranging from 5.6 to 8.0 to assess the pH dependence on GlyF uptake, and

181

treated at 4 or 30 °C to assess the effect of temperature on GlyF uptake. For the

182

inhibition experiments, the disks were preincubated with either 50 µM CCCP, 10 mM

183

glycine (Gly), 10 mM glutamic acid(Glu), 10 mM histidine (His), 10 mM

184

phenylalanine (Phe), or 1 mM pCMBS in the incubation medium for 30 min. Then

185

disks were incubated with 100 µM GlyF and the substrates for 1 h. All incubations

186

were conducted under mild agitation on a reciprocal shaker on 80 rpm at 30 °C. After

187

the full incubation period, the disks were rinsed using the incubation medium six

188

times to remove residual GlyF. The disks were smashed by freezing with liquid

189

nitrogen, and then ground with 10 mL of methanol and ultrasonicated for 30 min. The

190

extract solutions were centrifuged at 14000g for 10 min, and then filtered with 0.22-

191

µm filters. The final extract solution was analyzed by HPLC.

192

Determination of GlyF. Disk extracts and phloem sap were analyzed using an

193

HPLC system (Agilent 1100 series). Separations were done with a C18 reversed-

194

phase column. The solvent system consisted of acetonitrile and water containing 0.1%

195

TFA (60:40, v/v). The injection volume was 10 µL, and the flow rate was 1 mL/min.

196

All peaks with the same retention time as the GlyF standard were confirmed by

197

UPLC-HRMS (Agilent 1290-6540B Q-TOF).

198

Genome-Wide Identification of Amino Acid Transporters in R. communis.


Page 8 of 34

Page 9 of 34


199

Genome and annotation data were downloaded from the phytozome v9.1 database

200

(http://www.phytozome.net/ricinus.php). The genome annotation protein data set was

201

searched for proteins containing conserved sequences motifs for “amino acid

202

transporter” as defined by Pfam HMM (hidden Markov model) profiles (PF00324 for

203

ATF transporters and PF1490 for APC transporters) using the hmmsearch program, a

204

part of the HMMER package (version 3.1) (http://hmmer.org/). Following the

205

HMMER identification, all selected protein sequences were used in BlastP search

206

against all known Arabidopsis thaliana amino acid transporters to verify the

207

correctness of the annotation. Finally, all available ESTs for the selected genes were

208

gathered using a BLASTN search against NCBI R .communis dbEST data sets. Since

209

an integrated functional transport protein typically requires at least 300 amino acid

210

residues and is at least 40-50 kDa in melocular weight, protein sequence lengths of

211

less than 300 amino acid residues were excluded from further phylogenetic analysis26.

212

Phylogenetic Analysis. Multiple sequence alignment was generated by ClustalW2.

213

Phylogenetic trees were constructed by the neighbor-joining (NJ) method in MEGA

214

(version 5.0)27 with bootstrap values from 1000 replicates indicated at each node. The

215

names of RcAATs were based on the subfamily classification and their phylogenetic

216

relationships with the AtAAT and RcAAT. For example, the ten RcAAP members in

217

AAP class were named RcAAP1 to RcAAP10. The number of TMHs (Transmembrane

218

helices) of all genes for phylogenetics analysis were predicted by TMHMM Server

219

v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/).

220

Tissue Sampling and RNA Extraction. The cotyledons, from which the

221

endosperm had been removed, were incubated in buffer solution containing GlyF at a

222

concentration of 100 µM. Buffer solution containing 0.5% DMSO solvent, which is

223

the same concentration as in the GlyF incubation buffer, was set up as control. After



224

drug treatments, cotyledons were harvested at 1 h, 3 h, and 6 h in liquid nitrogen and

225

stored at −80°C for RNA isolation. Total RNA was extracted from the cotyledons

226

using Plant RNA Kit (OMEGA, USA). The quality of RNA was measured by

227

ultraviolet spectrophotometry for the ratio of absorbance at 260 nm and 280 nm.

228

qRT-PCR Analysis. iScript™ Reverse Transcription (Bio-Rad, USA) was used to

229

synthesize the first-strand complementary DNA (cDNA) by using 1 µg total RNA

230

which had digested by DNase I (NEB, USA) to remove contaminating genomic DNA.

231

Each 20 µL volume transcription system contains 4 µL 5×xiScriptTM 4 µL Reaction

232

Mix, 1 µL iScriptTM Reverse Transcriptase, and 5 µL Nuclease-free H2O. The

233

reverse transcription program was 25°C for 5 min, 42°C for 90 min, 85 °C for 5 min.

234

PCR was performed in optical 96-well plates with a CFX Connect Real-Time PCR

235

detection system (Bio-Rad, USA) with a total volume of 20 µL consisting of 1 µL of

236

cDNA, 0.5 µL of each of the two gene-specific primers (10 pmol µL-1) (Table S2),

237

and 10 µL 2Χ IQ SYBR Green Supermix reagent (Bio-Rad, USA) in a final volume

238

of 15 µL. Specific primers for RcActin gene were used as internal references (Mao et

239

al. 2015; Table S2). The relative expression levels of selected genes were normalized

240

to the reference gene RcActin with ∆∆Ct(Cycle threshold) method28. Significant

241

difference analysis was performed utilizing SPSS software carrying out ANOVA

242

analysis.

243

Primers specific for each gene were designed using Primer Premier (version 5.0)

244

software and confirmed by melting-curve analysis (Table S2). Ct values were

245

automatically determined by Optical System Software (Version 3.1, Bio-Rad, USA).

246

The PCR amplification efficiency of each primer pair was estimated by linear

247

regression, in which at least 5 points covering the Ct value were utilized based on the

248

highest correlation coefﬁcient29. The expression level of the different members of the


Page 10 of 34

Page 11 of 34


249

RcAAT gene family varied over a wide range, and genes with high Ct value indicated

250

low transcript abundance, which made it difficult for them to be covered by the linear

251

regression28. Thus, genes with Ct value higher than 26 were removed from further

252

analysis.

253

Result And Discussion

254

Membrane Potential Measurements. The measurement of protoplast membrane

255

potentials of R. communis seedlings after 6 h incubation of GlyF showed that there

256

was no significant difference in relative fluorescence compared with the control

257

(Figure 2). This result indicates that GlyF exhibit low toxicity to R. communis.

258

Therefore, R. communis seedlings were able to be used to conduct the GlyF phloem

259

mobility test and the further experiments24.

260

Detection of GlyF Phloem Mobility. The GlyF in the phloem sap of R. communis

261

seedlings has been measured during the experimental period and the concentration

262

reached 10.14 µM (almost 10% of incubation concentration) (Figure 3). GlyF in the

263

phloem sap was identified by the UPLC-HRMS and the exact mass of the standard

264

compound of GlyF (ESI+ ,m/z: 537.9585, [M + H]+ ) matched the detected GlyF in

265

phloem sap (ESI+ ,m/z: 537.9567, [M + H]+ ). Based on the results above, GlyF is

266

indeed phloem mobile.

267

The “Kleier model” is typically used to predict the mobility of xenobiotics based on

268

the physicochemical properties30, and we used it to predict the phloem mobility of

269

GlyF as well. However, the model predicted non phloem mobility, which was

270

inconsistent with our results (Figure 4). Taking into consideration that some

271

biological parameters are not included in the “Kleier model” there must be other

272

mechanisms for GlyF entering into the phloem (possibly carrier-mediated uptake) and

273

they should be further investigated.



274

Cotyledon Disk Uptake of GlyF. In order to fully understand the mechanism of

275

GlyF uptake, the time course, concentration dependence, pH dependence, and effector

276

inhibition experiments were performed on R. communis seedling cotyledon disks.

277

The time course of 0.1 mM GlyF uptake at pH 5.6 was characteristic of this

278

compound, in which the uptake of GlyF was at maximum velocity in the first

279

incubation hour (almost linear) and dramatically decreased afterwards (Figure 5). This

280

indicates that the uptake of GlyF reached equilibrium rapidly in 360 min of incubation

281

time.

282

Concentration dependence of GlyF, (0.01 to 1 mM) showed that there are two

283

distinctive stages of GlyF uptake (Figure 6A). The first saturable component between

284

0.01 and 0.2 mM indicated that this part of GlyF uptake is carrier-mediated. The

285

second component at the higher concentration was almost linear, suggesting that

286

passive diffusion dominates the uptake of GlyF in this regime. Using Lineweaver-

287

Burk plots we calculated the Km of GlyF uptake to be 0.295 mM and Vmax was 1.749

288

nmol/cm²/h (Figure S1A). CCCP is an inhibitor of oxidative phosphorylation and is

289

widely used to inhibit the transmembrane proton motive force. The saturable

290

component of GlyF uptake was significantly inhibited by 50 µM CCCP (Figure 6B).

291

Excluding the CCCP-insensitive component of GlyF uptake, the Km and Vmax of

292

CCCP-sensitive component were 0.164 mM and 0.424 nmol/cm²/h, respectively

293

(Figure S1B).

294

pH is an essential element for xenobiotics uptake. According to the calculation

295

result of GlyF, the net charge remains unchanged from pH 5.6 through pH 8.0 (Figure

296

S2), Thus 4 pHs (5.6 to 8.0) were chosen to conduct pH dependent experiment. The

297

result showed that the uptake of GlyF is pH dependent, which correspondingly

298

decreases with increasing pH of the incubation buffer (Figure 6C). The uptake of


Page 12 of 34

Page 13 of 34


299

GlyF at pH 8.0 is only 0.29 fold of that of pH 5.6.

300

pCMBS (p-chloromercuribenzenesulfonic acid) is a non or slowly permeant thiol

301

reagent which is used to inhibit transmembrane protein permeance31. pCMBS at 1

302

mM concentration inhibits 0.1 mM GlyF uptake by 41.3%. When the incubation

303

temperature shifted from 30°C to 4°C the uptake of GlyF dropped significantly and

304

only accounted 41.5% of the 30°C control). This result suggests that the uptake of

305

GlyF is sensitive to temperature. To investigate the substrate specificity of GlyF

306

uptake, 10 mM of Glu (acidic amino acid), Gly (neutral amino acid), His (basic amino

307

acid), and Phe (aromatic amino acid) were chosen to conduct competitive inhibition

308

of 0.1 mM GlyF uptake at pH 5.6, respectively. All chosen amino acids had a

309

significant inhibition effect on GlyF uptake: 30% (Glu), 28% (Gly), 45% (His), and

310

53% (Phe). Interestingly, Phe had the strongest inhibition, even though like Gly it is a

311

neutral amino acid; this observation is most likely due to its unique structure of the

312

benzene ring, which is a component of both Phe and GlyF (Figure 6D).

313

Collectively, these experiments demonstrated that the uptake of GlyF is sensitive to

314

pH, CCCP, temperature, pCMBS, and thus likely is mediated by an amino acid carrier

315

system at low concentration.

316

Genome-wide Identification and Phylogenetic Analysis of RcAAT Genes. To

317

further explore the roles of amino acid transporters in GlyF uptake, genome-wide

318

identifications for these gene families were conducted in silico. A total of 77 genes

319

were extracted using the conserved sequences Pfam PF00324 and PF1409 as seed

320

sequences to search against release v9.1 genome data with the HMMsearch program.

321

Candidate sequences were verified in the Pfam database to ensure the candidates

322

contained conserved transporter domains. After removing the sequences which did

323

not have conserved transporter domains and which were less than 300 amino acid



324

residues in length, 62 genes were obtained, which encode 45 ATF family proteins and

325

17 APC family proteins (Figure 7) 26. The RcATF family consists of five subfamilies

326

known as LHT, AAP, ProT, ANT, and AUX; the RcAPC family consists of two

327

subfamilies known as CAT and BAT. We named the RcAATs genes based on the

328

subfamily classification and their phylogenetic relationships with the AtAATs (Figure

329

7). The RcAATs were distributed across 43 scaffolds of the R. communis genome and

330

their encoded predicted protein lengths ranged from 376 to 643 amino acids (Table

331

S1). The transmembrane helices number of these 62 genes ranged from five to

332

fourteen.

333

Impact of GlyF on RcAATs Genes Expression. To determine the potential roles

334

of the RcAATs genes in the uptake of GlyF, qRT-PCR was performed to monitor the

335

expression dynamics in response to GlyF treatment. Primer pairs specific to the 62

336

RcAATs genes were designed and subjected to qRT-PCR analysis. Using the mixture

337

of cDNAs from all the samples (including the treatments and control) as PCR

338

templates, the Ct values for the 62 RcAAT genes ranged from 22.42 to 36.25,

339

indicating that the expression level of RcAATs varied over a wide range. Genes with

340

high Ct value resulted in qRT-PCR analysis deviation because the Ct value was not

341

covered by the regression of line. Therefore, we used a Ct value of 27 as the threshold

342

to select genes within the linear range, which led to 20 RcAATs (RcAAP2, RcAAP3,

343

RcANT7, RcANT11, RcANT15, RcProT2, RcLHT1, RcLHT7, RcAUX2, RcCAT2,

344

RcCAT5, RcCAT9, RcCAT10, RcCAT11 and RcCAT12.etc) being selected to analyze

345

the dynamics of expression in response to GlyF treatment.

346

Expression dynamics of the 20 selected RcAATs genes were monitored by qPCR

347

and analyzed with ∆∆Ct method. The results showed that 4 of the 20 RcAATs

348

(RcLHT6, RcANT15, RcProT2 and RcCAT2) showed significant up-regulation when


Page 14 of 34

Page 15 of 34


349

treated with GlyF, while the rest of the 20 ones did not show significant changes (data

350

not shown).

351

Generally, gene RcLHT6 was consistently upregulated upon GlyF treatment,

352

regardless of time point (Figure 8A). For RcANT15, there was no significant

353

difference between the DMSO control and GlyF treatment at 3 h. However, RcANT15

354

was also up-regulated at 1 h by GlyF treatment (Figure 8B). As for gene RcProT2, the

355

expression in GlyF treatment at 3 h and 6 h was lower than that in the control.

356

Interestingly, it is the opposite at 1 h, where GlyF treatment resulted in over 2-fold

357

increase of RcProT2 gene expression compared to the DMSO control (Figure 8C).

358

With RcCAT2, the expression profile was similar to RcProT2. And there was no

359

significant of gene expression difference between the control and GlyF treatments at

360

the later time points. However, gene expression of RcCAT2 was significantly

361

increased after 1 h of GlyF treatment, up nearly 4.5-fold (Figure 8D).

362

R. communus is a popular model for testing the uptake of conjugate compounds. In

363

previous studies, this test platform served quite well for screening phloem transport

364

and carrier-mediated conjugate compounds in our lab32,33. In our former research, we

365

added an endogenous monosaccharide to the pesticide parent compound fipronil in

366

order to develop a novel systemic pesticide; however, an amino acid conjugate is

367

another potential alternative. To reveal the uptake mechanism of new carrier-mediated

368

compounds, it is necessary to combine both physiological and molecular evidence.

369

Therefore, for the new conjugate GlyF, we have designed and performed phloem

370

mobility detection, cotyledon disk uptake, and qRT-PCR analysis for relative gene

371

expression profiles. In support of these studies, we first developed the phylogenetic

372

trees of AATs of ATF and APC families in R. communis.

373

Study of amino acid transporters has been mainly focused on Arabidopsis thaliana,



374

along with a few in other model or crop plants including tomato, potato, etc13,34.

375

Studies are ranged from gene identification, tissue and subcellular localization,

376

substrate selectivity, and molecular function in heterologous expression (e.g.

377

expression in S. cerevisieae and Xenopus) and in planta (e.g. mutant and

378

overexpression). Some amino acid transporters of these two ATF and APC families in

379

A. thaliana have been studied fairly well within different aspects16,7,11,35. The

380

subfamilies of these two families show similarities and diversities among each

381

other9,11,12. For example, AAPs recognize their substrates with moderate or low

382

affinity,7 with the exception of AtAAP6 having a high affinity to neutral amino acids

383

and glutamate13; in CATs, AtCAT1 and AtCAT5 have been reported as high affinity

384

transporters for cationic amino acids; while AtCAT6 was demonstrated to transport

385

both essential neutral amino acids and the basic amino acid lysine with moderate

386

affinities11. The same patterns could also be seen in tissue localization, expression

387

profiles, and relative molecular function, which indicates the tremendously

388

complicated and multidimensional relationships within these amino acid transporters

389

in plants, and therefore probably are important components of plant growth and

390

development.

391

From the qPCR gene expression profile with GlyF treatment, RcANT15, RcProT2,

392

RcCAT2, and RcLHT6 were selected. Three of them shared several similarities in their

393

expression profiles. Combining results with the GlyF uptake time course in foliar

394

Disks, which reached maximum velocity in the first hour and dramatically decreased

395

afterwards. The expression profile of candidate gene at this time point should be

396

significantly higher than the control group; this expectation is consistent with the

397

expression pattern of RcANT15, RcProT2, and RcCAT2. Although RcLHT6 exhibits a

398

different expression profile than these three genes, it is still significantly higher than


Page 16 of 34

Page 17 of 34


399

control group at the 1 h time point. Meanwhile, the plasma membrane potential of the

400

protoplasts of R. communis cotyledons did not remarkably change due to the addition

401

of 100 µM GlyF over 6 hours. Thus, we can assume GlyF did not generate a stress

402

response in R. communis seedlings, and the different expression profiles were caused

403

only by the additional GlyF in the buffer. Furthermore, as amino acids have been

404

reported being the trigger for the induces of amino acid transporter expression36, the

405

up-regulation of the four candidate transporters is also consistent with the R.

406

communis seedling cotyledon disks uptake results, which indicates the possible

407

involvement of an amino acid carrier system. Taken together, we can conclude that

408

RcLHT6, RcANT15, RcProT2, and RcCAT2 belonging to ATF or APC superfamilies

409

are the primary candidates for glycinergic-fipronil uptake or phloem loading in castor

410

bean seedlings.

411

At the same time, we could not rule out the possibility that the amino acid

412

transporters non-induced by GlyF but expressing abundantly could be involved in the

413

uptake or phloem loading of GlyF, especially those genes belonging to AAP

414

subfamily. RcAAP2 and RcAAP3 were expressed abundantly in R. communis seedling

415

according to our result (Table S1). And it was reported AtAAPs could transport a wild

416

range of amimo acid and participate in phloem loading37. Our result demonstrated

417

four abundantly expressed AATs genes were regulated by GlyF, but it doesn’t

418

necessarily mean that those four genes are involved in the uptake and phloem loading

419

of GlyF. Further studies such as function complementation tests and tissue

420

localization and subcellular localization of these GlyF-induced AATs genes need to be

421

conducted to fully understand how AATs participate in the uptake process as well as

422

phloem loading in R. communis seedlings.

423



424 425 426 427 428

Supporting Information Available：：

429

Lineweaver-Burk plots of GlyF uptake·············································Figure S1

430

The net charge of GlyF in different pH conditions·································Figure

431

S2

432

Amino acid transporter genes for phylogenetic trees analysis·····················Table

433

S1

434

Primers used in this study·······························································Table

435

S2

436

This material is available free of charge via the Internet at http://pubs.acs.org.

437

Funding

438

Financial support was from the National Natural Science Foundation of China (Grant

439

31171886), the Specialized Research Fund for the Doctoral Program of Education of

440

China (Grant 20134404130003) and Natural Science Foundation of Guangdong

441

Province (Grant 2014A030311044)

442

Notes

443

The authors declare no competing financial interest.

444

REFERENCES

445

(1)

Wu, H.-X.; Yang, W.; Zhang, Z.-X.; Huang, T.; Yao, G.-K.; Xu, H.-H. Uptake

446

and phloem transport of Glucose-fipronil conjugate in Ricinus communis

447

involve a carrier-mediated mechanism. J. Agric. Food Chem. 2012, 60, 6088–

448

6094.


Page 18 of 34

Page 19 of 34


449

(2)

Chollet, J.-F.; Delétage, C.; Faucher, M.; Miginiac, L.; Bonnemain, J.-L.

450

Synthesis and structure-activity relationships of some pesticides with an α-

451

amino acid function. Biochim. Biophys. Acta. 1997, 1336, 331–341.

452

(3)

Delétage-Grandon, C.; Chollet, J.-F.; Faucher, M.; Rocher, F.; Komor, E.;

453

Bonnemain, J.-L. Carrier-mediated uptake and phloem systemy of a 350-Dalton

454

chlorinated xenobiotic with an α-amino acid function. Plant Physiol. 2001, 125,

455

1620–1632.

456

(4)

Frommer, W. B.; Hummel, S.; Riesmeier, J. W. Expression cloning in yeast of

457

a cDNA encoding a broad specificity amino acid permease from Arabidopsis

458

thaliana. Proc. Natl. Acad. Sci. 1993, 90, 5944–5948.

459

(5)

Hsu, L.-C.; Chiou, T.-J.; Chen, L.; Bush, D. R. Cloning a plant amino acid

460

transporter by functional complementation of a yeast amino acid transport

461

mutant. Proc. Natl. Acad. Sci. 1993, 90, 7441–7445.

462

(6)

463 464

Rentsch, D.; Schmidt, S.; Tegeder, M. Transporters for uptake and allocation of organic nitrogen compounds in plants. FEBS Lett. 2007, 581, 2281–2289.

(7)

Fischer, W.N.; Loo, D.D.F.; Koch, W.; Ludewig, U.; Boorer, K. J.; Tegeder,

465

M.; Rentsch, D.; Wright, E.M.; Frommer, W. B. Low and high affinity amino

466

acid H+-cotransporters for cellular import of neutral and charged amino acids.

467

Plant J, 2002. 29,717-731.

468

(8)

Lee, Y.-H.; Tegeder, M. Selective expression of a novel high-affinity transport

469

system for acidic and neutral amino acids in the tapetum cells of Arabidopsis

470

flowers: Tapetum cell-specific amino acid transport processes. Plant J. 2004,

471

40, 60–74.

472 473

(9)

Fischer,W,N; André,B; Rentsch,D; Krolkiewicz,S; Tegeder,M; Breitkreuz,K and Frommer,W.B. Amino acid transport in plants. Trends Plant Sci. 1998,



474 475

3,1360 - 1385. (10)

476 477

Lopez,A.O.; Chang, H.-C.; Bush,D,R; Amino acid transporters in plants. Biochim Biophys Acta. 2000,1465, 275–280.

(11) Su, Y.-H. Molecular and functional characterization of a family of amino acid

478

transporters from Arabidopsis. Plant Physiol. 2004, 136, 3104–3113.

479

(12) Wipf, D.; Ludewig, U.; Tegeder, M.; Rentsch, D.; Koch, W.; Frommer, W. B.

480

Conservation of amino acid transporters in fungi, plants and animals. Trends

481

Biochem. Sci. 2002, 27, 139–147.

482

(13) Liu, X.; Bush, D. R. Expression and transcriptional regulation of amino acid

483 484 485

transporters in plants. Amino Acids. 2006, 30, 113–120. (14)

Chen, L.; Bush, D. R. LHT1, a lysine-and histidine-specific amino acid transporter in Arabidopsis. Plant Physiol. 1997, 115, 1127–1134.

486

(15) Rentsch D, Hirner B, Schmelzer E, Frommer,W.B. Rentsch, D. Salt stress-

487

induced proline transporters and salt stress-repressed broad specificity amino

488

acid permeases identified by suppression of a yeast amino acid permease-

489

targeting mutant. Plant Cell. 1996, 8, 1437–1446.

490 491

(16) Chen, L.; Ortiz-Lopez, A.; Jung, A.; Bush, D. R. ANT1, an aromatic and neutral amino acid transporter in Arabidopsis. Plant Physiol. 2001, 125, 1813–1820.

492

(17) Bennett, M. J.; Marchant, A.; Green, H. G.; May, S. T.; Ward, S. P.; Millner, P.

493

A.; Walker, A. R.; Schulz, B.; Feldmann, K. A. Arabidopsis AUX1 gene: A

494

permease-like regulator of root gravitropism. Science. 1996, 273, 948–950.

495

(18) Wipf, D.; Benjdia, M.; Tegeder, M.; Frommer, W. B. Characterization of a

496

general amino acid permease from Hebeloma cylindrosporum. FEBS Lett.

497

2002, 528, 119–124.

498

(19) Jack, D. L.; Paulsen, I. T.; Saier, M. H. The amino acid/polyamine/organocation


Page 20 of 34

Page 21 of 34


499

(APC) superfamily of transporters specific for amino acids, polyamines and

500

organocations. Microbiology. 2000, 146, 1797–1814.

501

(20) Chan, A. P.; Crabtree, J.; Zhao, Q.; Lorenzi, H.; Orvis, J.; Puiu, D.; Melake-

502

Berhan, A.; Jones, K. M.; Redman, J.; Chen, G. Draft genome sequence of the

503

oilseed species Ricinus communis. Nat. Biotechnol. 2010, 28, 951–956.

504

(21) Bick, J.-A.; Neelam, A.; Hall, J. L.; Williams, L. E. Amino acid carriers of

505

Ricinus communis expressed during seedling development: molecular cloning

506

and expression analysis of two putative amino acid transporters, RcAAP1 and

507

RcAAP2. Plant Mol. Biol. 1998, 36, 377–385.

508

(22)

Neelam, A.; Marvier, A. C.; Hall, J. L.; Williams, L. E. Functional

509

characterization and expression analysis of the amino acid permease RcAAP3

510

from castor bean. Plant Physiol. 1999, 120, 1049–1056.

511

(23) Bustin, S. A. Quantification of mRNA using real-time reverse transcription PCR

512

(RT-PCR): trends and problems. J. Mol. Endocrinol. 2002, 29, 23–39.

513

(24) Chollet, J.-F.; Rocher, F.; Jousse, C.; Deletage-Grandon, C.; Bashiardes, G.;

514

Bonnemain, J.-L. Synthesis and phloem mobility of acidic derivatives of the

515

fungicide fenpiclonil. Pest Manag. Sci. 2004, 60, 1063–1072.

516

(25) Wang, J.; Lei, Z.; Wen, Y.; Mao, G.; Wu, H.; Xu, H. A novel fluorescent

517

conjugate applicable to visualize the translocation of Glucose–fipronil. J. Agric.

518

Food Chem. 2014, 62, 8791–8798.

519

(26) Hirner, A. Arabidopsis LHT1 is a high-affinity transporter for cellular amino

520

acid uptake in both root epidermis and leaf mesophyll. Plant cell. 2006, 18 ,

521

1931–1946.

522 523

(27)

Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood,



524

evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol.

525

2011, 28, 2731–2739.

526

(28) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods. 2001, 25, 402–408.

527 528

(29) Ramakers, C.; Ruijter, J. M.; Deprez, R. H. L.; Moorman, A. F. Assumption-free

529

analysis of quantitative real-time polymerase chain reaction (PCR) data.

530

Neurosci. Lett. 2003, 339, 62–66.

531

(30) Hsu, F. C.; Kleier, D. A. Phloem mobility of xenobiotics VIII. A short review. J.

532

Exp. Bot. 1996, 47, 1265–1271.

533

(31) Rocher, F.; Chollet, J.-F.; Legros, S.; Jousse, C.; Lemoine, R.; Faucher, M.;

534

Bush, D. R.; Bonnemain, J.-L. Salicylic acid transport in Ricinus communis

535

involves a pH-dependent carrier system in addition to diffusion. PLANT

536

Physiol. 2009, 150, 2081–2091.

537

(32) Yang, W.; Wu, H.-X.; Xu, H.-H.; Hu, A.-L.; Lu, M.-L. Synthesis of Glucose–

538

fipronilconjugate and its phloem mobility. J. Agric. Food Chem. 2011, 59,

539

12534–12542.

540

(33) Lei, Z.; Wang, J.; Mao, G.; Wen, Y.; Tian, Y.; Wu, H.; Li, Y.; Xu, H. Glucose

541

positions affect the phloem mobility of Glucose–fipronil conjugates. J. Agric.

542

Food Chem. 2014, 62, 6065–6071.

543

(34) Waterworth, W. M. Enigma variations for peptides and their transporters in

544 545

higher plants. Ann. Bot. 2006, 98, 1–8. (35) Tegeder, M.; Rentsch, D. Uptake and partitioning of amino acids and peptides.

546 547 548

Mol. Plant. 2010, 3, 997–1011. (36)

Pratelli, R.; Pilot, G. Regulation of amino acid metabolic enzymes and transporters in plants. J. Exp. Bot. 2014, 65, 5535–5556.


Page 22 of 34

Page 23 of 34


549 550

(37) Tegeder, M. Transporters for amino acids in plant cells: some functions and many unknowns. Curr. Opin. Plant Biol. 2012, 15, 315–321.

551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569

570



GlyF

571 572

Figure1. Schematic representation of GlyF

573 574 575 576 577 578 579 580 581 582 583 584 585


Page 24 of 34

Page 25 of 34


NC

SOCF3

N

NC N

NH 2

N

SOCF3 O

Cl

Cl

Cl

CF 3

NC

CF3

SOCF3 O

NC

N Cl

N H Cl

N H

b

N

c

N Cl

CF3

O

SOCF3 O

COOH

N

N H Cl

N

a

N H Cl

O N H

O

CF3

586 587

Scheme 1. Reagents and conditions: (a) phenyl chloroformate, acetone, KOH, 0 °C,1

588

h and overnight RT, (b) tert-Butyl glycine hydrochloride, DBU, 65 °C, overnight. (c)

589

TFA/CH2Cl2=20mL/20mL, 2 h.

590 591 592 593 594 595 596 597 598



Counts

A

GlyF Control

B

599 600

Figure2. Membrane potential of protoplasts after 6 h of GlyF treatment. (A)

601

Fluorescence intensity histogram. (B) Relative fluorescence of the treatment (GlyF

602

and control). The protoplasts were suspended in a buffered solution at pH 5.8 with or

603

without (control) tested compounds at 100 µM for 6 h. The protoplasts were analyzed

604

by flow cytometry for DiBAC4(3) fluorescence. The data [mean ± SE; n = 3] within a

605

column are not significantly different, as determined by Kruskal−Wallis test (p>0.05).

606 607 608


Page 26 of 34

Page 27 of 34


609 610 611

612 613

Figure 3. Concentration of GlyF in phloem sap of R. communis seedlings over five

614

hours. Each column is the mean of 12 seedlings ± SE (n = 4).

615 616 617 618 619 620 621



622 623

Figure 4. Prediction of phloem mobility of GlyF via Kleier map; GlyF is located in

624

the non-phloem mobile area. Log Kow and pKa were calculated by the ACD

625

Laboratories Percepta program, version 14.0

626


Page 28 of 34

Page 29 of 34


627 628

Figure 5. Time course of 0.1 mM GlyF uptake at pH 5.6 by leaf disks. Each point was

629

the mean of 12 disks ± SE (n = 4).

630 631 632 633 634 635 636 637 638 639 640 641



642 643

Figure 6. Different factors of GlyF uptake by cotyledon disks acquired from Ricinus

644

communis seedlings. (A) Concentration dependence of GlyF uptake. Disks were

645

incubated in a buffer at pH 5.6 with GlyF concentration ranging from 0.01 mM to 1

646

mM. (B) CCCP inhibits saturable component of GlyF uptake between 0.01 and 0.2

647

mM concentration. Disks incubated with 50 µM CCCP were set as the complementary

648

set of A. (C) pH dependence of 0.1 mM GlyF uptake. (D) Effect of inhibitors, amino

649

acids, and low temperature (4°C) on the uptake of 0.1 mM GlyF at pH 5.6. All data

650

shown are the averages of 12 disks ± SE (n = 4). Statistics were analyzed by ANOVA

651

test, followed by Dunnett’s test to identify the differences in the mean of each group

652

with the control group. ***, P < 0.001.


Page 30 of 34

Page 31 of 34


653 ACS Paragon Plus Environment


654 655

Figure 7. Phylogenetic relationships of the deduced protein sequences of R.

656

communis Amino Acid Transporter (AATs) gene family with the known Arabidopsis

657

thaliana AAT genes. The tree is reconstructed using the protein-coding sequences of

658

the amino acid transporter genes of ATF (A) and APC (B) superfamilies calculated by

659

hmmsearch program from R. communis whole genome data using MEGA5.0 in

660

poisson mode with the Neighbor-Joining (NJ) algorithm. The optimal trees were

661

statistically evaluated by bootstrap analysis with 1000 replications. Bootstrap support

662

values are indicated on each node. The information on the identified RcAAT genes are

663

listed in Table S1. Branches with black dot indicated member induced by the GlyF

664

treatment.

665 666 667


Page 32 of 34

Page 33 of 34


668 669

Figure 8. Expression pattern of four ATTs genes, (A) RcLHT6, (B) RcANT15, (C)

670

RcProT2, (D) RcCAT2, in response to GlyF treatment in R. communis seedlings. The

671

expression levels of the genes were determined using qRT-PCR using gene-specific

672

primers and were normalized by the RcActin internal control gene. The data were

673

obtained and processed from three experimental replicates. The data (mean ± SE, n =

674

3) with a column topped by different letters are significantly different from each

675

other; same letters indicate no difference, as determined by Duncan’s multiple-range

676

test (P > 0.05). CK means DMSO solvent control.

677 678 679 680 681



682 683

TOC graphic

684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709


Page 34 of 34

GlycinergicâFipronil Uptake Is Mediated by an Amino Acid Carrier

Recommend Documents

GlycinergicâFipronil Uptake Is Mediated by an Amino Acid Carrier