Metabolic Resistance to Acetolactate Synthase Inhibiting Herbicide

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Agricultural and Environmental Chemistry

Metabolic resistance to acetolactate synthase (ALS)inhibiting herbicide tribenuron-methyl in Descurainia sophia L. mediated by cytochrome P450 enzymes Qian Yang, Jinyao Li, Jing Shen, Yufang Xu, Hongjie Liu, Wei Deng, Xuefeng Li, and Mingqi Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05825 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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Journal of Agricultural and Food Chemistry

Metabolic

resistance

to

acetolactate

synthase

(ALS)-inhibiting

herbicide

tribenuron-methyl in Descurainia sophia L. mediated by cytochrome P450 enzymes

Qian Yang†, Jinyao Li†, Jing Shen†, Yufang Xu†, Hongjie Liu†, Wei Deng†, Xuefeng Li† and Mingqi Zheng*, † †

Department of Applied Chemistry, College of Science, China Agricultural University,

Beijing 100193, P. R. China.

*Corresponding Author Mingqi Zheng Phone: 86 10 62733924. E-mail: [email protected]

1 ACS Paragon Plus Environment


1

ABSTRCT: D. sophia is one of the most notorious broadleaf weed in China, and has

2

evolved extremely high resistance to ALS-inhibiting herbicide tribenuron-methyl. The

3

target-site resistance due to ALS gene mutations was known well, while the

4

non-target-site resistance is not yet well-characterized. Metabolic resistance, which is

5

conferred by enhanced rates of herbicide metabolism, is the most important NTSR. To

6

explore the mechanism of metabolic resistance underlying resistant (R) D. sophia

7

plants, tribenuron-methyl uptake and metabolism levels, qPCR reference gene stability,

8

and candidate P450 genes expression patterns were investigated. The results of liquid

9

chromatography-mass spectrometry (LC-MS) analysis indicated that the metabolic

10

rates of tribenuron-methyl in R plants was significantly faster than in susceptible (S)

11

plants, and this metabolism differences can be eliminated by P450 inhibitor malathion.

12

18S rRNA and TIP41-like were identified as the most suitable reference genes using

13

programs of BestKeeper, NormFinder and geNorm. The P450 gene CYP96A146

14

constitutively overexpressed in R plants compared to S plants, this overexpression in R

15

plants can be suppressed by malathion. Taken together, higher expression level of P450

16

genes, leading to higher tribenuron-methyl metabolism, appears to be responsible for

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metabolic resistance to tribenuron-methyl in R D. sophia plants.

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KEYWORDS: metabolic resistance, cytochrome P450 monooxygenases, Descurainia

20

sophia L., tribenuron-methyl, CYP96A146, gene expression

21


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INTRODUCTION

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Weed resistance is the consequence of weed evolutionary adaption to herbicide

24

selection, and can be categorized into target-site based resistance (TSR) and

25

non-target-site based resistance (NTSR). TSR is usually conferred by gene mutations

26

of target enzymes, leading to the reduction of herbicide binding ability. NTSR is

27

achieved by mechanisms of reducing herbicide concentration reaching the target-site,

28

which included mechanisms of enhanced herbicide metabolism and sequestration,

29

reduced penetration and translocation.1-3 As the most important NTSR, metabolic

30

resistance is usually caused by cytochrome P450 monooxygenases (P450s) ，

31

glutathione S-transferases (GSTs), glycosyltransferases (GTs) and ATP-binding

32

cassette (ABC) transporters.4-5

33

The P450s are a superfamily of heme-containing enzymes involved in both

34

anabolic and catabolic pathways, and can catalyze kinds of plant reactions including

35

hydroxylations, epoxidations, dealkylations, isomerizations, decarboxylations and

36

deaminations.4,6-7 As metabolic enzymes, P450s in plants play important roles in

37

herbicide resistance which have been reviewed in details elsewhere.1,4,8 Most

38

evidences on P450s involvement in herbicide resistance were indirect and mainly

39

obtained by the synergism of P450 inhibitors9-10 or other indirect ways including

40

RNA-seq.11-13 Synergisms of P450 inhibitors only demonstrated the participation of

41

one or more P450s in resistance, but fail to offer any information regarding specific

42

P450. The RNA-seq easily identified a large number of differentially expressed contigs,

43

while it is time-consuming and labor-intensive process to eliminate ‘false positive’, to



44

clone full length genes and validate the roles of candidate alleles in herbicide

45

resistance. Moreover, it is very difficult to purify individual P450 or clone randomly

46

specific P450 alleles conferred herbicide resistance from so much potential

47

candidates.8,14 Hence, there was little direct evidence on the individual P450

48

involvement in herbicide resistance.

49

Descurainia sophia L. is one of the most notorious weed infesting winter wheat in

50

China, and evolved extremely high resistance to ALS-inhibiting herbicide

51

tribenuron-methyl. Our previous work has confirmed that TSR (ALS gene mutations)

52

and NTSR (enhanced metabolism) mechanisms were responsible for D. sophia

53

resistance to tribenuron-methyl.3,15-18 Resistance mutations were identified in positions

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197 (Pro substituted by Leu, Ser, Thr or Tyr), 376 (Asp to Glu) or 574 (Trp to Leu) in

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ALS of tribenuron-methyl-resistant D. sophia.15-18 The experiments of P450 inhibitor

56

and RNA-seq also demonstrated that P450 and other metabolic enzymes may mediate

57

the NTSR in D. sophia. For example, P450 inhibitor malathion greatly reversed the

58

tribenuron-methyl resistance in D. sophia, which indicated that one or more P450s

59

could be involved in resistance to tribenuron-methyl. In addition, up-regulation of four

60

P450s (CYP96A146, CYP96A147, CYP96A15-like, CYP71A1-like), three GTs and

61

one ABC transporter were identified and confirmed in resistant D. sophia by RNA-Seq

62

and qPCR.3 The CYP96A146 and CYP96A147 genes were completely new P450 gene

63

and significantly up-regulated in R D. sophia. However, these evidences on metabolic

64

enzymes involving in NTSR are indirect. Yet, identifying NTSR genes is very

65

important for understanding, diagrnosing and managing herbicide resistance. Given the

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66

importance and the originality, we expect to identify the direct evidences on P450s

67

involvement in tribenuron-methyl-resistant D. sophia by a series of experiments. These

68

experiments aim to (1) compare absorption and metabolism rates of tribenuron-methyl

69

in susceptible (S) and resistant (R) D. sophia during different periods by LC-MS

70

analysis; (2) study the effects of P450 inhibitor malathion on absorption and

71

metabolism rate of tribenuron-methyl in S and R plants; (3) assess reference gene

72

expression stability in S and R plants, before or after pesticide application; (4)

73

investigate the different expression of P450 genes in S and R plants before and after

74

tribenuron-methyl treatment; (5) investigate the impacts of P450 inhibitor malathion on

75

tribenuron-methyl-induced expression of P450 genes in S and R plants.

76 77

MATERIALS AND METHODS

78

Plant materials. Seeds of the resistant (R) population N11 were collected from

79

winter wheat fields at Baoding of Hebei province in China (N38°36’32.80’’,

80

E115°01’52.50’’), where tribenuron-methyl was using for controlling D. sophia more

81

than twenty years. The susceptible (S) population SD8 was collected from roadsides at

82

Linyi

83

tribenuron-methyl or other herbicides had been applied. In order to minimize the

84

differences of genetic background, SD8 and N11 were purified by individual plant

85

reproduction and genotyping according to the methods described in previous

86

research.18

87

in

Shandong

province

(N35°05’45.00’’,

E118°09’3.78’’),

where

no

D. sophia seeds were immersed in 20% H2O2 and rinsed with distilled water 30



88

min later, then soaked in 0.3 % gibberellin solution stay overnight in 4 °C. The seeds

89

were rinsed with distilled water and placed in climate chamber to germinate under

90

conditions of 25 °C/22 °C day/night temperatures, 16 h photoperiod with light intensity

91

of 20,000 Lux. Germinating seedlings were transported into 9-cm diameter plastic pots

92

containing moist loam soil, and kept in climate chamber. When the plants grow up to

93

4-leaf period, the seedlings were thinned to 2 plants per pot.

94

Tribenuron-methyl or malathion treatment. D. sophia at stage of 5 to 6-leaf (50

95

days after transplant) was treated by tribenuron-methyl in the absence and presence of

96

malathion. Malathion at concentration of 1200 mg L-1 was applied 60 min prior to

97

tribenuorn-methyl treatment using a moving-boom cabinet sprayer delivering 600 L

98

ha-1 water at a pressure of 0.4 MPa by a flat fan nozzle. Total 15 µL tribenuron-methyl

99

solution with concentration of 20 mg L-1 (dissolving in acetone) was applied on leaf

100

surface of each plant by a micro applicator (Hamilton PB 600 dispenser, Hamilton Co.,

101

USA). In total, 130 S and 130 R plants (40 for LC-MS analysis and 90 for qPCR) were

102

used for tribenuron-methyl treatment and tribenuron-methyl plus malathion treatment,

103

respectively. After herbicide application, plants were returned to climate chamber with

104

the same conditions as above.

105

Tribenuron-methyl extraction and clean up. In order to compare the uptake of

106

tribenuron-methyl in S and R D. sophia plants, the residual tribenuorn-methyl on leaf

107

surface was determined. Above-ground parts of S and R plants were harvested at 0, 1,

108

3, 5 and 7 days after treatment (DAT) with tribenuron-methyl respectively. For each

109

time point, 2 plants were harvested as one replicate per population, and total four

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110

replicates were applied. The residual tribenuron-methyl on surface of two D. sophia

111

plants was washed with 10 mL acetonitrile solution containing 1% acetic acid. The

112

elution solution was filtrated by 0.22 µm syringe filter, and tribenuron-methyl in the

113

solution was quantified by LC-MS (Shimadzu LCMS-8030, Japan).

114

To analyze the metabolic differences of tribenuron-methyl in S and R D. sophia

115

plants, tribenuron-methyl in S and R plants was extracted and quantified following the

116

QuEChERS method (AOAC Official Method 2007.01). Above-mentioned plant tissues

117

after washing off tribenuron-methyl were used for tribenuron-methyl metabolism

118

determination. The plant tissues (about 0.3g) were ground into powder with liquid N2,

119

and transferred into 50 mL centrifuge tubes. The homogenate was sonicated for 10 min

120

after adding 5 mL acetonitrile with 1% acetic acid (v/v). After adding 0.5 g NaCl, the

121

homogenate was shaken vigorously for 2min and centrifuged at 5000 rpm for 5 min.

122

Then 1 mL of supernatant acetonitrile layer was transferred into a 2 mL centrifuge tube

123

and vortexed for 1 min after adding 25 mg primary secondary amine (PSA, 40-60 µm),

124

7 mg graphitized carbon black (GCB, 120-400 mesh) and 50 mg MgSO4. The extract

125

was centrifuged at 12000 rpm for 3 min. Finally, 1 mL of the supernatant was filtered

126

into an autosampler vial with 0.22 µm syringe filters and then analyzed by LC-MS

127

without further cleanup. The followed results indicated more than 90%

128

tribenuron-methyl were extracted from R and S plants.

129

LC-MS analysis. The separation and quantitation of tribenuron-methyl was

130

conducted by LC-MS with a SHIMADZU HPLC packed colum Shim-pack XR-ODS

131

II (75 mm × 2.0 mm i.d., 2.2 µm) maintained at 30 °C. The mobile phase was



132

composed of 35% water with 0.1% formic acid (v/v) and 65% methanol, and the flow

133

rate was 0.2 mL min-1. The injection volume was 2 µL and total run time was 2.5 min.

134

The MS run at conditions of DL temperature of 250 °C, heat block temperature of

135

400 °C, nebulizing gas flow of 3.0 L min-1, and drying gas flow of 15.0 L min-1. The

136

multiple reaction monitoring mode (MRM) for tribenuron-methyl was optimized at

137

396.0 > 155.0 under the above conditions.

138

LC-MS analysis method validation. Linearity, recovery, precision were evaluated

139

to ensure the quality of the analytical method. Linearity was determined by injecting

140

tribenuron-methyl working standard solutions at concentration of 0.005, 0.01, 0.02,

141

0.05 and 0.1 mg L-1 followed by linear regression analysis. Precision was calculated as

142

relative standard deviation (RSD) from recovery studies with standard-spiked samples

143

(n=4) at levels of 0.016, 0.16 and 0.8 mg L-1. Both the spiked and unspiked samples

144

were extracted, cleaned up and subjected to LC-MS analysis.

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145

RNA extraction and cDNA synthesis. Total RNA extraction was performed using

146

RNApre Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s

147

instructions. Pooled samples with leaves from 6 individual plants were used for RNA

148

extraction. Nucleic acid concentration was measured at 260 nm using a DeNovix

149

DS-11 spectrophotometer (DeNovix, USA). RNA with A260/A280 and A260/A230

150

absorption ratio values of 1.8 - 2.2 and 2 - 2.2 can be used for cDNA synthesis. RNA (1

151

µg) of each sample was used for first-strand cDNA synthesis using the FastQuant RT

152

Kit (Tiangen, Beijing, China).

153

qPCR programs. qPCR was carried on the ABI 7500 real time PCR system


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154

(Applied Biosystems, Foster city, USA) with SuperReal PreMix Plus (SYBR Green)

155

(Tiangen, Beijing, China) according the MIQE criteria.19 Reactions were conducted in

156

a 20 µL volume (10 µL 2 × SuperReal PreMix Plus, 1 µL 6-fold diluted cDNA, 0.6 µL

157

primers, 0.4 µL 50 × ROX reference dye, and 7.4 µL RNase-free ddH2O) with four

158

replicates for each cDNA sample. qPCR programs consisted of 15 min incubation at

159

95 °C, 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 32 s. At the end of the

160

amplification cycle, a melting analysis was carried out to verify the absence of

161

non-specific amplification. Assessment of qPCR efficiency was performed using five

162

points with four-fold cDNA dilution series. Data was analyzed with 7500 Software

163

v2.3 (Applied Biosystems, Foster city, CA, USA).

164

Expression stability analysis of candidate reference genes. Gene expression

165

stability was assessed in plants resistant or sensitive to tribenuron-methyl, subjected or

166

not to pesticide (tribenuron-methyl or malathion) stress. The candidate reference genes

167

were 18S rRNA, GAPDH, UBC, ACT7, SAND family, TIP41-like, F-box family and

168

ACT2, which were proved stable in D. sophia20 or A. thaliana.21,22 The primer

169

information was list in Table 1. For every candidate reference gene, the mean

170

quantification cycle (CT) value of every RNA sample from each of the two samplings

171

were used for stability analysis. The expression stability of each candidate reference

172

gene was comprehensively assessed using programs of BestKeeper, NormFinder and

173

geNorm.23-25

174

Effect of tribenuron-methyl on P450s expression with or without malathion.

175

The information for primers and expected amplicon of each P450 gene were given in



Page 10 of 33

176

Table 2. Relative expression levels of P450 genes were measured before (BT) and 1, 3,

177

5, 7 days after tribenuron-methyl or malathion treatment (DAT), which was the same

178

collection time as tribenuron-methyl uptake and metabolism experiments.

179

Relative expression ratio (as 2-△△CT) was calculated by the comparative CT

180

method,26 where △CT = [CT target gene-CT mean of two internal control genes]. Three

181

biological replicates and four technical replicates were performed for each time point

182

of S and R populations.

183

Statistical analysis. Data of tribenuron-methyl uptake and metabolism was analyzed

184

by independent-samples t-test (P < 0.05). Data of gene expression levels was subjected

185

to One-way analysis of variance (ANOVA) with Duncan test (P < 0.05).

186 187

RESULTS

188

Validation of LC-MS analysis method. The retention time of tribenuron-methyl

189

was 1.36 min. The determination coefficient (R2) of linear curve was 0.9998. Recovery

190

was 107.3%, 105.3% and 99.7% with an RSD of 1.66%, 1.26% and 1.08% at addition

191

level of 0.08, 0.8 and 4 µg, respectively. These indicated that the LC-MS analysis

192

method was appropriate for determination of residual tribenuron-methyl in D. Sophia.

193

Foliar uptake of tribenuron-methyl by S and R plants with or without

194

malathion treatment. The reduced dose between applied on and washed off leaf

195

surface was considered as the tribenuron-methyl uptaked by leaves of S or R D. sophia

196

plants. The uptake of tribenuron-methyl by S and R plants displayed no significant

197

differences at 0, 1, 3, 5 and 7 DAT without malathion treatment. The average


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198

absorption percentage by S and R plants was about 9.0% (0 DAT), 34.2% (1 DAT),

199

52.2% (3 DAT), 60.9% (5 DAT) and 66.4% (7 DAT) (Figure 1A).

200

Compared with treatment only by tribenuron-methyl, malathion significantly

201

increased the average tribenuron-methyl absorption percentage by S and R plants about

202

1.3- to 2.0-fold during the testing periods. Nevertheless, the average tribenuron-methyl

203

absorption percentage between S and R plants exhibited no significant differences at

204

the same time point (Figure 1A).

205

Tribenuron-methyl metabolism in S and R plants with or without malathion

206

treatment. Tribenuron-methyl metabolism was calculated by the differences of

207

tribenuron-methyl absorption and residues in plants. In absence of malathion, the R

208

plants metabolizes tribenuron-methyl significantly faster than did the S plants at 3, 5

209

and 7 DAT. The metabolism percentage of tribenuron-methyl in R plants was 65.7%,

210

78.9% and 87.8% at 3, 5, 7 DAT respectively, which was significant higher than that of

211

56.9%, 70.3% and 72.6% in S plants (Figure 1B).

212

By contrast, malathion significantly reduced the tribenuron-methyl metabolism

213

both in S and R plants. The malathion decreased the tribenuron-methyl metabolism

214

percentage about 5.2-, 2.4-, 2.0- and 1.2-fold in R plants, and 2.2-, 1.9-, 1.8- and

215

1.4-fold in S plants at 1, 3, 5, and 7 DAT, respectively. The tribenuron-methyl

216

metabolism in S and R plants showed no significant difference at 1, 3 and 5 DAT,

217

when treated with malathion. Therefore, the tribenuron-methyl metabolism in R plants

218

was 1.1-fold higher in R plants than did S plants at 7 DAT (Figure 1B).

219

Selection of reference genes with stable expression. All eight candidate reference



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220

genes displayed high specificity with single-peak dissociation curves (Figure S1), and

221

good amplification efficiency (94% to 104%) (Table 1). The results of three

222

complementary approaches (BestKeeper, NormFinder and geNorm) showed that all

223

candidate genes were found suitable for normalization in both of tribenuron-methyl

224

(sampling Tri) and tribenuron-methyl plus malathion (sampling Tri+Mal) treatment

225

plants (SD

Metabolic Resistance to Acetolactate Synthase Inhibiting Herbicide

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