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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
Journal of Agricultural and Food Chemistry
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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
17
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
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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
54
197 (Pro substituted by Leu, Ser, Thr or Tyr), 376 (Asp to Glu) or 574 (Trp to Leu) in
55
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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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
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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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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
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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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(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
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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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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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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