Metabolic resistance to acetolactate synthase (ALS)-inhibiting

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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]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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INTRODUCTION

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Weed resistance is the consequence of weed evolutionary adaption to herbicide

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

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

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

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

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which included mechanisms of enhanced herbicide metabolism and sequestration,

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

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

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

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cassette (ABC) transporters.4-5

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

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

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hydroxylations, epoxidations, dealkylations, isomerizations, decarboxylations and

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

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

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evidences on P450s involvement in herbicide resistance were indirect and mainly

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

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

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

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

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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

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

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specific P450 alleles conferred herbicide resistance from so much potential

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

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involvement in herbicide resistance.

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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

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

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

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

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Linyi

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

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

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reproduction and genotyping according to the methods described in previous

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research.18

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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the same conditions as above.

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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tribenuron-methyl were extracted from R and S plants.

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

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

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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

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

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

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

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

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396.0 > 155.0 under the above conditions.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Kit (Tiangen, Beijing, China).

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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)

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

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

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

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

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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

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

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

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

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

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Expression stability analysis of candidate reference genes. Gene expression

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

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

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

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

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

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

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

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gene was comprehensively assessed using programs of BestKeeper, NormFinder and

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geNorm.23-25

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

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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,

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

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collection time as tribenuron-methyl uptake and metabolism experiments.

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

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

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

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of S and R populations.

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

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

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

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RESULTS

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

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

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

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

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

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

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

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

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

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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),

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

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Compared with treatment only by tribenuron-methyl, malathion significantly

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

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

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

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the same time point (Figure 1A).

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

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

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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

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

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

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

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By contrast, malathion significantly reduced the tribenuron-methyl metabolism

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

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

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

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

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

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

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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

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

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

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

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

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plants (SD