Biodegrading two pesticide residues in paddy plant and environment

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Food Safety and Toxicology

Biodegrading two pesticide residues in paddy plant and environment by a genetically engineered approach Xiang Ning Su, Jing Jing Zhang, Jintong Liu, Nan Zhang, Li Ya Ma, Feng Fan Lu, Zhao Jie Chen, Zhan Shi, Wen Jing Si, Chang Liu, and Hong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07251 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Journal of Agricultural and Food Chemistry

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Title: Biodegrading two pesticide residues in paddy plant and environment by a genetically

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engineered approach

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Running head: Paddy plants enhanced degradation of pesticides

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Authors: Xiang Ning Sua,b, Jing Jing Zhangc, Jin Tong Liua, Nan Zhanga, Li Ya Maa, Feng

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Fan Lua, Zhao Jie Chena,b, Zhan Shia, Wen Jing Sia, Chang Liua, Hong Yanga,b*

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Institute: aJiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing

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Agricultural University, Nanjing 210095, China; bState & Local Joint Engineering Research

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Center of Green Pesticide Invention and Application, Nanjing Agricultural University,

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Nanjing 210095, China; cCollege of Plant Protection, Henan Agricultural University,

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Zhengzhou 450002, China

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Mailing address: Weigang No.1, Chemistry Building, College of Sciences, Nanjing

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Agricultural University, Nanjing 210095, China

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*Corresponding author: Hong Yang

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Telephone number: +86-25-84395204

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Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Accumulating pesticide (or herbicide) residues in soils become one of the seriously

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environmental problems. This study focused on identifying the removal of two widely-used

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pesticides isoproturon (IPU) and acetochlor (ACT) by a genetically developed paddy (or rice)

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plant overexpressing an uncharacterized glycosyltransferase (IRGT1). IRGT1 conferred plant

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resistance to isoproturon/acetochlor, which was manifested by attenuated cellular injure and

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alleviated toxicity of rice growth under isoproturon/acetochlor stress. A short-term study

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showed that IRGT1-transformed lines removed 33.3−48.3% and 39.8−53.5% from the growth

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medium and remained only 59.5−72.1% and 58.9−70.4% isoproturon and acetochlor in plants

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compared to untransformed rice, respectively. This phenotype was well confirmed by

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IRGT1-expression in yeast (Pichia pastoris) which grew better and contained less

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isoproturon/acetochlor

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isoproturon/acetochlor concentrations at all developmental stages were significantly lower in

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the transformed rice containing only 59.3-69.2% (isoproturon) and 51.7-57.4% (acetochlor) of

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those in wild-type. In contrast, UPLC/Q-TOF-MS/MS analysis revealed that more

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isoproturon/acetochlor metabolites were detected in the transformed rice. Sixteen metabolites

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of isoproturon (19 for acetochlor) for Phase I reaction and 9 isoproturon conjugates (13 for

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acetochlor) for Phase II reaction in rice were characterized, of which 7 isoproturon and 6

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isoproturon metabolites or conjugates were reported in plants for the first time.

than

control

cells.

A

long-term

study

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KEYWORDS: Phase II component; isoproturon; acetochlor; rice; degradation

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showed

that

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INTRODUCTION

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Environmental concern about accumulative pesticide (or herbicide) residues in soils is rising.

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Many types of pesticides do not vanish during the current season of their application. They

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may accumulate in soils from season to season or year to year, leading to the great

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environmental challenge to the successive crop production. Isoproturon is a phenylurea

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herbicide widely used for weeding in the farmland across China, Europe and other areas of

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the world.1,2 Recent studies show that isoputruon is detected in surface and ground water and

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its concentration exceed the limit level of 0.1 μg L−1 EU drinking water (WHO and European

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Union).3,4 Isoproturon is highly toxic to aquatic organisms and potentially carcinogenic, and

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has been categorized into the endocrine disruptor that impairs the fertility system of

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mammals.5 Acetochlor is another worldwide applied herbicide for pre-emergence control of

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grasses or weeds.6,7 In China and United States, more than 10 million kilogram of acetochlor

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has been used annually since 1997, making its residues ubiquitously detectable.8 Acetochlor is

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water-soluble, and it migrates in soils and runs off to streams, lakes and rivers. The U.S.

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Environmental Protection Agency classified acetochlor as a B-2 carcinogen and suspected

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endocrine disrupters.9 Because isoproturon and acetochlor residues are medium-to-long-term

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persistent depending on soil types and environmental conditions,10 developing strategies for

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facilitating environmental removal and degradation of isoproturon/acetochlor in crops is

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critical not only for bioremediation and safe crop production as well.

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The use of plants or other organisms to phytoextract and phytodegrade organic

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contaminants for environmental remediation is an appealing strategy and represents a

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low-cost and sustainable solution to environmental improvement.11,12 Plants have the ability

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to remove and catabolize surrounding organic xenobiotics by root systems through

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multi-pathways or specific mechanisms involved in a variety of genes encoding detoxifying

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enzymes or proteins.13 One of them is the glycosyltransferases that effectively neutralize the 3



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toxic effect of organic contaminants by transformation of these foreign compounds.14-17

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Recent studies have shown that the pesticide degradation can be facilitated by glycosylation

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in plants.18-20 UDP-glycosyltransferases from Arabidopsis were shown to detoxify

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deoxynivalenol.15,21 Furthermore, several types of genes encoding oxidoreductases, laccases,

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hydrolases P450 and peroxidases actively participate in the pesticide metabolism in

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plants.22-25 Those metabolic enzymes provide valuable resources to develop engineered plants

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for bioremediation.

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The widespread contamination of soil pesticides makes it difficult for crops to avoid

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absorbing these compounds. In this case, selecting the plants (or crops) that are naturally

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gifted with special mechanisms for high uptake but lower accumulation of pesticides is

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critically important. Rice (Oryza sativa) is an excellent plant species for environmental

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research owning to its abundant germplasm resources. Many genotypes or cultivars bear

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special traits that contribute to the strong adaption to various environmental stresses including

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those to eliminate organic contaminants from soil, water and plant.18,19,26 We previously

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isolated a group of Phase II reaction genes encoding glycosyltransferases responding to

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pesticides,24 but the environmental significance relevant to the degradation of pesticides is yet

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to be investigated. In this study, we investigated the capability of a genetically engineered rice

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plant which expressed an uncharacterized glycosyltransferase gene (here referred as

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ISOPROTURON-RESPONSIVE GLYCOSYLTRANSFEARSE1, IRGT1, LOC_Os10g40640),

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to remove isoproturon and acetochlor from plant growth medium and degrade the two

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pesticides. We further identified their transformation pathways by characterizing metabolites

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and degradation products. Our work may help understand the mechanisms by which the rice

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plants extract the pesticide residues from its surroundings and catabolize the residues in rice.

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MATERIALS AND METHODS

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Rice Growth Conditions

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The uniformly rice (Oryza sativa. Japonia. cv. Nipponbare) seeds were sowed on a net

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floating in the 1/2 strength Hoagland nutrient solution and grown in a growth chamber under

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the condition of 30/25 °C (day/night), 200 μmol m-2 s-1 illumination with a photoperiod of

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14/10 h (light/dark) for 7 days.18 Practically, the field application dosage of isoproturon is

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under 3.48 mg/kg and acetochlor is 0.52-0.98 mg/kg.5,27 Plants were exposed to 0.05 and 2

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mg/L isoproturon (96.9% purity) or 0.005 and 0.1 mg/L acetochlor (92% purity), which are

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between the environmentally realistic concentrations and the maximum application dosage in

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the field. All growth media and containers were thoroughly sterilized prior to use. Plants in

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the short-term experiments were exposed to 2 mg/L isoproturon or 0.1 mg/L acetochlor

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(below the maximum application dosage) for 6 d. In the long-term experiments plants were

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exposed to 0.05 mg/L isoproturon or 0.005 mg/L acetochlor (environmentally realistic

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concentrations) for 30, 45, 60, 90, 100 and 120 d. The growth and treatment solutions were

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changed every two days for both experiments.

116 117

Transcript Analysis by qRT-PCR

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Total RNA was extracted using Trizol (Invitrogen). One μg of RNA was incubated with 1

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unit of RNase-free DNase I (Takara) at 37 °C for 30 min. The reverse transcription reaction

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was initiated with a kit (Beijing TransGen Biotech). The resultant cDNA was diluted 5-fold

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for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.28 Total

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reaction mixture (20 μL) contained 1 μL template cDNA, 10 μL of 2 × SYBR Premix Ex Taq

123

(TaKara), 200 nM primers and 9 μL ddH2O (SI Table S1) and was subjected to following

5



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reaction: 1 cycle of 94 °C for 30 s for denaturation, 40 cycles of 95 °C for 5 s and 60 °C for 30

125

s for annealing and extension.

126 127

Development of IRGT1 Transformed Rice

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The PCR-prducts IRGT1 were digested at Bgl II and Spe I (SI Table S1) and inserted into the

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corresponding sites of pCAMBIA1304 with CaMV35S as a promoter. The constructed vector

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was introduced into Agrobacterium tumefaciens EHA105.29 The embryonic callus of rice was

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induced by mature embryo infected by A. tumefaciens. More than twenty 35S::IRGT1

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transgenic lines (T3 homozygotes) were developed. Three lines were randomly selected for

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functional identification.

134 135

Assessment of Physiological Responses

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Chlorophyll in fresh leaves (0.1 g) was extracted with 8 mL of 80% acetone (pH 7.8). The

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extracting solution was centrifuged at 5,000 g for 10 min, and the supernatant was used to

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determine the content of chlorophyll.30 Fresh tissues (0.1 g) were cut into 5 mm length and

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immersed in tubes with 20 mL deionized water for incubating at 30 °C for 2 h. The sample

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medium (EC1) was measured using an electrical conductivity meters. The samples were

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boiled at 100 °C for 30 min and cooled down to 25 °C. The final conductivity (EC2) of the

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solution was determined.22 Fresh tissues (0.5 g) were ground and dissolved in 3 mL of 0.67%

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(w/v) trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 10,000 g for 10

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min. Two mL of the supernatant was mixed with 2 mL of 0.5% thiobarbituric acid (TBA) in

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20% TCA. The mixture was remained in boiling water for 30 min, cooled to the room

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temperature immediately and centrifuged at 12,000 g for 10 min. The supernatant absorbance

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was measured at 450, 532 and 600 nm respectively. 22

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Assay of Glycosyltransferase Activity

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Plant samples (0.5 g) were ground and extracted with 50 mM NaCl, 1 mM EDTA, 1% (w/v)

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polyvinylpyrrolidone in a 50 mM Tris-HCl (pH 8.0) solution. The homogenate was

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centrifuged at 8,500 g and 4 °C for 30 min. The supernatant (100 μL) was mixed with 0.04

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mM p-nitrophenol and 2 mM UDP-glucose and incubated at 30 °C for 2 h.17 The mixture was

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filtered through a 0.22 μm nylon membrane and detected by high performance liquid

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chromatography (HPLC) (Waters 515) under the following condition: Hypersil reversed

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phase C18 column (Thermo, 250 mm × 4.6 mm i.d.) and mobile phase methanol: water (3:2;

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v/v) mobile phase, 0.6 mL min-1 flow rate and 317 nm wavelength. One unit of the GTs

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activity was defined as the consumption of 1 μmol p-nitrophenol per minute under detection

159

condition.

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Determination of Isoproturon/Acetochlor Accumulation in Rice

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Fresh tissues were separately harvested depending on experiments required. The shoot,

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root and grain tissues were ground to powder using liquid nitrogen. The samples were

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extracted ultrasonically with 25 mL of mixed acetone-water (3:1, v:v) for 30 min. The extract

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was centrifuged at 4,000 g for 8 min and the supernatant was collected. The step was repeated

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in triplicate. The supernatant was concentrated to remove acetone in a vacuum rotary

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evaporator at 40 °C. The remaining water was placed onto the LC-C18 solid phase extraction

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(SPE) column. The SPE column was washed with 4 mL methanol. Eluents were collected.

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The content of isoproturon was determined by HPLC under the condition: Hypersil reversed

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phase C18 column (Thermo, 250 mm × 4.6 mm i.d.); mobile phase, methanol: water (68:32,

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v:v), flow rate, 0.6 mL/min and ultraviolet (UV) detection at a wavelength of 241 nm. 17

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For acetochlor, 5.0 g of rice sample powder was ultrasonic-extracted with 40 mL

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dichloromethane for 1 h. The supernatant was filtered through a filter, which was washed with 7



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20 mL dichloromethane twice. The extracts were collected, combined and concentrated to 0.5

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mL by rotary evaporation. The left 0.5 mL solution was added to the Florisil SPE column,

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which was washed with 10 mL dichloromethane. The eluent was collected and concentrated

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in a vacuum rotary evaporator at 35 °C. The residue was dissolved in 2 mL chromatographic

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n-hexane. The mixture was passed through a 0.22 μm polytetrafluoroethylene filter membrane

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for analysis by gas chromatography (GC) (System 7820A, Agilent) with electron capture

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detector (ECD) under the condition: capillary column (internal diameter: 0.25 mm, film

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thickness: 0.25 mm); column and detector temperature, 230 °C; injection port temperature,

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250 °C; carrier nitrogen gas (99.999% purity) and constant flow, 0.6 mL min-1. 7

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The isoproturon/acetochlor concentrations in growth medium were measured by

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purifying 10 mL solution through LC-18 SPE column. The column was washed with

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methanol. The eluent was collected. The concentration of isoproturon was determined as

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indicated above. For acetochlor, the washed methanol was collected and concentrated to dry.

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The residue in flask was dissolved with 4 mL chromatographic n-hexane for GC analysis. The

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spiked recoveries of determination of isoproturon/acetochlor and acetochlor in rice tissues and

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nutrient solution were shown in SI Table S2.

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Subcellular localization of IRGT1

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The IRGT1 cDNA was inserted into pCAMBIA 1305-GFP vector with a 35S promoter. The

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IRGT1-GFP fusion vector was transformed with the ER-marker into tobacco leaves.31 The

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fluorescence was detected using the confocal laser scanning microscopy (Confocal

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System-UitraView VOX, Perkin Elmer).

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Characterization of Isoproturon/Acetochlor Metabolites in Rice

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Analysis of isoproturon/acetochlor and their metabolites in rice was performed by a Shimadzu

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LC 20ADXR connected to an AB SCIEX Triple TOF 5600 mass spectrometer. The condition

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of UPLC was set up as follows: the injection volume, 20 μL; autosampler temperature, 4 °C;

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Poroshell 120 EC-C18 column (Aglient). Mobile phase for isoproturon contained solvent A

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(water + 0.1% formic acid) and solvent B (acetonitrile). A linear gradient program of mobile

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phase was performed for 25 min at a flow rate of 0.3 mL/min under the following conditions:

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(1) 5% B for 1 min, 1-5 min from 5%-15% B, 5-20 min from 15%-35% B, 20−22 min from

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35%-100% B, and 100% B for 3 min, (2) Back to the initial conditions, and (3) equilibrating

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for 1 min before the next sample injection. Mobile phase for acetochlor consisted of solvent A

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(acetonitrile + 0.1% formic acid) and solvent B (H2O + 0.1% formic acid) at a flow rate of 0.3

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mL/min, and the linear gradient program used as 40% A from 0 to 11.0 min, 100% A from

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11.1 to 20.0 min and 40% A from 20.1 min to 30.0 min. The mass spectrometer (MS) was

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operated in the positive product ion mode. TOF-MS parameters included ion source gas 1, 65

211

psi; ion source gas 2, 65 psi; curtain gas, 30 psi; source temperature, 550°C; and ionspray

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voltage floating, 5500 V. The APCI positive calibration solution for calibration delivery

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system (UPLC-Triple TOF 5600) was employed to ensure a working mass accuracy of < 5

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ppm.19 Atrazine was used as an internal standard. MultiQuant and software Peak View

215

softwares were used for data processing.

216 217

Transformation of IRGT1 in Yeast Cells

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The pPICZaC-IRGT1 plasmid construct was inserted into yeast (Pichia pastoris strain

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X-33).32 Twenty-five positive colonies were cultivated in 50 mL of Buffered Glycerol

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Complex Medium at 30 °C, shaking at 200 rpm for 48 h until the OD600 reached 2.0. Methanol

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was added to the culture to a final concentration of 1% every 24 h.23,24 The yeast cells were

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transferred to the fresh YPD medium supplemented with 0-8 mg L−1 isoproturon or 0-0.5 mg 9



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L−1 acetochlor for treatment. The harvested cells were dried and dissolved in 2 mL methanol

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for HPLC (isoproturon) analysis or dissolved by 2 mL n-hexane for GC (acetochlor) analysis.

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The recoveries of isoproturon/acetochlor in yeast were summarized in SI Table S3.

226 227

Statistical Analysis

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All experiments were independently performed at least in triplicate. Data were expressed

229

standard deviation. The statistical significance of each result was assessed by the variance

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post hoc test (ANOVA, Tukey’s test) at the 95% confidence level. Statistical analysis was

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performed using SPSS 22.

232 233 234

RESULTS

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IRGT1 Transcripts Were Increased in Rice Exposed to Isoproturon and Acetochlor

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The length of IRGT1 DNA sequence in rice genome is 4067 bp, with 5 exons interrupted by 4

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introns (SI Figure S1A). IRGT1 encodes a putative glycosyltransferase with 492 amino acids

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(SI Figure S1B-D). To identify IRGT1 expression in rice, the wild-type plants were exposed

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to isoproturon at 0−8 mg/L for 4 d, and qRT-PCR was used to assess the transcripts. In shoots,

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expression of IRGT1 was progressively induced under isoproturon stress; compared to the

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control, a 3.8-6.0 fold increase in transcripts was detected (Figure 1A). In roots, treatments

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with lower levels of isoproturon upregulated the IRGT1 expression, while the higher level

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reduced the transcripts (Figure 1A). A similar expression pattern was observed for acetochlor

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(Figure 1B).

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To better understand the role of IRGT1 in plant cells, we examined its subcellular

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localization by generating a recombinant plasmid connecting the green fluorescent protein

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(GFP) to IRGT1. The IRGT1-GFP fusion was transformed and expressed in the tobacco leaf 10


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epidermal cells.30 Several subcellular localization markers were tested and only the

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endoplasmic reticulum (ER) marker did work. The clear signals of co-localization of the

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fusion proteins and marker around the ER area were probed (Figure 1C-E), indicating that the

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IRGT1 was localized to ER.

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IRGT1 Overexpression Improved Rice Growth and Reduced isoproturon/acetochlor

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Toxicity

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To identify the role of IRGT1 in mediating rice resistance to isoproturon/acetochlor, the

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IRGT1 DNA fragments were genetically transformed into rice (see M&M). qRT-PCR

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analyses revealed that three transgenic lines expressed 14.1 to 40.3 fold higher transcripts of

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IRGT1 than untransformed wild-type (SI Figure S2). Compared to the wild-type, the

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transgenic lines overexpressing (OE) IRGT1 significantly enhanced glycosyltransferase

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activities in roots and shoots (Figure 1F, G), indicating that the transformed lines were

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reliable for the following study.

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We used the overexpressing lines to measure elongation and biomass of rice under

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isoproturon/acetochlor exposure. There were no significant differences of the growth between

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the wild-type and overexpressing lines without isoproturon (Figure 2A-C). In the presence of

265

2 mg/L isoproturon, the growth of the overexpressing lines appeared better than that of

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wild-type (Figure 2A-C). Like isoproturon, the overexpressing lines had elevated growth

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under 0.1 mg/L acetochlor stress. The shoot elongation of the overexpressing lines was

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28.5−41.5% greater than wild-type (Figure 2D-E). Chlorophyll is an important growth

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biomarker commonly used for responding to environmental xenobiotics.30,33 Under

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isoproturon stress, the chlorophyll concentration increased in the transgenic lines compared to

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wild-type (Figure 3A). The increased chlorophyll concentration was also found in

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overexpressing lines under acetochlor (Figure 3B), confirming that IRGT1 overexpression

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could confer the resistance to isoproturon/acetochlor toxicity in rice.

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The electrolyte leakage of plant reflects the damage of cellular membrane under

275

environmental stress.22 Compared to wild-type, the electrolyte leakage of the transformed rice

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was significantly reduced by 33.6−41.3% and 29.7−38.5%, respectively, under isoproturon

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and acetochlor stress (Figure 3C, D). The thiobarbituric acid reactive substances (TBARS)

278

level is also a biomarker representing the final lipid peroxides in response to toxic

279

compounds.33 Likewise, the thiobarbituric acid reactive substances concentration in the

280

overexpressing lines was reduced by 30.3−48.8% and 40.2−45.1%, respectively, under

281

isoproturon and acetochlor compared to the wild-type. (Figure 3E, F), indicating that IRGT1

282

overexpression was able to detoxify the isoproturon/acetochlor-induced toxicity.

283 284

IRGT1-Overexpressing

285

Isoproturon/Acetochlor from Growth Medium

286

We further determined the accumulation of isoproturon/acetochlor in the overexpressing and

287

wild-type plants. Both isoproturon and acetochlor concentrations in overexpressing lines were

288

significantly lower. For example, the former concentration in the shoots of overexpressing

289

lines had 59.5−67.0% of the wild-type, while the latter had 58.9−68.3% (Figure 4A, B). The

290

lower concentrations of isoproturon/acetochlor in overexpressing lines might be the result of

291

the restricted influx of the pesticides into rice or taking up more by the overexpressing lines.

292

To clarify it, we determined the isoproturon/acetochlor residues in the growth medium and

293

calculated their removal rates. It was estimated that the overexpressing lines removed

294

isoproturon and acetochlor by 33.3−48.3% and 39.8−53.5% over the wild-type, respectively

295

(Figure 4C, D). These results suggested that the reduction of isoproturon/acetochlor in the

Rice

Accumulated

Less

12


and

Extracted

More

Page 13 of 33


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overexpressing lines was most likely promoted by the uptake and degradation by the

297

overexpressing lines, rather than the restriction of isoproturon/acetochloruptake.

298

The role of IRGT1 in mediating the reduced accumulation of isoproturon/acetochlor in

299

rice was confirmed by the long-term experiment, in which young plants were exposed to 0.05

300

mg/L isoproturon or 0.005 mg/L acetochlor for 30 d (tillering stage), 45 d (maximum tiller

301

number stage), 60 d (panicle initiation), 90 d (florescence), 100 d (immature grain) and 120 d

302

(mature grain). Compared to wild-type, the overexpressing lines constantly accumulated less

303

isoproturon in shoots and roots during the entire developmental stage (Figure 4E-G; SI Figure

304

S3A-C). A similar result was found with acetochlor (Figure 4H-J; SI Figure S3D-F). For

305

example, the acetochlor concentrations in the immature grains of the three overexpressing

306

lines decreased by 36.7−43.9%, compared to the wild-type (SI Figure S3C, F); similarly, the

307

acetochlor concentrations in the mature grains of the transgenic lines decreased by 42.6−48.4%

308

(Figure 4G, J).

309 310

Expression of IRGT1 in Yeast Promoted Cell Detoxification of Isoproturon/Acetochlor

311

To verify the functional role of IRGT1 in rice resistance to isoproturon/acetochlor stress, the

312

pPICZαC-IRGT1 vector was transformed into the yeast strain. The transformants were

313

incubated in the YPD medium supplemented with isoproturon or acetochlor. Both empty

314

vector and IRGT1-transformed cells showed no difference in growth under normal condition

315

(Figure 5A, B). Upon isoproturon/acetochlor exposure, particularly at higher levels, the

316

IRGT1-transformed cells grew better than the control cells (Figure 5A, B). The accumulation

317

of isoproturon/acetochlor in the yeast cells was inspected. An extra degradation of

318

isoproturon/acetochlor in the transformed cells was detected, compared to the control cells

319

(Figure 5C, D). The percentage of isoproturon dissipation increased by 1.9 (24 h), 2.0 (48 h)

320

and 2.4 (72 h) folds and the acetochlor dissipation increased by 2.5 (24 h), 1.9 (48 h) and 2.2 13



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321

(72 h) folds, respectively (Figure 5C, D). These results confirmed that IRGT1 was able to

322

mediate isoproturon/acetochlor degradation.

323 324

IRGT1 Overexpression Promoted Formation of Isoproturon/Acetochlor-Degradation

325

Products

326

To confirm that IRGT1 overexpression reduced isoproturon and acetochlor in rice, the

327

isoproturon/acetochlor-degradation products were characterized using UPLC-Q-TOF-MS/MS.

328

Based on the high resolution MS datasets, we have successfully characterized 16 metabolites

329

and 9 conjugates of isoproturon (SI Table S4), along with 19 metabolites and 13 conjugates of

330

acetochlor from rice (SI Table S5). The details of extracted ion chromatograms and MS2

331

spectra of isoproturon/acetochlor metabolites and conjugates were presented in Figure S6-9.

332

While the reduced parent compounds isoproturon/acetochlor (m/z 207/270) were observed in

333

the overexpressing line, the increased isoproturon/acetochlor-degradation products were

334

detected (Figure 6A-D; SI Figure S10). The concentrations of 1,1-dimethyl-3-phenyl-urea

335

(m/z 165) in shoots and roots were 1.73 and 1.19 fold higher in the overexpressing line than in

336

the wild-type. The formation of m/z 299 in shoots, 237 and 281 in roots of the

337

isoproturon-exposed OE-3 line was two times or more than those of the wild-type. Also, the

338

relative content of glucosylated-metabolites of m/z 371, 385, 299 and 401 in shoots of the

339

isoproturon-exposed OE-3 line was significantly more than those of the wild-type. For

340

acetochlor, metabolites SEMEMA-H2O+Cys (m/z 355) were 1.63 times higher in the

341

overexpressing

342

glucosylated-metabolites of m/z 442, 356, 299 in shoots and 285, 299 and 400 in roots of the

343

acetochlor-exposed OE-3 line was significantly more than those of the wild-type. Of all

344

isoproturon/acetochlor metabolites, 9 isoproturon and 6 acetochlor metabolites were

345

characterized in plants for the first time (SI Table S4-5).

line

than

those

of

the

wild-type.

14


The

relative

content

of

Page 15 of 33


346

Based on the data of MS2 identifying degradation products, these isoproturon/acetochlor

347

metabolites can be classified into two mechanisms of biological transformation. There were

348

16 isoproturon metabolites undergoing Phase I reaction and 9 conjugates going through Phase

349

II reaction. For acetochlor, 19 metabolites and 13 conjugates went through Phase I and Phase

350

II reaction, respectively (Figure 7; SI Figure S11).

351 352 353

4. DISCUSSION

354

Elimination of environmental pesticide residues is critically important for crop production and

355

food

356

isoproturon/acetochlor removal from rice growth medium and intensified disappearance of

357

isoproturon/acetochlor in a genetically developed paddy plant. Our work provided an insight

358

into the mechanism underlying the isoproturon/acetochlor-degradation concerning the

359

catabolized

360

isoproturon/acetochlor stress. It also demonstrated the perspective regarding the potential

361

application of the strategy to phytoextraction and phytodegradation of pesticide-contaminated

362

soils and plants.11,13

safety.

This

products

study

and

highlighted

putative

the

pathways

environmental

in

plants

importance

when

of

challenged

the

to

363

Our data showed that IRGT1-overexpressing rice under isoproturon/acetochlor exposure

364

displayed attenuated toxic symptoms in term of decreased cellular injury and peroxidative

365

stress, with concomitant enhancement of total biomass, plant elongation and chlorophyll

366

concentration. The improved growth and physiological responses to isoproturon/acetochlor

367

stress were confirmed by the ectopic expression of IRGT1 in the yeast. The IRGT1-mediated

368

resistant trait was associated with low accumulation of isoproturon/acetochlor in rice. The

369

short-term study showed that the transformed rice had a lower level of isoproturon/acetochlor

370

in both plant tissues and growth medium. The reduced isoproturon/acetochlor concentration in 15



371

rice was most likely attributed to the intensified degradation of isoproturon/acetochlor, while

372

the low level of the pesticides in the root growth environment could be the result of more

373

isoproturon/acetochlor extraction by the rice plants.

374

The removal efficiency may have environmental implications in potential use of the

375

transformed rice growing on the pesticide-polluted soils, particularly in the wetland suitable

376

for rice growth.13 Compared to the short-term study, the long-term exposure led to the

377

constant uptake of isoproturon/acetochlor from environment and allocation to a wide range of

378

tissues or organs of rice. In this case, the transformed rice was also found to accumulate a

379

lower level of isoproturon/acetochlor throughout the life span, suggesting that the transgenic

380

rice had a strong ability to catabolize isoproturon/acetochlor in the plants. Due to the limited

381

land resources, crops such as rice and wheat must be planted in soils with mild pesticide

382

contamination,19 developing genetically engineered plants that effectively degrade pesticide

383

residues can be an efficient way to minimizing the environmental risks.

384

The biodegradation of pesticides in higher plants has been proposed to be three main

385

phases, conversion (Phase I), conjugation (Phase II) and compartmentalization (Phase III).13

386

In phase II, glycosylation is one of major processes that modify the xenobiotic molecules by

387

conjugating with sugars, glutathione or amino acids, thus making them easier for access to

388

degradation machinery.15 Because a lower concentration of isoproturon/acetochlor in the

389

transformed rice was detected, we inferred that the enhanced degradation would be associated

390

with enhanced accumulation of isoproturon/acetochlor metabolites in plants. Our analysis

391

showed that more isoproturon/acetochlor conjugates and other metabolites in the transformed

392

rice were detected (SI Table S4-5). By UPLC-Q-TOF-MS/MS analysis, we have

393

characterized a total of 25 isoproturon and 32 acetochlor metabolites; of these, 9 isoproturon

394

and 13 acetochlor conjugates were identified. Xenobiotics conjugation may occur in several

395

forms such as glucosyl, malonyl-glucosyl and acetyl-glucosyl moieties in plants.34 The 16


Page 16 of 33

Page 17 of 33


396

malonyl-glucosyl conjugation is favorable of transporting the conjugate into vacuoles through

397

anionic transporter, which is proposed to be a major pathway to xenobiotics

398

detoxification .13,19 Sixteen isoproturon-metabolites were generated through methylation,

399

demethylation, hydroxylation or hydrolyzation. Five of the components were introduced into

400

Phase II reaction and formed nine isoproturon-conjugated products (Figure 7). For example,

401

1-isopropenyl-IPU-N-malonylglucoside (m/z 471) was transformed from N-OH-demethyl-IPU

402

through

403

2-methoxyl-IPU-N-acetylglucoside (m/z 443) and 2-IPU-N-acetylglucoside (m/z 413) were

404

generated from 2-methoxyl-IPU and N-OH-demethyl-IPU through N-glycosylation and

405

acetylation. Monodemethyl-IPU was previously reported in wheat.17 Isopropenyl-IPU (m/z

406

205) and its metabolites were derived from OH-isopropyl-IPU (m/z 223) in rice root, which

407

was also detected in wheat and soybean cell cultures.35

N-glucosylation

and

malonylation;

the

compounds

408

Nineteen acetochlor-metabolites were generated via dealkylation, dehydrogenation,

409

substitution or hydrolysis reaction through Phase I reaction, suggesting other transformation

410

and degradation systems were involved. 2-Chloro-N-(2-methyl-6-ethylphenyl) acetamide

411

(CMEPA, m/z 212), 2-Hydroxy-2’-ethyl-6’-methyl-N-(ethoxymethyl) acetanilide (HEMEMA,

412

m/z 252) and 2-Methyl-6-ethylphenol (MEP, m/z 137) were the main metabolic products of

413

acetochlor in rice. N-Chloroacetyl-7-ethyl-2,3-dihydroindole (CED) (m/z 224) and CMEPA

414

(m/z 212) and HEMEMA (m/z 252) were previously reported as acetochlor-photodegraded

415

products in water.6,37 Two acetochlor degradation products HEMEMA and MEP can be

416

yielded by soil bacteria.38 N, N-Diethylaniline (DA) (m/z 150), 2-Methyl-6-ethylaniline (MEA)

417

(m/z 136), 4, 8-Dimethyl-2-oxo-1, 2, 3, 4-tetrahydroquinoline (DOT) (m/z 176) were detected

418

by photodegradation of acetochlor in water in the previous research.37 In this study, they are

419

also identified in rice. We also found that some of Phase I reaction components were integral

420

to

the

formation

of

conjugates

in

Phase

II

reaction.

17


Seven

intermediates


Page 18 of 33

421

2-Chloro-N-(2-methyl-6-ethylphenyl) acetamide (CMEPA), 2-Methyl-6-ethylaniline (MEA),

422

2-Hydroxy-2’-ethyl-6’-methyl-N-(ethoxymethyl)

423

methylphenol (MP), 2-Methyl-6-ethylphenol (MEP), 2-Sulfydryl-2’-ethyl-6’-methyl-N-

424

(ethoxymethyl) acetanilide (SEMEMA) and 2-Methyl-4-hydroxy-6-ethylaniline (MEAOH)

425

enter Phase II reaction (SI Figure S11). Some intermediates conjugate with sugar in Phase II

426

mechanism for biotransformation of plants (SI Table S5).17 Since acetochlor contains chlorine,

427

its degradation products tend to react with sulfydryl of glutathione (GSH), hydroxymethyl

428

glutathione (hmGSH) and dipeptide or cysteine to produce six conjugates containing sulfydryl

429

(SI Table S5).36 The production of conjugation reduces the quantity of intermediate, and are

430

less toxic to plants.

acetanilide

(HEMEMA),

2-Methyl-6-

431

Overall, our work provided the genetic, physiological and chemical evidence that IRGT1

432

overexpression facilitated the dissipation of isoproturon/acetochlor in rice. The increased

433

accumulation of the isoproturon/acetochlor metabolites reflected the detoxification of

434

isoproturon/acetochlor in rice. The degradation products can be further transported into

435

vacuoles or other machinery for deep catabolism.11,13 The enhanced degradation of

436

isoproturon/acetochlo in rice plants would drive the removal of the pesticides from the growth

437

medium.

438 439 440

ACKNOWLEDGEMENTS

441

The authors acknowledge the financial support of National Key R&D Program of China ，

442

2018YFD0200100 (No. 2018YFD0200100, 2016YFD0200201) and National Natural Science

443

Foundation of China (No. 21377058).

444

18


Page 19 of 33


445

Supporting Information Available

446

This information is available free of charge via the Internet at http://www.sciencedirect.com.

447 448

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and cloning of the catechol 1, 2-dioxygenase gene from these strains. World J. Microbiol.

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(36) Barr, D. B.; Hines, C. J.; Olsson, A. O.; Deddens, J. A.; Bravo, R.; Striley, C. A. F.;

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Norrgran, J.; Needham, L. L. Identification of human urinary metabolites of acetochlor in

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(37) Zheng, H. H.; Ye, C. M. Photodegradation of acetochlor in water and UV photoproducts identified by mass spectrometry. J. Environ. Sci. 2003, 15, 783–790.

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(38) Xu, J.; Yang, M.; Dai, J. Y.; Cao, H.; Pan, C.; Qiu, X. H.; Xu, M. Q. Degradation of

559

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560 561 23



562

563 564

Figure 1. Transcriptional expression, subcellular localization and activity of IRGT1 in rice

565

wild-type (WT) and overexpression lines (OEs) under isoproturon (IPU) and acetochlo (ACT)

566

exposure. Ten day-old young rice plants were exposed to 0-8 mg/L IPU for 4 d or 0-0.8 mg/L

567

ACT for 6 d. Expression of IRGT1 transcripts in WT under IPU (A) and ACT (B) stress.

568

Subcellular localization of IRGT1-GFP fusion proteins with endoplasmic reticulum (ER) 24


Page 24 of 33

Page 25 of 33


569

markers in tobacco leaf epidermal cells (C-E) detected by Confocal images: IRGT1-GFP (C),

570

ER marker RFP-KDEL (D), and merged image of C and D (E). Scale bars = 20 μm. IRGT1

571

activities in the WT and OE lines under IPU (F) and ACT (G) stress. Values are the means ±

572

SD. Means followed by different lowercase are significantly different between the biotypes or

573

treatments (p < 0.05). OE-1, OE-3 and OE-5 represented three independent transgenic lines

574

overexpressing IRGT1.

575 576 577 578 579 580 581 582 583 584 585 586 587

25



588 589

Figure 2. Effects of isoproturon (IPU) and acetochlo (ACT) on the growth of rice wild-type

590

(WT) and overexpressing (OE) lines. Ten day-old young rice plants were exposed to 0 and 2

591

mg/L IPU for 4 d or 0 and 0.1 mg/L ACT for 6 d. Phenotypes of the WT and OE lines plants

592

exposed to IPU (A, B) and acetochlor (E, F) (Scale bar = 5 cm). Elongation of rice shoots and

593

roots exposed to IPU (C) and acetochlor (G). Dry weight of rice shoots and roots exposed to

594

IPU (D) and acetochlor (H). Values are the means ± SD. Means followed by different

595

lowercase are significantly different between biotypes (p < 0.05).

26


Page 26 of 33

Page 27 of 33


596 597

Figure 3. Effect of isoproturon (IPU) or acetochlo (ACT) on chlorophyll concentration,

598

membrane permeability and TBARS production of the wild-type (WT) and overexpressing

599

(OE) lines. Ten day-old rice young plants were exposed to 0 and 2 mg/L IPU for 4 d or 0 and

600

0.1 mg/L ACT for 6 d. Chlorophyll concentration of rice exposed to IPU (A) and ACT (B).

601

Membrane permeability of rice shoots and roots exposed to IPU (C) and ACT (D). TBARS

602

concentration in rice shoots and roots exposed to IPU (E) and acetochlor (F). Values are the

27



603

means ± SD. Means followed by different lowercase are significantly different between

604

biotypes (p < 0.05).

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620

28


Page 28 of 33

Page 29 of 33


621 622

Figure 4. Accumulation of isoproturon (IPU) and acetochlo (ACT) in rice wild-type (WT)

623

and overexpressing (OE) lines. For short-term experiments: ten day-old young plants were

624

exposed to 2 mg/L IPU for 4 d (A) and 0.1 mg/L ACT (B) for 6 d; the removal of IPU (C) and

625

ACT (D) from plant growth media for 2 d. For long-term study: the rice plants grew in 0.05

626

mg/L IPU and 0.005 mg/L ACT for 60, 90 and 120; accumulation of IPU and ACT at 60 (E,

627

H), 90 (F, I) in shoot and root, and 120 (G, J) d in grain was determined. Values are the means

628

± SD. Means followed by different lowercases indicate the significant difference between WT

629

and OE lines (p < 0.05). 29



630

631 632

Figure 5. Expression of IRGT1 in yeast (Pichia pastoris X-33). Both empty and transformed

633

yeast cells were cultured in YPD medium supplemented with IPU at 0, 0.5, 2 and 8 mg/L or

634

ACT (0, 0.02, 0.1 and 0.5 mg/L) for 0-72 h. Phenotypes of cell growth under IPU (A) and

635

ACT (B). Degradation rate of IPU and ACT in the empty vector and IRGT1 transformed cells

636

with 2 mg/L IPU (C) or 0.1 mg/L ACT (D). The collected yeast cells were diluted to

637

OD600=10-2, 10-3, 10-4 and 10-5. Asterisks indicate the significant difference of degradation

638

between the empty vector and transformed cells (p < 0.05).

639

640 641 642 30


Page 30 of 33

Page 31 of 33


643

644 645

Figure 6. The relative content of isoproturon (IPU) derivatives in wild-type (WT) and OE-3

646

Line. (A) Relative content of IPU metabolites in shoots. (B) Relative content of IPU

647

conjugates in shoots. (C) Relative content of IPU metabolites in roots. (D) Relative content of

648

IPU conjugates in roots. The 10-day old rice seedlings were exposed to 2 mg/L IPU for 4 d.

649

After that, the derivatives were extracted and analyzed. Values are the means ± SD. Asterisks

650

mean significant difference between WT and OE-3 line (p < 0.05).

651 652 653 31



654

655 656 657

Figure 7. Proposed metabolic pathway of isoproturon (IPU) in rice shoot and root. The

658

metabolites and conjugates of IPU were detected using AB SCIEX Triple TOF 5600 mass

659

spectrometer.

660

32


Page 32 of 33

Page 33 of 33


Graphics


Biodegrading two pesticide residues in paddy plant and environment

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