Degrading and Phytoextracting Atrazine Residues in Rice (Oryza

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Degrading and Phytoextracting Atrazine Residues in Rice (Oryza sativa) and Growth Media Intensified by A Phase II Mechanism Modulator Jing Jing Zhang, Shuai Gao, Jiang-Yan Xu, Yi Chen Lu, Feng Fan Lu, Li Ya Ma, Xiang Ning Su, and Hong Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02346 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Environmental Science & Technology

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Title: Degrading and Phytoextracting Atrazine Residues in Rice (Oryza sativa) and Growth

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Media Intensified by a Phase II Mechanism Modulator

3

Running head: OsARGT1 facilitates ATZ transformation in rice

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Authors: Jing Jing Zhanga,b, Shuai Gaoc,d, Jiang Yan Xua, Yi Chen Lua,e, Feng Fan Lua, Li Ya

5

Maa, Xiang Ning Sua,b, 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; cDepartment of Biochemistry and Molecular Biology, College of Life

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Science, Nanjing Agricultural University, Nanjing 210095, China; dCollege of Life Sciences,

11

Fudan University, Shanghai, 200433 China; eCollege of Food Science and Light Industry,

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Nanjing Tech University, Nanjing 211800, 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: +86-25-84395207

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Email: [email protected]

18 19 20 21 22 1

ACS Paragon Plus Environment


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Abstract

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Atrazine (ATZ) residue in farmland is one of the environmental contaminants seriously

25

affecting crop production and food safety. Understanding the regulatory mechanism for ATZ

26

metabolism and degradation in plants is important to help reduce ATZ potential toxicity to

27

both plants and human health. Here, we report our newly developed engineered rice

28

overexpressing a novel Phase II metabolic enzyme glycosyltransfearse1 (ARGT1) responsible

29

for transformation of ATZ residues in rice. Our results showed that transformed lines, when

30

exposed to environmentally realistic ATZ concentration (0.2-0.8 mg/L), displayed

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significantly high tolerance, with 8-27% biomass and 36-56% chlorophyll content higher, but

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37-69% plasma membrane injury lower than untransformed lines. Such results were well

33

confirmed by ARGT1 expression in Arabidopsis. ARGT1-transformed rice took up 1.6-2.7

34

fold ATZ from its growth medium compared to its wild type (WT) and accumulated ATZ

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10%-43% less than that of WT. A long-term study also showed that ATZ in the grains of

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ARGT1-transformed rice was reduced by 30-40% compared to WT. The ATZ-degraded

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products were characterized by UPLC/Q-TOF-MS/MS. More ATZ metabolites and conjugates

38

accumulated in ARGT1-transformed rice than in WT. Eight ATZ metabolites for Phase I

39

reaction and ten conjugates for Phase II reaction in rice were identified, with three

40

ATZ-glycosylated conjugates that have never been reported before. These results indicate that

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ARGT1 expression can facilitate uptake of ATZ from environment and metabolism in rice

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plants.

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Keywords: glycosyltransferase, atrazine, rice, detoxification, transformation

44 2


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Introduction

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Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine, ATZ) is a broadleaf weed

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control herbicide widely used for production of gramineous crops such as corn, sorghum and

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rice.1 Due to its low cost and high effectiveness, ATZ has been used for nearly 60 years across

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70 countries such as United States, China and other parts of the world. From 1980 to 1999,

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the annual spray amount of ATZ in the United States was up to 29,000 tons.2 The annual

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consumption of ATZ in China was up to 15,000 tons from the early 1980s.3 In the waters

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adjacent to the farmland with its application, the concentration of ATZ is as high as one

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mg/L.4 The widespread ATZ residues are detected in surface and groundwater, leading to the

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environmental risks to wild life and human health.5,6,7 Thus, minimizing ATZ residues in

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farmland and other ecosystems is crucial for safe crop production.8,9

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Many strategies have been proposed in recent years to remedy ATZ-contaminated

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environments.10,11 Studies examined the ability of indigenous microbial communities to

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degrade organic toxicants in soil.12,13 Due to their low abundance in soils, inoculation of the

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indigenous microbes may strengthen the degradation of the toxicants. However, maintaining a

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long-lasting activity of microorganisms inoculated in the soil is technically challenging.12,13

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The physicochemical treatments, known as electrochemistry, chemical oxidation and

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UV-ozonation, are applicable to removal of toxicants only on a small scale. Nevertheless,

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these approaches are usually complicated, costly and may produce secondary effects.14,15

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Relatively, phytoremediation is an ideal technology to remediate moderately polluted soil in a

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large scale. In this way, selection of desirable plants is essential for the proposed goal

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achievement. To fortify toxicant uptake and degradation in plants, efforts have been made to 3



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express microbial catabolic enzymes in target plants.16,17 The transformation of plants

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carrying a mammalian liver P450s (cytochrome P450 monooxygenases) CYP1A1 was

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reported to degrade herbicides chlorotoluron and norflurazon in potato plants.18

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Plants are naturally gifted with multiple mechanisms for eliminating organic xenobiotics

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through enzymatic and non-enzymatic degrading systems.19 One of the mechanisms is the

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conjugation of toxicants with sugars catalyzed by a group of special glycosyltransferases

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(GTs), by which a lipophilic aglycone is converted into a hydrophilic compound ready for

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catabolism.20 Recently, several glycosyltransferases for detoxification of polluted compounds

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were reported in the model plant Arabidopsis.21-24 For example, transgenic Arabidopsis

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overexpressing a Populus glycosyltransferase displayed a high capacity of removing

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trichlorophenols from media.25 Furthermore, a bifunctional O- and C-glucosyltransferase in

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Arabidopsis was identified to detoxify 2, 4, 6-trinitrotoluene from environment.26 However,

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such reports are still rare. Recent genome-wide profiling of transcriptome identified many

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genes encoding specific enzymes (e.g. GTs, laccase and P450) associated with transformation

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of pesticides in plants.27-31 They provide valuable sources of proteins to develop the

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engineered crops ready for transformation of residual pesticides in plants.

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Rice (Oryza sativa L.) is a staple food providing the major calories for human diet in

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Asia and many other parts of the world. Rice is a short-lived plant, with large biomass and

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rich genotypes (or cultivars). Importantly, many wild-type cultivars bear strong adaption to

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various environmental stresses. Although rice is a food crop, it can be also a desirable plant

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candidate for phytoremediation, especially for polluted wetland. For instance, rice has

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exhibited a great potential for arsenic and trace element removal.32,33 In this case, rice as 4


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phytoremdeiation plants and contaminated with toxicants is not suitable for food crops. Our

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previous studies and others have demonstrated that the rice is easy to absorb ATZ from its

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growth media but accumulate low levels of ATZ in plants.29,30,34-36. The uptake of ATZ by rice

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was measured by high performance liquid chromatography (HPLC). To get an insight into the

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mechanism for ATZ metablism in rice, we recently isolated a group of GTs-coding genes

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responding to ATZ from rice genome.28,30,37 This allowed us to identify these genes in

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metabolizing ATZ in detail. In this study, we characterized one of the putative

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glycosyltransferase

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GLYCOSYLTRANSFEARSE1, ARGT1) responsible for ATZ transformation and detoxification

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in rice. The significance of the study is to provide a better understanding of mechanisms for

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the specific detoxification and disappearance of ATZ residues in rice growing in

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genes

(here

referred

as

ATAZINE-RESPONSIVE

environmentally relevant ATZ-contaminated soils.

101 102 103

Materials and Methods

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Plant Growth and Treatment.

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Uniform rice (Oryza sativa Japonia. cv. Nipponbare) seeds were surface-sterilized with 3%

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H2O2 for 30 min and thoroughly washed. The germinating seeds were sowed on a net floating

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

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condition of 200 µmol m−2 s−1 light intensity, 14/10 h day/night cycle and 30/25 °C (day/night)

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for 10 d. For Arabidopsis thaliana (ecotype Colubia, col-0), sterilized seeds were sown on

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1/2-strength Murashige and Skoog (MS) agar medium. One week later, the germinating 5



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seedlings were transferred to a net floating on 1/2× MS nutrient solution and grew in a growth

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chamber with 250 µmol m-2 s-1 light intensity, 14/10 h light/dark cycle and 22/20 °C

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(day/night) for 12 d. Both rice and Arabidopsis plants were exposed to 0, 0.01, 0.2, 0.4 or 0.8

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mg/L ATZ (99% purity) in the nutrient solutions and grown for a certain period of time

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depending on the need of studies. The ATZ concentration used in this study was limited below

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the level of environmentally realistic contamination reported previously.4,12,35 All growth

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media and containers were thoroughly sterilized prior to use. The growth and treatment

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solutions were changed daily.

119 120

Transcript Analysis by qRT-PCR.

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Rice and Arabidopsis were hydroponically cultured for 16 and 24 d, respectively. Total RNA

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was extracted from harvested plants using Trizol (Invitrogen, Carlsbad, CA). One µg of total

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RNA was incubated at 37 °C for 30 min with 1 unit of RNase-free DNaseI (Takara) and 1 µL

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10 × reaction buffer to remove the contaminant DNA. The reverse transcription reaction was

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initiated with a TransScript First-Strand cDNA Synthesis SuperMix kit (Beijing TransGen

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Biotech Co., Ltd). The resultant cDNA was diluted to 5 fold with sterile water and kept at

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−20 °C for quantitative RT-PCR analysis (qRT-PCR), which was performed on a MyiQ Single

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Color real time PCR system (Bio-Rad). A final 20 µL volume solution contained 1 µL cDNA,

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10 µL of 2×SYBR Premix Ex Taq (TaKara) and 200 nM primers (SI Table S1). The thermal

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cycling condition was set as 1 cycle of 95 °C for 30 s for denaturation, 40 cycles of 95 °C for

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5 s and 60 °C for 34 s for annealing and extension, respectively. Reactions were run in

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triplicate. 6


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Generation of Transformed Rice and Arabidopsis Plants.

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The coding sequence (CDS) of OsARGT1 (LOC_Os04g40520) was PCR-amplified using

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primers containing BglII and SpeI restriction sites (SI Table S1). The synthesized OsARGT1

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was digested with BglII and SpeI and inserted into the BglII and SpeI sites of pCAMBIA1304

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with CaMV35S as a promoter. The constructed vector was introduced into Agrobacterium

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tumefaciens EHA105 by thermal activation.38 To obtain transgenic rice, the embryonic callus

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of rice induced by mature embryo was infected by Agrobacterium carrying OsARGT1. The

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validated vector was transformed into Arabidopsis thaliana by the floral dip method.39 All

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transgenic plants (T3 homozygotes) were genetically identified using the following method:

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the transgenic plants were grown in 25 µm/mL hygromycin. Following two weeks, seedlings

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that could grow normally were considered as the positive transformants. DNA from the

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positive seedlings was extracted for PCR validation, with the forward primers:

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CAGAACTCGCCGTAAAGAC and the reverse primers: CCCAATACCCATGTAAAGC.

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Only homozygotes identified were used in this study.

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Measurement of Plant Growth and Membrane Permeability.

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Fresh tissues were dried in an air-forced oven (105 °C for 20 min and 70 °C for 60 h) and

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weighed. Chlorophyll content was quantified according to the method described previously.40

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Fresh leaves (0.1 g) were extracted with 8 mL of 80% acetone (pH 7.8). The supernatant was

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collected with the extracting solution and centrifuged at 5,000 g for 10 min. The chlorophyll

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content was spectrophotometrically determined. 7



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To determine plasma membrane permeability of tissues, 0.1 g fresh shoots or roots were

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cut into 5 mm length and placed in 10 mL deionized water and incubated at 32 °C for 2 h. The

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sample medium (EC1) was measured using an electrical conductivity meters (METTLER

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TOLEDO FE30-FiveEasy™). The sample was boiled at 121 °C for 20 min and cooled to

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25 °C. The second conductivity of the killed tissue extracts (EC2) was measured again. The

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electrolyte leakage (EL) was expressed with the formula EL=EC1/EC2×100.41

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Assay of Glycosyltransferase Activity.

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Activity of glycosyltransferases (GTs; EC 2.4.x.y) was determined by a modified protocol as

164

described previously.42 Fresh tissues (1 g) were ground and extracted with 1 mM EDTA, 50

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mM NaCl, 1% (w/v) polyvinylpolypyrrolidone in a 50 mM Tris–HCl (pH 8.0) buffer solution.

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The homogenate was centrifuged at 10,000 g and 4 °C for 30 min. Supernatant (100 µL) was

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mixed with 2 mM UDP-glucose and 0.04 mM p-nitrophenol. The mixture was incubated at

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30 °C. The reaction was terminated after 2 h by addition of 250 µL methanol. The mixture

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was chilled at −20 °C for 0.5 h. The supernatant was filtered through a 0.22 µm nylon

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membrane and analyzed using high performance liquid chromatography (HPLC) with a

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ultraviolet (UV) detection under the condition: hypersil reversed phase C8 column (Thermo,

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250 mm × 4.6 mm i.d.), 317 nm wavelength, methanol: water (3:2; v/v) mobile phase, and 0.6

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mL/min flow rate. One unit of the GT activity was defined as the consumption of 1 µmol

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p-nitrophenol per minute under assay condition.

175 176

Quantification of ATZ in Rice and Arabidopsis. 8


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Rice seedlings were grown in the half strength Hoagland nutrient solution containing ATZ at

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0.2, 0.4 and 0.8 mg/L for 6 d. For a long term exposure experiment, seedlings were exposed to

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0.01 mg/L ATZ for 30, 60, 100 and 120 d, respectively. For Arabidopsis, seedlings were

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treated with 0.4 mg/L ATZ for 4 d after growing in the half strength MS liquid media for 20 d.

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Fresh tissues were separately harvested and ground using liquid nitrogen. The sample powder

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(3.0 g) were dissolved in 25 mL of acetone−water (3:1, V:V) by ultrasonic extraction for 30

183

min. The mixture was centrifuged at 4,000 g for 8 min and the supernatant was collected in a

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flat bottom flask. This step was repeated in triplicate. The pooled supernatant was vaporized

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at 42 °C to remove acetone using a rotary vacuum evaporator. The residual water was

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liquid-liquid extracted by petroleum ether thrice. The extract was concentrated to dryness. The

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residue was dissolved in methanol (0.5 mL) and diluted by purified water. The mixture was

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purified using LC-18 SPE column. Methanol (2 mL) was used to wash the ATZ sample. The

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content of ATZ was determined by HPLC (Waters 515; Waters Technologies Co. Ltd., USA)

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with UV detector under the condition: Hypersil reversedphase C18 column (Thermo, 250 mm

191

× 4.6 mm i.d.); mobile phase, methanol:water (65:35, V:V); wavelength, 225 nm; and flow

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rate, 0.6 mL/min.35

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To determine the concentration of ATZ in each pot, 20 mL nutrient solution was

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extracted by petroleum ether for three times, each time with 20 mL. The extract was

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concentrated to dryness using a rotary concentrator at 40 °C. The residue was dissolved in 2

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mL methanol. The method for measuring ATZ concentration in the nutrient solution by HPLC

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was the same as that of plant tissues indicated above. To assess the removal of ATZ by plants,

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the ATZ concentration and solution volume of initial and post-exposure were determined. To 9



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eliminate the effect of natural degradation, the nutrient solution containing ATZ without

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growing plants was set as the control. The formula of ATZ removal was shown as below:

201 202

RemovalATZ= (VInitial×CInitial-Vpost-exposure×Cpost-exposure+ Vnatural degradation×Cnatural degradation) / numberseedlings.

203 204

Characterization of Atrazine Metabolites and Conjugates in Rice.

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LC-MS analysis was performed on a Shimadzu LC 20ADXR LC system in-line with an AB

206

SCIEX Triple TOF 5600 mass spectrometer. The autosampler temperature was set at 4 °C,

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and the injection volume was set as 20 µL. LC was performed using an Poroshell 120 EC-C18

208

column (2.7 µm, 2.1×50 mm, Aglient) and a gradient system with the mobile phase consisting

209

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

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mL/min. The gradient program was followed: (1) 5% B for 1 min, 1-5 min from 5%-15% B,

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5-20 min from 15%-35% B, 20-22 min from 35%-100% B, and 100% B for 3 min, (2) Back

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to the initial conditions and (3) equilibrating for 1 min before the next sample injection. The

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MS experiments were performed using AB Sciex Triple TOFTM 5600 system with Accelerator

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TOFTM Analyzer and electrospray ionization source. The mass spectrometer was operated in

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

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

217

floating 5500 V. The APCI positive calibration solution for the AB SCIEX Triple TOFTM

218

systems on calibration delivery system was employed once every 2 samples to ensure a

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working mass accuracy of old leaves

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≥ new leaves > roots, confirming that ATZ is easily transported from roots to the aerial parts

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of rice. Such a trait is advantageous to extracting ATZ from environment because the large

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bulk shoot can hold more ATZ. The efficient root to shoot translocation of ATZ was likely

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attributed to the great capability of xylem transport in rice.45 The enhanced ATZ metabolism

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in the transgenic rice possibly resulted from the intensified transformation of ATZ through

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Phase II mechanism, because the relative contents of conjugates in the ARGT1-transformed

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rice were higher than those in WT. Alternatively, the glycosylated-ATZ molecules were

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translocated from roots to shoots because glycosylation can change the solubility of the

394

targeted molecules by increasing their hydrophilic properties, making them easier to

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translocate to shoots.37,46 As the ARGT1-transformed rice enhanced the capacity of

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phytoextracting ATZ from environment, this suggests that ARGT1 overexpression could 18


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contribute to the removal of ATZ from soil. Meanwhile, the enhanced ATZ transformation in

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the transgenic rice implies the detoxification of cellular ATZ and thus promotes plant resistant

399

to the adverse environment.

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While the lower content of ATZ was accumulated in the ARGT1-transformed rice than in

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WT, a higher level of ATZ metabolites were examined in the transformed rice. We specified

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the ATZ-degraded products using UPLC-LTQ-MS/MS. There were eight ATZ degraded

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products including deisopropyl atrazine (DIA), hydroxy atrazine (HA), deethyl atrazine

404

(DEA),

405

demethylated atrazine (DMA) and dehydrogenated atrazine (DHA). These reactions belong to

406

the phase I reaction, by which some toxicants can be activated through oxidoreduction and

407

hydrolysis reaction.47 From the MS2 spectrum, hydroxyisopropylatrazine (HIA) was ionized

408

to generate characteristic ions of m/z 214 (loss of H2O) and ion m/z 174 by loss of the

409

N-hydroxyethyl

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non-phytotoxic to both plants and vertebrates.48,49 Recent studies with animals rat and rabbit

411

also demonstrates that a hydroxylatrazine (HA) and three chlorometabolites of ATR including

412

DEA, DIA and diaminochlorotriazine (DACT) had no statistical and significant effects on the

413

rat and rabbit embryo-fetal development and weight even at dose levels producing maternal

414

toxicity.50,51 However, a report also pointed out the toxic effect of ATZ metabolites such as

415

DEA and DIA on amphipods (Hyalella azteca and Diporeia spp), although their toxic degree

416

was lower than that of ATZ.52 In this study, a relatively higher level of the ATZ metabolites

417

and a lower ATZ content was detected the OsOX plants. The reduced ATZ toxicity and

418

improved growth of OsOX plants under ATZ stress should be attributed to the combined

hydroxyisopropyl

moiety

atrazine

(SI

Figure

(HIA),

S7F).30

atraton,

The

hydroxyethyl

atrazine

ATZ-hydroxylated

(HEA),

metabolite

is

19



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effects. Currently, we are not sure whether these ATZ metabolites in rice are toxic to the

420

plants or human beings. The presence of ATZ metabolites in the rice grain used as food must

421

be avoided. It should be interesting if these compounds will be ecotoxicologically

422

investigated in the future.

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The conjugation of xenobiotics (e.g. isoproturon in wheat) with sugars is Phase II

424

mechanism for biotransformation in plants.18,53 Similarly, the glycosylated xenobiotics such as

425

2,

426

6-hydroxybentazone were reported in Arabidopsis.24,25 In this study, three glycosylated

427

conjugates were characterized, including HA+Glc-H2O, DIHA+Glc-H2O and DIA-HCl+Glc,

428

which were transformed through HA and DIA reaction with D-glucose reaction. The former

429

two compounds are the O-glucosylation type, while the third one belongs to the

430

C-glucosylation. A similar reaction was reported for 2, 4, 6-trinitrotoluene toxicant, in which

431

the C-glucosylation occurred on the carbon of benzene ring in Arabidopsis.26 As a secondary

432

reaction, other compounds that undergo phase 1 reactions may enter this pathway. Since ATZ

433

contains chlorine, it tends to react with sulfydryl of glutathione (GSH), hydroxymethyl

434

glutathione (hmGSH), dipeptide, or cysteine to generate ATZ-HCl+GSH, ATZ-HCl+hmGSH,

435

ATZ-HCl+Cys&Ser,

436

respectively. The double bond of α-carbon formed on the N-ethyl group acts as an

437

electrophilic Michael acceptor, which covalently binds to cysteine,54 to produce DHA+Cys.

438

ATZ-HCl+Cys&Glu, ATZ-HCl+Cys&Gly and ATZ-HCl+Cys were likely generated by losing

439

amino

440

ATZ-HCl+hmGSH was identified in this study. In legume, its homologue γGlu-Cys-βAla

4,

5-trichlorophenol,

acids

from

pentachlorophenol

ATZ-HCl+Cys&Glu,

the

corresponding

and

pesticides

ATZ-HCl+Cys&Gly

parent

molecules.

metabolites

and

Notably,

like

ATZ-HCl+Cys,

a

conjugate

20


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(homoglutathione; hGSH) was identified involving phase II detoxification.55 The consumed

442

hGSH and hmGSH can be regenerated from the oxidised form by relavent glutathione

443

reductase.56

444

Figure 5 manifests the proposed multiple metabolic pathways of ATZ based on the

445

specified ATZ metabolites. The eight products DIA, HA, DEA, HIA, Atraton, HEA, DMA,

446

and DHA were generated through dealkylation, dehydrogenation or hydrolysis reaction by

447

Phase I reaction. Three intermediates DHA, HA and DIA were brought into Phase II reaction,

448

while the rest of them were subjected to unknown pathways. These components along with

449

ten other ATZ-conjugates from Phase II reaction could be access to active membrane transport

450

systems for further catabolism. Among the ten Phase II ATZ-conjugates, three conjugates

451

HA+Glc-H2O, ATZ-HCl+hmGSH and DIA-HCl+Glc were described here for the first time in

452

plants. Further charactering these components will help understanding the precise catabolic

453

pathways leading to the complete disappearance of ATZ in rice plants.

454 455 456

ACKNOWLEDGEMENTS

457

The authors acknowledge the financial support of the National Key Research and

458

Development Project of China (No. 2016YFD0200201) and the National Natural Science

459

Foundation of China (No. 21377058, 21577064).

460 461 462

Supporting Information Available 21



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

464 465 466

References

467

(1) Arora, S.; Mukherjee, I.; Kumar, A.; Garg, D. K. Comparative assessment of pesticide

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residues in grain, soil, and water from IPM and non-IPM trials of basmati rice. Environ.

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Monit. Asses. 2014, 186, 361–366.

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(2) Delorenzo, M. E.; Lauth, J.; Gi, P. P. S.; Ross, P. E. Atrazine effects on the microbial food web in tidal creek mesocosms. Aquat. Toxicol. 1999, 46, 241-251.

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(3) Deng, J. C.; Jiang, X.; Wang, D. C.;, Lu, X.; Gao, H.; Wang, F. Research advance of

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614

(56) Okumura, R.; Koizumi, Y.; Sekiya, J. Synthesis of hydroxymethylglutathione from

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434-437.

617 618 619

Table 1. Summary of all mass spectrometer data for metabolites and conjugates of ATZ in

620

rice. No.

Acronym

Chemical formula

tR

Tho m/z,

Exp m/z,

[M+H]+

[M+H]+

△ ppm

MS2 main fragments (m/z)

1

Prometryn

C10N19N5S

14.13

242.14339

242.14343

0.1

200, 158, 116, 110, 91, 85, 74, 68

2

ATZ

C8H14ClN5

13.24

216.10105

216.10108

0.1

174, 146, 138, 132, 104

174.05398

-0.7

146, 132, 104

Metabolites 1

DIA

C5H8ClN5

3.60

174.0541

2

HA

C8H15N5O

4.44

198.13494

198.135

0.3

156, 128, 114

3

DEA

C6H10ClN5

5.84

188.06975

188.06971

-0.2

146, 104, 79, 68, 61

4

HIA

C8H14ClN5O

6.65

232.09596

232.09587

-0.4

174, 146, 132, 104

5

Atraton

C9H17N5O

7.02

212.15059

212.15048

-0.5

170, 142

6

HEA

C8H14ClN5O

8.12

232.09596

232.09579

-0.8

172, 146, 130, 104

7

DMA

C7H12ClN5

9.47

202.0854

202.08551

0.6

174, 104, 96, 68

8

DHA

C8H12ClN5

9.65

214.0854

214.08523

-0.8

172, 130

No

Conjugates

1

DIHA+Glc-H2O

C11H19N5O6

1.89

318.14081

318.14106

0.8

170, 156

2

HA+Glc-H2O*

C14H25N5O6

2.51

360.18776

360.18745

1.0

198, 156

3

ATZ-HCl+Cys&Ser

C14H25N7O4S

3.99

388.17615

388.17596

-0.5

301, 214, 172

4

ATZ-HCl+hmGSH*

C19H32N8O7S

5.28

517.21875

517.21828

-0.9

388, 301, 267, 249, 197, 155

5

ATZ-HCl+Cys&Glu

C16H27N7O5S

5.98

430.1867

430.18678

0.2

341, 301, 214, 172, 130

6

DIA-HCl+Glc *

C11H19N5O5

6.02

302.1459

302.14619

1.0

214, 172, 144, 138

7

ATZ-HCl+GSH

C18H30N8O6S

6.17

487.20818

487.20846

0.6

214, 172, 130

8

ATZ-HCl+Cys&Gly

C13H23N7O3S

6.28

358.16559

358.16588

0.8

214, 172, 130, 102

9

ATZ-HCl+Cys

C11H20N6O2S

6.46

301.14412

301.14414

0.0

214, 172, 144, 130, 102

10

DHA+Cys

C11H19ClN6O2S

9.97

335.10515

335.10535

0.6

214, 172, 146, 130, 104

621

Prometryn, internal standard; tR, retention time; Tho m/z, thoretical m/z; Exp m/z, experimental m/z;

622

*, Compounds that have been reported for the first time.

623 29



Page 30 of 38

624 625

Figure captions

626

Figure 1. The transcriptional expression and activity of OsARGT1 in transgenic lines and

627

wild-type (WT) of rice. (A) Ten day-old WT seedlings were exposed to ATZ (0–0.8 mg/L) for

628

6 d and the transcripts of OsARGT1 were determined by qRT-PCR. (B) Transcripts of

629

OsARGT1 in rice transgenic lines (OsOX lines1/4/5) determined by qRT-PCR under

630

ATZ-free condition. (C) and (D) OsARGT1 activities in shoots (C) and roots (D) of rice with

631

or without ATZ treatment. Ten day-old rice seedlings were exposed to 0 and 0.4 mg/L ATZ for

632

6 d. Values are the means ± SD (n=3). Lowercases and capital letters indicate the significant

633

difference in shoots and roots, respectively (A). Means followed by different letters are

634

significantly different within each biotype or treatment (p< 0.05, ANOVA).

635 636

Figure 2. Growth analysis of transgenic lines and WT of rice and Arabidopsis. (A)

637

Photographs of three lines of rice and WT. (B, C) Elongation of rice shoots and roots. (D) Dry

638

mass of rice roots. (E) Chlorophyll content of rice. Membrane permeability of rice shoots (F)

639

and roots (G). (H) Photographs of transgenic lines of Arabidopsis and WT. (I) Dry weight of

640

Arabidopsis. Ten day-old rice seedlings were exposed to 0, 0.2, 0.4 and 0.8 mg/L ATZ for 6 d.

641

Twenty day-old Arabidopsis seedlings were exposed to 0 and 0.4 mg/L ATZ for 4 d. Values

642

are the means ± SD (n=3). Means followed by different lowercases were significantly

643

different between each biotype (p < 0.05, ANOVA).

644 645

Figure 3. Accumulation of ATZ in OsOX lines and WT. ATZ accumulation in shoots (A) and 30


Page 31 of 38


646

roots (B) of 10 day-old rice following exposure 0.2, 0.4 and 0.8 mg/L ATZ for 6 d. (C)

647

Removal of ATZ in growing media by planting OsOX lines and WT. Ten day-old seedlings

648

were exposed to ATZ for 2 d. (D) Illustration of different tissues of 30 d and 60 d rice. Old

649

leaves refer to the first four leaves; mature leaves refer to the later seven leaves with pulvinus;

650

and new leaves refer to three young leaves by tillering. (E)-(G) Distribution of ATZ in

651

different tissues of rice following exposure to 0.01 mg/L ATZ for 30 d (E), 60 d (F), 100 d (H)

652

and 120 d (I). (G) Illustration of rice flag leaf and grain.

653 654

Figure 4. Relative contents of ATZ derivatives in WT and OsOX-1 line. (A) ATZ metabolites

655

in shoots. (B) ATZ conjugates in shoots. (C) ATZ metabolites in roots. (D) ATZ conjugates in

656

roots. Ten-day old rice seedlings were exposed to 0.8 mg/L ATZ for 6 d. After that, the

657

derivatives were extracted and analyzed. Compounds reported for the first time were indicated

658

by the red arrow. Values are the means ± SD with three replicates. Asterisks mean

659

significantly different between WT and Line OsOX-1 (p < 0.05, ANOVA).

660 661

Figure 5. Proposed metabolic pathways of ATZ in rice. Eight metabolites and ten conjugates

662

in rice were characterized using AB SCIEX Triple TOF 5600 mass spectrometer. The putative

663

pathways among S-conjugates were indicated by green arrows.

664

31



Page 32 of 38

665 666 667

Figure 1.

668 669

32


Page 33 of 38


670 671 672

Figure 2.

673 674 33



Page 34 of 38

675 676 677

Figure 3.

678 679 680 681 34


Page 35 of 38


682

683 684 685

Figure 4.

686 687 688

35



Page 36 of 38

689 690 691

Figure 5.

692 693 694 695 696 697 698 699 700 701 702 36


Page 37 of 38

703


TOC

704 705 706 707

37



84x47mm (300 x 300 DPI)


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Degrading and Phytoextracting Atrazine Residues in Rice (Oryza

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