A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants

Ecotoxicology and Environmental Safety 119 (2015) 25–

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants Li Rong Tan a,b,1, Yi Chen Lu a,b,1, Jing Jing Zhang a,b, Fang Luo a, Hong Yang a,n a b

Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China

art ic l e i nf o

a b s t r a c t

Article history: Received 31 December 2014 Received in revised form 25 April 2015 Accepted 27 April 2015

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. & 2015 Elsevier Inc. All rights reserved.

Keywords: Atrazine P450s Deep-sequencing Microarray Degradation Rice

1. Introduction Herbicides such as atrazine are widely used for killing weeds to improve crop production. However, the wide-use and persistent residues of herbicides in soils have brought about great concerns on toxic impacts on plant growth, development and crop productivity (Su et al., 2005; Sathiakumar et al., 2011; Lu et al., 2013). Fortunately, plants have evolved sophisticated strategies to cope with the adverse effects of herbicide by attenuating the phytotoxicity (Kawahigashi, 2009). It is proposed that in higher plants the herbicide metabolism comprises three major active phases including conversion (Phase I), conjugation (Phase II), and compartmentalization (Phase III); one of the major components responsible for the herbicide degradation is cytochrome P450 monooxygenases (cytochrome P450s), which is thought of playing a critical role in the phase I metabolism (Siminszky, 2006). n Correspondence to: Weigang No.1, Chemistry Building, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China. E-mail address: [email protected] (H. Yang). 1 The authors made equal contribution to the study.

http://dx.doi.org/10.1016/j.ecoenv.2015.04.035 0147-6513/& 2015 Elsevier Inc. All rights reserved.

Plant cytochrome P450s (EC 1.14.1.4.1) are membrane-integrated and heme-containing protein enzymes depending on an NADPH–P450 oxidoreductase complex to transfer reducing equivalents from NADPH to the cytochrome P450 (Jensen and Møller, 2010). It is actually an enzymatic complex, whose activities are composed of cytochrome P450 protein and NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4). CPR transfers electrons from NADPH to diverse P450 monooxygenases that participate in a broad range of reactions such as biosynthesis of secondary metabolites, signaling molecules, defense-related chemicals and plant hormones (Ro et al., 2002; Nelson et al., 2004). Recent studies have identified a number of plant P450 genes, making this group become one of the largest gene families in the plant kingdom (Mizutani and Ohta, 2010). To date, there are about 246 P450 genes in Arabidopsis thaliana and 356 in rice, but most of the P450 genes have not been functionally characterized (http://drnelson.uthsc. edu/CytochromeP450.html) (Nelson et al., 2004; Mizutani and Ohta, 2010). Pioneer studies on cytochrome P450s mainly focused on the herbicide selectivity and resistance in weeds (Shirakura et al., 1995; Werck-Reichhart et al., 2000), while recent investigations have extended to the crop contamination and

26

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

environment-related toxicology of herbicides (Shirakura et al., 1995; Powles and Yu, 2010; Zhang et al., 2012). The metabolism of herbicide mediated by cytochrome P450s may impact crop growth and production (Singh et al., 1998; Deng and Hatzios, 2002). Recently, the N-dealkylation and hydroxylation metabolites of atrazine mediated by cytochrome P450s have been reported in vertebrate species such as rat (Hanioka et al., 1998) and human (Joo et al., 2010; LeBlanc and Sleno, 2011), and its action model has been proposed. However, little is known about atrazine metabolism and detoxification in crops. Therefore, genome-wide investigation on the herbicide metabolism-related P450s in plants will help understanding the mechanism for its detoxification and degradation. Atrazine (2-chloro-4-ethyamino-6-isopropylamino-s-triazine, ATR) belongs to the triazine herbicide family that controls many broadleaf and grassy weeds in areas of graminaceous crop production such as corns and sorghums (Su et al., 2005). The action mode of atrazine is the selective repression of photosynthesis II by blocking the electron transport to generate reactive oxygen species (Anderson and Zhu, 2004). Because of its persistence in soils and intensive use, atrazine has become a contaminant in ground/surface water and soils worldwide (Jablonowski et al., 2011). Some crops such as rice were reported to absorb atrazine readily from environments (Su et al., 2005; Zhang et al., 2014). Atrazine blocked seed germination and reduced elongation of coleoptile of rice (Moore and Kröger, 2010). Accumulation of atrazine induced oxidative stress by modifying antioxidant enzymes and gene expression (Ramel et al., 2007; Lu et al., 2013; Zhang et al., 2014), and affect DNA organization in rice and other plants (Besplug et al., 2004). Rice is a major food crop in many regions of the world, especially in Asia, because it provides the major portion of calories in the human diet (Khush, 2005). Crop contamination with herbicides not only affects the cereal production, but also serves as a food chain pollutant, threatening to human health (Hintz et al., 1998; Arora et al., 2008; Lu et al., 2014). There is an urgent need to assess the possible modification and degradation of atrazine in rice crops and to develop a strategy to minimize its accumulation in edible part (e.g. grain). In this study, we have performed a transcriptome analysis of cytochrome P450 genes in rice plants exposed to atrazine. The sequenced P450 genes were validated by microarray and further by quantitative real time PCR (qRT-PCR). We also assayed the cytochrome P450s activity in the atrazineexposed plants and assessed the correlation between the activity and rice growth, atrazine accumulation or physiological response. The modified products of atrazine were precisely characterized by UPLC/MS. Therefore, the objectives of this study were to (a) investigate P450 genes from rice that respond to atrazine and (b) develop an effective way to characterize atrazine-metabolized or atrazine-degraded products mediated by the cytochrome P450s in the rice crop.

2. Materials and methods 2.1. Plant materials and treatment Rice (Oryza sativa, Japonica) seeds were sterilized in 3% H2O2 solution, followed by washing with distilled water. The seeds were germinated in an incubator at 30 °C for 48 h under darkness. The germinating seeds were transferred and grown hydroponically in a black polyvinylchloride pot containing 1/2 strength Hoagland nutrient solution (Lu et al., 2014). Two-leaf stage rice seedlings were treated with 0.4 mg L
1 atrazine in 1/2 strength nutrient solution (Zhang et al., 2012). Each pot contained 20 seedlings which were grown in a growth chamber (PGX-350D, SAFE Co.)

under the condition of 25/20 °C (day/night), 200 mmol m
2 s
1 artificial illumination with a photoperiod of 14/10 h (light/dark) and 70% relative humidity. The etiolated rice seedlings were grown in dark for testing cytochrome P450 monooxygenase activity. The pH of nutrient solution was adjusted to 5.6–5.8. The culture solution was renewed every 2 d. 2.2. RNA isolation and transcriptome analysis Total RNA was extracted from shoots and roots using Trizol (Invitrogen, Carlsbad, CA). Four libraries were constructed, including two libraries of shootþAtr (shoot treated with atrazine) and Rootþ Atr (root treated with atrazine), which were generated from the RNA pool derived from samples treated with 0.4 mg L
1 atrazine for 2, 4 and 6 d. The two other libraries of Shoot–Atr (shoot control, atrazine-free) and Root–Atr (root control, atrazinefree) were generated from the RNA pool derived from untreated samples. One microgram RNA samples was incubated for 30 min at 37 °C with 1 unit of RNase-free DNaseI (Takara) to remove the genomic DNA and incubated with 1 mL of 50 mM EDTA for DNaseI inactivation for 10 min at 65 °C. The 1% agarose gel, stained by ethidium bromide, was run to indicate the integrity of the RNA. All RNA samples were quantified and examined for protein contamination (A260 nm/A280 nm ratios) and reagent contamination (A260 nm/A230 nm ratios) by a Nanodrop ND-1000 spectrophotometer. RNA samples were selected based on 28S/18S rRNA band intensity (2:1) and A260/A280 nm ratios between 1.8 and 2.0, A260/A230 nm ratios greater than 1.5 (Wang et al., 2010). The highthroughput transcriptome analysis was described previously (Zhou et al., 2013). 2.3. Microarray analysis The microarray analysis was performed based on the method described previously (Lu et al., 2013). Briefly, total RNA was isolated as described above and sent to GenScript (Nanjing, China) for hybridization to the Agilent whole genome microarray chip (version 2.0) using the single channel hybridization design. Total RNA (500 ng) was converted into labeled cRNA with nucleotides coupled to fluorescent dye Cy3 using the Quick Amp Kit according to the manufacturer's instructions. The qualified Cy3-labeled cRNA (1.65 mg) was hybridized to 2.0 4  44 k microarrays. The hybridized array was washed and scanned, and the data were extracted from the scanned image using Feature Extraction version 10.2 (Agilent Technologies). An error-weighted average signal intensity of two probes within a chip was used for normalization with Lowess normalization module implanted in JMP Genomics software. An average expression of all probes among 16 data sets was used as the baseline. Pairwise comparison between treatments was conducted to get the expression profiles of each probe. For each sample, three hybridizations were carried out and each probe had four replicate spots on a microarray. Genes were regarded as differentially expressed if their average signal intensity among 4 replicates was above 20 in a minimum of one condition and expression ratio is greater than 2 fold with p o0.05 (Student’s t-test). 2.4. Quantitative real time RT-PCR The 20 mL reverse transcription reaction mixture contained 1 mg RNA, 1 mL of 100 mM oligo (dT) primers, 1 mL of 10 mM dNTP (deoxyribonucleotide triphosphate) mixture, 0.5 mL of 40 U/mL of RNase inhibitor, 0.5 mL of 200 U/mL of M-MLV reverse transcriptase (TaKaRa Biochemical) and 4 mL of 5  M-MLV buffer. The mixture was maintained at 42 °C for 10 min and heated at 95 °C for 2 min to stop the reverse transcription reaction. The resultant cDNA was

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

27

diluted 5 fold with sterile water and kept at
20 °C for real time PCR analysis. Quantitative real time PCR was performed according to the established method (Zhou et al., 2012) using the My-IQ Single Color Realtime PCR system (Bio-Rad) and analyzed using IQ5 software (Bio-Rad). A total reaction mixture (25 mL) included 2 mL of template cDNA, 12.5 mL of the 2  TransStartTM Top Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.) and 200 nM of primers. The thermal cycling conditions were 1 cycle of 94 °C for 30 s for denaturation, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. All reactions were repeated in triplicate. The relative gene ΔΔ expression level was calculated by 2
Ct algorithm. The gene ubiquitin (LOC_Os03g13170) was used as a internal standard for normalization. The primers of chosen genes were listed in Supplementary Data S1. All reactions in qRT-PCR were run in triplicate by monitoring the dissociation curve to detect and eliminate the possible primer-dimer and nonspecific amplifications (Supplementary Fig. S1).

The reaction mixture contained 100 mL of 2 mM p-nitroanisole and 90 mL microsomes in 96-well microplate and incubated for 3 min at 30 °C. The reaction was initiated by the addition of 10 mL of 10 mM NADPH. The absorbance at 405 nm was recorded every 30 s for 15 min at 30 °C using a microplate reader. Varying concentrations of ρ-nitrophenol was used to make a standard curve. The activity of PNOD was presented as nmol p-nitrophenol min
1 mg
1 protein. The reaction was repeated three times. For in vitro treatment of cytochrome P450 inhibitors, microsomes were prepared from rice shoots. The cytochrome P450 inhibitors PBO and MAL were dissolved in DMSO and 10 μL of varied concentrations of inhibitor was added in the reaction mixture and preincubated for 2 min. The reaction was initiated by adding NADPH. Enzyme assay conditions were as described above. Each assay was conducted with three replicates.

2.5. Phylogenetic tree and chromosomal location of P450 genes in rice

The contents of inhibitors (10 mg L
1 PBO and 20 mg L
1 MAL) were chosen according to numerous literatures (Burnet et al., 1993). Two-leaf stage seedlings grew in the 1/2 strength Hoagland nutrient solution with PBO (10 mg L
1) or MAL (20 mg L
1) for 24 h and transferred to the same 1/2 strength Hoagland nutrient solution with atrazine (0.4 mg L
1) and PBO (10 mg L
1) or MAL (20 mg L
1) for another 2, 6 and 10 d, respectively. After that, the elongation of shoots and roots was measured with a rule and the dry weight was measured according to the method of Li and Yang (2013). The electrolyte leakage of rice tissues was determined by the method (Dionisio-Sese and Tobita, 1998). For quantify the atrazine in rice, fresh shoot (1 g) and root (0.5 g) tissues were ground and extracted with 15 mL acetone–water (3:1, v/v) shaking for 30 min on a shaker (BS-31, Jeio TECHCO., Ltd.). The homogenate was centrifuged at 4000  g for 10 min and filtered. The extraction step was repeated in triplicate. The supernatants were pooled and vaporized to remove acetone at 40 °C in a rotary vacuum evaporator. The residual water was extracted with 20, 15 and 5 mL petroleum for three times, respectively and petroleum ether was evaporated to dryness. The residue was dissolved in 0.5 mL methanol and 10 mL water. The dissolved solution was passed through a pre-activated LC-C18 solid phase extraction (SPE) column. After discarding the eluents, the column was eluted with 2 mL HPLC-grade methanol. The solution was collected and analyzed by high performance liquid chromatography (HPLC) with a Waters 515 pump and 2487 ultraviolet (UV) detector (Waters Technologies Co. Ltd.). The operating conditions were: column, Hypersil reversed phase C8 column (Thermo, 250 mm  4.6 mm i. d.); UV detection wavelength, 235 nm; mobile phase, methanol: water (65:35, v/v); flow rate, 0.6 mL min
1; injection volume, 20 mL. The retention time of atrazine was 16.5 min. The method of atrazine analysis above was validated by testing the limit of detection (LOD), accuracies, precisions and recoveries of atrazine in tissues (Supplementary Data S2).

The phylogenetic tree for the P450 genes was constructed with neighbor-joining (NJ) algorithm using MEGA (version 5.1) program (Lu et al., 2013). The amino acid sequences of rice (O. sativa, Japonica) and A. thaliana were retrieved from http://www.rice. plantbiology.edu/ and http://www.arabidopsis.org/. Alignment of amino acid sequences was performed using CLUSTALW (version 2.0). Chromosomal location of the rice P450 genes was got using the Oryzabase: http://www.shigen.nig.ac.jp/rice/oryzabaseV4/. 2.6. Assay of cytochrome P450 monooxygenase activity Etiolated seedlings at two-leaf stage grew in the 1/2 strength Hoagland nutrient solution containing 0.4 mg L
1 atrazine for 2 d. Shoot and root tissues (about 20 g) were ground to powder in liquid nitrogen and extracted with 1.5 volumes of 100 mM potassium phosphate buffer (pH 7.5) containing 250 mM sucrose, 40 mM ascorbate acid, 5 mM DTT and 1 mM EDTA. The crude homogenized tissues were filtered through several layers of chilled cheesecloth and centrifuged at 12,000g for 15 min. The supernatant was transferred and centrifuged at 100,000g for 90 min (Beckman ultracentrifuge Optima XE-100). Finally, the pellet was resuspended in 100 mM potassium phosphate buffer (pH 7.5), containing 20% (v/v) glycerol and 10 mM mercaptoethanol. The microsomal sample was stored at -70 °C prior to use. The protein content of microsome was measured by the method of Bradford with bovine serum albumin (BSA) as a standard (Bradford, 1976). NADPH-cytochrome P450 reductase (CPR) activity was conducted by the method described previously with some modification (Brankova et al., 2007; Guengerich et al., 2009). The 200 mL reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 5 mg mL
1 cytochrome c and microsomal suspension (0.5–1 mg mL
1 protein). Ten microliter of 10 mM NADPH was last added to start the reaction. The increase of absorbance at 550 nm was recorded for 5 min at 25 °C using a microplate reader (Molecular Devices SpectraMax M5). The control reaction mixture contained the same components except for potassium phosphate buffer replace the microsomal suspension. The enzyme activity was expressed as nmol min
1 mg
1 protein using a millimolar extinction coefficient of 21.1 cm
1. p-Nitroanisole O-demethylase (PNOD) activity was determined using the procedure described previously (Yang et al., 2004). p-Nitroanisole was dissolved in dimethyl sulfoxide (DMSO) to make a 400 mM stock solution and diluted in 100 mM potassium phosphate buffer (pH 7.5) to make a final concentration of 2 mM.

2.7. Determination of growth, electrolyte leakage and atrazine accumulation in rice

2.8. Analysis of atrazine metabolites in rice by UPLC-LTQ-Orbitrap MS/MS The tissue samples were extracted three times with acetone– water (3:1, v/v) for 30 min (10 mL of solution per extraction), followed by centrifugation at 4000  g for 15 min. The supernatant was concentrated to remove acetone in a vacuum rotary evaporator at 40 °C. The residual water was loaded onto an LC-C18 SPE column. The elute was discarded and the column was washed with 2 mL HPLC-grade methanol. The solutione was filtered through a 0.22 μm springe filter for analysis of LC/MS. LC/MS analysis was performed with an Ultra Performance

28

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

Liquid Chromatography apparatus (UHPLC) (Dionex, Thermo, USA) equipped with a linear ion trap-orbitrap hybrid (LTQ Orbitrap XL) mass spectrometer. Instrument control was through Tune 2.6.0 and Cheomeleon programs. The analysis was performed using Hypersil GOLD C18 column (100  2.10 mm2, granulation of 3 mm, Thermo Fisher Scientific). Two mobile phase were used with acetonitrile (A) and 0.1% formic acid aqueous (B). Chromatographic separation was performed at a flow rate of 0.2 mL min
1 under the following conditions: 0–1 min, 5% A; 1–15 min 5–35% A; 15– 25 min 35–95% A; 25–30 min 95% A; 30–31 min, 5% A; 31–36 min, 5% A. The injection volume was 5 mL. Column oven temperature was set at 35 °C, and autosampler temperature was set at 10 °C. Ion source was equipped with an electrospray ionization (ESI) probe which set in positive mode. The external mass calibration of the Orbitrap was performed to ensure a working mass accuracy o5 ppm. The capillary temperature was set at 300 °C and the source voltage was set at 4 kV. The flow of the sheath gas and auxiliary gas was set at 35 and 10 L/min respectively. Accurate mass spectra of atrazine and its metabolites [Mþ H] þ ions for the m/z were run in a full scan mode from 50 to 250. The biggest signal intensity ions were applied for MS3 fragmentation in the data dependent scan mode. The product ions were generated by the LTQ ion trap at normalized collision energy of 35%, activation Q value of 0.25 using an isolation width of 2 Da. All the operations, data acquisition and analysis of data were controlled by Thermo Xcalibur software (version 2.2.0). 2.9. Statistical analysis The biological studies were carried out independently for three times. Each result in this study was the mean of at least three replicated treatments. Significant difference between the treatment and control was statistically evaluated by standard deviation and Student's t-test methods. Values were considered to be significantly different at p o0.05.

3. Results 3.1. Identification of cytochrome P450 genes in atrazine-treated rice plants The false discovery rates (FDRs) o0.001 and the absolute value of log2Ratio Z1 were used as a criterion to estimate the significant difference of gene expression (Wang et al., 2010). Based on the criteria, a total of 201 genes encoding cytochrome P450 were identified and showed differential responses to atrazine (Supplementary Data S3). Profiling of the 201 gene expression revealed that 56 (27.9%) were up-regulated (log2Ratio4 0), 64 (31.8%) were down-regulated (log2Ratio o0), and 81 (40.3%) were unchanged in shoots. In roots, 57 (28.4%) were up-regulated, 68 (33.8%) were down-regulated, and 76 (37.8%) genes remained unchanged. Apparently, expression of most of the P450 genes in rice could be altered by atrazine. The highest induced gene in shoots was LOC_Os02g09320, which had a 3.58-fold transcript abundance relative to the control. The following inducible genes were LOC_Os03g55240 and LOC_Os04g48460, each encoding a putative cytochrome P450. In roots, the gene LOC_Os01g43774 was mostly induced, with a 6.10-fold higher than the control. The second and third induced genes belong to LOC_Os07g11739 and LOC_Os12g16720, respectively. 3.2. Microarray and qRT-PCR validation of P450 genes from atrazineexposed rice In order to proof the atrazine-responsive rice cytochrome P450

genes from deep-sequencing, a microarray analysis with the same samples from deep-sequencing was undertaken. With two-fold or more change in differential expression, 69 cytochrome P450 genes were significantly identified (Supplementary Fig. S2). Of these, 50 genes were from roots and 29 were from shoots, in which 10 genes were co-expressed in both roots and shoots. Under atrazine exposure, 21 genes were up-regulated and 29 were down-regulated in roots, from which, 4 genes were expressed only in the RootþAtr library and 12 were expressed only in Root–Atr library, suggesting that treatment with atrazine led to null expression of some P450 genes in roots. Among the 29 genes in shoot, there were 15 genes up-regulated and 14 down-regulated. Also, 4 genes were expressed only in the ShootþAtr library and one was expressed in the Shoot–Atr library. Besides, we randomly selected 8 genes. As shown in Fig. 1, all genes tested were expressed in shoots and roots, showing a similar expression pattern to the genes from deep-sequencing and microarray analysis. 3.3. Phylogenetic analysis of cytochrome P450 genes from atrazineexposed rice plants A phylogenetic tree is a branching diagram that is implicated in the evolutionary relationship of genes among the interspecies or different species based on sequence similarity. Using the 69 rice P450 gene sequences, a rooted phylogenetic tree was created (Supplementary Fig. S3A). These genes could be divided into two clusters, containing 38 and 31 genes, respectively. Each cluster was further categorized into many more subgroups. We further examined the relationships of P450s between rice and Arabidopsis, a dicot model plants. Mapping the amino acid sequences of the 69 cytochrome P450 genes to those of Arabidopsis resulted in identification of 257 matches, with an identity from 40% to 76% (Supplementary Data S4). There were 76 genes that had an identity more than 50%. Although some P450 genes had a high similarity between rice and Arabidopsis, such as AT2G26710, most of them had a low degree of evolutionary relationship. Only 20 out of 69 rice P450 genes could be clustered with Arabidopsis. To profile the P450 genes on rice genome, the chromosome location of 69 P450 genes was analyzed. These genes were widely distributed on the 12 chromosomes, and chromosome 1, 2, 3, and 6 harbor eight or more of the cytochrome P450 genes (Supplementary Fig. S3B). 3.4. Identification of atrazine metabolites by LC/MS To get an insight into the chemical modification and consequent degradation of atrazine in rice plants, we characterized its metabolites by LC/MS. The extracted ion chromatograms (EIC) of the target masses within 5 ppm accuracy were extracted from the total ion chromatogram (TIC) of the control and sample for a given exact mass. Our analysis showed several biotransformations of atrazine including N-dealkylations products: desethylatrazine (DEA), desisopropylatrazine (DIA), desmethylatrazine (DMA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA) ( Table 1, Supplementary Figs. S4 and S5). The major metabolites of atrazine were DEA, DMA and HA, whereas DIA, HEA and HIA were identified as minor metabolites. Under positive ionization mode, the atrazine chromatogram showed a retention time at 19.18 min (Supplementary Fig. S4A). The m/z 216 precursor ions upon MS/MS experiments gave rise to the m/z 174 and m/z 188 fragment ions, respectively (Supplementary Fig. S5A). With the supporting data of MS2 and MS3 fragment ions (Supplementary Figs. S5B and S5C), the characteristic peaks of m/z 174 and 188 at 8.64 and 11.85 min were identified as DIA and DEA, respectively (Supplementary Figs. S4B and S4C). A new N-dealkylation product, named DMA, was found with the peak of m/z 202 at 15.85 min, and the precursor ion yielded the

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

29

Fig. 1. Quantitative RT-PCR validation of selected deep-sequencing and microarray cytochrome P450 genes from rice (Oryza sativa). These genes ID are as follows. A: LOC_Os03g12260; B: LOC_Os03g12500; C: LOC_Os04g01140; D: LOC_Os05g25640; E: LOC_Os06g02019; F: LOC_Os10g16974; G: LOC_Os10g17260; H: LOC_Os12g16720. TPM: transcripts per million clean tags. ATR: atrazine. Vertical bars represent standard deviation of the mean (n¼3) between the treatment and control. PCR, Quantitative RT-PCR; DGEþ MA, Digital gene expression þ Microarray.

fragment ions of m/z 174 and 132 (Supplementary Figs. S4D and S5D). The peaks of m/z 198 at 9.52 min were identified as HA (Supplementary Figs. S4E and S5E). In addition, the two peaks of m/z 232 with the retention time at 12.66 and 14.07 min were the isomeride of hydroxylation atrazine (Supplementary Fig. S4F). These two oxidation products were identified respectively as HEA (adding an oxygen atom onto the ethyl side chain) and HIA (adding an oxygen atom onto the isopropyl side chain). The

accurate molecular weights were set for further optimization in MS/MS quantitative analysis. HEA and HIA were both ionized to yield characteristic product ions of m/z 214 (loss of H2O) under the MS/MS conditions, but form different ions of m/z 174 and 188 through the loss of CH2 ¼CH–CH2–OH and CH2 ¼CH–OH parts from the N-hydroxyisopropyl and N-hydroxyethyl moiety respectively. In subsequent MS3 analysis of the precursor ion at m/z 214, they led to different ions (Supplementary Figs. S5F and S5G). Thus,

30

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

Table 1 Summary of mass spectra data for atrazine and its metabolites. Generic name

Acronym

[Mþ H] þ (m/z)

Retention time (min)

MS/MS fragments

MS/MS/MS fragments

Atrazine Desethylatrazine Desisopropylatrazine Desmethylatrazine* Hydroxyatrazine Hydroxyethylatrazine* Hydroxyisopropylatrazine*

ATR DEA DIA DMA HA HEA HIA

216.10 188.07 174.05 202.08 198.13 232.09 232.09

19.18 11.85 8.64 15.85 9.52 14.07 12.66

173.91, 187.95, 145.86 145.84 131.89, 145.97 173.92, 123.85, 131.85 155.91 214.04, 187.95 214.04, 173.93

173.91-145.85, 131.95 145.84-103.88 131.89-103.83 173.92-131.83, 145.92 155.91-85.71, 113.94, 127.94 214.04-172.02 214.04-135.87, 186.06

*

Compounds that have been reported in rice for the first time.

two hydroxylation products were distinguished. Most of metabolites could be found in both shoot and root tissues, except DIA that was not detected in root tissues (Table 1). HIA and HEA had been found as atrazine metabolites mediated by cytochrome P450s from human liver microsomes (Joo et al., 2010). In rice, DMA, HIA and HEA have been found and reported from this study.

3.5. Induction of cytochrome P450 monooxygenase activity by

atrazine To support that atrazine metabolites generated in plants were linked to cytochrome P450s, we measured the activities of NADPH-cytochrome P450 reductase (CPR) and p-Nitroanisole O-demethylase (PNOD), both of which have been reported to play important roles in detoxifying toxic compounds (Wang et al., 2013). The CPR activity in atrazine-exposed rice plants was significantly higher than the control, with the activities of CPR in roots and shoots being increased by 1.7 and 5.9 fold, respectively (Fig. 2A). Similarly, the PNOD activity in atrazine-treated rice

Fig. 2. Effect of atrazine (A and B) and piperonyl butoxide (PBO) and malathion (MAL) (C and D) on the activities of NADPH-cytochrome P450 reductase (CPR) (A and C) and p-nitroanisole O-demethylase (PNOD) (B and D) in rice microsomes. Two-leaf stage etiolated seedlings grew in the 1/2 strength Hoagland nutrient solution containing 0.4 mg L
1 atrazine for 2 d. Vertical bars represent the means 7 SD (n¼ 3). The asterisk indicates the significant differences between the treatments and the controls (po 0.05). The different letter indicates the significant differences between the treatments and the controls (p o0.05).

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

31

Fig. 3. Effect of atrazine (ATR), piperonyl butoxide (PBO) and malathion (MAL) on accumulation of atrazine in rice shoot (A) and root (B). Two-leaf stage seedlings grew in the 1/2 strength Hoagland nutrient solution with PBO (10 mg L
1) and MAL (20 mg L
1) respectively for 24 h and transferred to the same 1/2 strength Hoagland nutrient solution with atrazine (0.4 mg L
1) þPBO (10 mg L
1) and atrazine (0.4 mg L
1) þ MAL (20 mg L
1) for 2, 6 and 10 d. The treatment solution was renewed every 2 d. Values are the means 7 SD (n ¼3). The different letter indicates the significant differences between the treatments and the controls (p o 0.05).

plants was elevated by 2.59 fold in roots and 4.08 fold in shoots compared to the control, respectively (Fig. 2B). These results indicated that cytochrome P450 activity was induced by atrazine. To validate the result, we determined in vitro inhibition of cytochrome P450 activity with the two P450 specific inhibitors piperonyl butoxide (PBO) and malathion (MAL) (Varsano et al., 1992; Singh et al., 1998). As shown in Fig. 2C, D. Increasing the concentration of both PBO and MAL resulted in reduction of CPR activity, although the CPR and PNOD activities showed different responses to PBO and MAL. 3.6. Effects of cytochrome P450 activity on atrazine accumulation, growth and electrolyte leakage We further determined whether atrazine accumulation was associated with cytochrome P450 activity. The rice plants usually accumulated more atrazine in shoots than in roots (Fig. 3). There was a time-dependent change in atrazine accumulation. Using the cytochrome P450 inhibitors, the change in atrazine contents in plants was monitored. At the first 6 d, there was no significant difference of atrazine contents between the treatments of atrazine (ATR) and ATRþPBO or ATR þMAL. After treatment for 10 d, however, more atrazine was accumulated in shoots with PBO than in those with MAL or atrazine alone (Fig. 3A). The similar tendency in roots was also found in presence of PBO (Fig. 3B). The elongation and biomass of rice were greatly inhibited when treated with 0.4 mg L
1 atrazine for 10 d (Fig. 4). Compared to the control (atrazine-free), the elongation of shoot and root was decreased by 33.2% and 46.3% (Fig. 4C) and the dry weight (DW) of shoot and root per plant was reduced by 61.2% and 63.3%, respectively, when treated with atrazine (Fig. 4D). The cytochrome P450 inhibitor PBO alone did not significantly affect on rice growth. But in the presence of atrazine, its effect on rice tissues was evident. Atrazine plus PBO significantly inhibited the elongation of rice shoots and roots as compared to the atrazine provision alone. To further understand effects of P450 activities on the physiological response of rice to atrazine, we measured the electrolyte leakage, which represents the cellular membrane damage due to the toxicant exposure (Varsano et al., 1992). As shown in Fig. 4E and F, treatment with atrazine at 0.4 mg L
1 progressively increased the percentage of electrolyte leakage in shoots and roots.

Application of PBO (10 mg L
1) in the presence of atrazine led to further elevation of electrolyte leakage, suggesting that depression of P450 activities was able to damage the cellular membrane.

4. Discussion A global analysis of transcriptome helps to understand diverse gene expression and mechanisms for detoxification and degradation of toxic compounds in plants (Lu et al., 2013). Atrazine has been widely used in China, India and other parts of the world. Its residues and contamination in environments is a major problem for crop production and food safety (Su et al., 2005; Li et al., 2012). Therefore, it is of great importance to minimize its concentration in plants. Understanding of the atrazine-responsive gene expression is the first step to dissect the genetic and the molecular basis for rice seedlings to detoxify atrazine. The present study identified 201 genes coding for cytochrome P450s from rice exposed to atrazine. Thus, our study represents so far the deepest dissection of plant P450 genes in response to toxic compounds. After analysis by microarray, there were 69 cytochrome P450 genes validated. Under atrazine exposure, the cytochrome P450 genes showed different response in expression. The altered transcription implies that the gene expression can be reprogrammed by atrazine. Among the genes validated, 21 and 15 were up-regulated in roots and in shoots, respectively. Plant cytochrome P450s participate in numerous biochemical pathways associated with biosynthesis of plant secondary products (e.g. phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates), growth regulation (e.g. salicylic acid, abscisic acid, gibberellins, jasmonic acid, and brassinosteroids) and resistance to toxic compounds (Jensen and Møller, 2010; Mizutani and Ohta, 2010; Cui et al., 2010),. For example, salicylic acid (SA), a regulator of plant response to various environmental stresses (Yang et al., 2003; Liang et al., 2012), has been reported to improve plant resistance to herbicide napropamide toxicity in rapeseed (Brassica napus) (Cui et al., 2010). Cytochrome P450s are known to be involved in detoxification of xenobiotics in the phase I metabolism in plants (Siminszky, 2006; Zhang et al., 2014; Kebeish et al., 2014). But, how atrazine in plants is detoxified and chemically modified by cytochrome P450s is largely unknown. We specified atrazine metabolites in rice plants to evidence the

32

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

Fig. 4. Effect of atrazine (ATR) and piperonyl butoxide (PBO) on growth (A–D) and electrolyte leakage (E and F). Two-leaf stage seedlings grew in the 1/2 strength Hoagland nutrient solution with PBO (10 mg L
1) for 24 h and transferred to the same 1/2 strength solution with atrazine (0.4 mg L
1) and PBO (10 mg L
1) for 10 d (A–D) or 2, 6, and 10 d (E and F). Treatment solution was renewed every 2 d. A and B: phenotypes of shoots and roots with ATR and/or PBO. C: elongation of roots and shoots. D: biomass of plant tissues. E: shoots. F: roots. Values are the means 7 SD (n¼ 3). The different letter indicates the significant differences between the treatments and the controls (po 0.05).

involvement of cytochrome P450s in the biochemical catalytic process. Metabolites of atrazine regarding to cytochrome P450s catalysis were detected by LC-MS. Analysis of shoot extracts from atrazine-treated rice revealed the presence of six derivatives. DEA,

DMA, and HA were successfully detected as major metabolites, while DIA, HEA and HIA were identified as minors. Metabolites of hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA) have been reported in human microsomes (Joo et al., 2010;

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

33

Fig. 5. Proposed cytochrome P450-mediated metabolites of atrazine in rice seedlings.

LeBlanc and Sleno, 2011). Interestingly, two hydroxylation species (HEA and HIA) were also detected in the rice plants. We distinguished the two isomerides of hydroxylation products by profiling MS/MS data. HEA and HIA were ionized to yield characteristic product ions of m/z 214 (loss of H2O) in common, but formed different ions of m/z 174 and 188 through the loss of the N-hydroxyisopropyl and N-hydroxyethyl moiety, respectively. This is the first report of HEA and HIA in rice plants. Furthermore, the atrazine-hydroxylated products were proved to be non-phytotoxic to plants, as several studies in vertebrates (Li et al., 2006), human (LeBlanc and Sleno, 2011) and plants (Topal et al., 1996) indicated that detoxification in these systems also involves hydroxylation reactions, which was likely catalyzed by cytochrome P450 enzymes. The cytochrome P450 system is one of the most important enzymatic pathways for detoxification of xenobiotics in monocots (including rice) and has been described as a common atrazine tolerance mechanism (Burnet et al., 1993; Marcacci et al., 2006). In addition, the atrazine hydroxylation may occur partly due to the production of benzoxazinones, which are synthesized by CYP71 family P450 enzymes and are considered as defense substances against bacteria in crops such as corn and sorghum (Bailey and Larson, 1991; Merini et al., 2009). However, the mono N-dealkylation products of atrazine are partially detoxified, while the double dealkylated and hydroxylated products have no phytotoxicity towards plants (Shimabukuro, 1968). Several studies have identified desethylisoprophylatrazine (DIDEA) relevant to cytochrome P450s in tulip, rat and human plasma exposed to atrazine and DIDEA was assumed as a significant metabolite (Topal et al., 1996; Brzezicki et al., 2003; Panuwet et al., 2008). In this study, DIDEA was not detected, possibly due to the low abundance or below the detection. Overall, based on the current knowledge and data from this study, a metabolism of atrazine relevant to

cytochrome P450s in rice was proposed (Fig. 5). Cytochrome P450s are the major integral membrane proteins mediating many aspects of plant growth, development and environmental responses. Several model substrates such as p-nitroanisole were employed to assay the dealkylation activities catalyzed by cytochrome P450s (Qiu et al., 2003; Yang et al., 2004; Wang et al., 2013). To figure out the connection between the atrazine metabolism and activity of cytochrome P450s, the activities of both NADPH-cytochrome P450 reductase and p-Nitroanisole O-demethylase were assesed. CPR and PNOD activities were significantly induced in atrazine-treated plants. The activity was higher in root than shoot, which could be the result of direct exposure of root tissues to atrazine. Furthermore, two P450s inhibitors of PBO and MAL were used to identify the correlation between cytochrome P450s and atrazine accumulation and toxicity. The activities of CPR and PNOD were inhibited by PBO and MAL, respectively. Because of this, PBO in the presence of atrazine exerted a damage effect on cellular membrane, and as a consequence the contents of atrazine in roots and shoots were increased. These results indicated that CPR was possibly directly or indirectly involved in atrazine metabolism or detoxification in rice. Further genetic and molecular studies will be required to characterize cytochrome P450 genes and proteins that are responsible for the metabolism and detoxification of atrazine.

Author contributions Conceived and designed the experiments: HY. Performed the experiments: LRT YCL. Analyzed the data: LRT YCL JJZ FL. Contributed reagents/materials/analysis tools: HY. Wrote the paper: HY LRT YCL.

34

L. Rong Tan et al. / Ecotoxicology and Environmental Safety 119 (2015) 25–34

Acknowledgements The authors acknowledge the financial support of the National Natural Science Foundation of China (No. 21377058), the Fundamental Research Fund for the Central Universities of China (KYZ201223) and the Special Fund for Agro-scientific Research in the Public Interest from the Ministry of Agriculture of China (No. 201203022).

Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04. 035.

References Anderson, T.D., Zhu, K.Y., 2004. Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera, Chironomidae). Pestic. Biochem. Physiol. 80, 54–64. Arora, S., Mukherjee, I., Trivedi, T.P., 2008. Determination of pesticide residue in soil, water and grain from IPM and non-IPM field trials of rice. Bull. Environ. Contam. Toxicol. 81, 373–376. Bailey, B.A., Larson, R.L., 1991. Maize microsomal benzoxazinone N-monooxygenase. Plant Physiol. 95, 792–796. Besplug, J., Filkowski, J., Burke, P., Kovalchuk, I., Kovalchuk, O., 2004. Atrazine induces homologous recombination but not point mutation in the transgenic plant-based biomonitoring assay. Arch. Environ. Contam. Toxicol. 46, 296–300. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brankova, L., Ivanov, S., Alexieva, V., 2007. The induction of microsomal NADPH, cytochrome P450 and NADH, cytochromereductases b5 by long-term salt treatment of cotton (Gossypium hirsutum L.) and bean (Phaseolus vulgaris L.) plants. Plant Physiol. Biochem. 45, 691–695. Brzezicki, J.M., Andersen, M.E., Cranmer, B.K., Tessari, J.D., 2003. Quantitative identification of atrazine and its chlorinated metabolites in plasma. J. Anal. Toxicol. 27, 569–573. Burnet, M.W.M., Loveys, B.R., Holtum, J.A.M., Powles, S.B., 1993. Increased detoxification is a mechanism of simazine resistance in Lolium rigidum. Pestic. Biochem. Physiol. 46, 207–218. Cui, J., Zhang, R., Wu, G.L., Zhu, H.M., Yang, H., 2010. Salicylic acid reduces napropamide toxicity by preventing its accumulation in rapeseed (Brassica napus L.). Arch. Environ. Contam. Toxicol. 59, 100–108. Deng, F., Hatzios, K.K., 2002. Characterization of cytochrome P450-mediated bensulfuron-methyl O-demethylation in rice. Pestic. Biochem. Physiol. 74, 102–115. Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant responses of rice seedlings to salinity stress. Plant Sci. 135, 1–9. Guengerich, F.P., Martin, M.V., Sohl, C.D., Cheng, Q., 2009. Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase. Nat. Protoc. 4, 1245–1251. Hanioka, N., Jinno, H., Kitazawa, K., Tanaka-Kagawa, T., Nishimura, T., 1998. In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes. Chem-Biol. Interact. 116, 181–198. Hintz, R.L., Harmoney, K.R., Moore, K.J., George, J.R., Brummer, E.C., 1998. Establishment of switchgrass and big bluestem in corn with atrazine. Agron. J. 90, 591–596. Jablonowski, N.D., Schäffer, A., Burauel, P., 2011. Still present after all these years, persistence plus potential toxicity raise questions about the use of atrazine. Environ. Sci. Pollut. Res. 18, 328–331. Jensen, K., Møller, B.L., 2010. Plant NADPH-cytochrome P450 oxidoreductases. Phytochemistry 71, 132–141. Joo, H., Choi, K., Hodgson, E., 2010. Human metabolism of atrazine. Pestic. Biochem. Physiol. 98, 73–79. Kawahigashi, H., 2009. Transgenic plants for phytoremediation of herbicides. Curr. Opin. Biotechnol. 20, 22–230. Kebeish, R., Azab, E., Peterhaensel, C., El-Basheer, R., 2014. Engineering the metabolism of the phenylurea herbicide chlortoluron in genetically modified Arabidopsis thaliana plants expressing the mammalian cytochrome P450 enzyme CYP1A2. Environ. Sci. Pollut. Res. 21, 8224–8232. Khush, G.S., 2005. What it will take to feed 5.0 billion rice consumers in 2030. Plant Mol. Biol. 59, 1–6. LeBlanc, A., Sleno, L., 2011. Atrazine metabolite screening in human microsomes, detection of novel reactive metabolites and glutathione adducts by LC-MS. Chem. Res. Toxicol. 24, 329–339. Li, A., May, M.P., Bigelow, J.C., 2006. Identification of a metabolite of atrazine, N-ethyl-6methoxy-N’-1-methylethyl.-1,3,5-triazine-2,4-diamine, upon incubation with rat liver microsomes. J. Chromatogr. B 836, 129–132.

Li, X., Wu, T., Huang, H., Zhang, S., 2012. Atrazine accumulation and toxic responses in maize Zea mays. J. Environ. Sci. 24, 203–208. Li, Y.Y., Yang, H., 2013. Bioaccumulation and degradation of pentachloronitrobenzene in Medicago sativa. J. Environ. Manag. 119, 143–150. Liang, L., Lu, Y.L., Yang, H., 2012. Toxicology of isoproturon to the food crop wheat as affected by salicylic acid. Environ. Sci. Pollut. Res. 19, 2044–2054. Lu, Y.C., Yang, S.N., Zhang, J.J., Zhang, J.J., Tan, L.R., Yang, H., 2013. A collection of glycosyltransferases from rice (Oryza sativa) exposed to atrazine. Gene 531, 243–252. Lu, Y.C., Zhang, S., Yang, H., 2014. Acceleration of the herbicide isoproturon degradation in wheat by glycosyltransferases and salicylic acid. J. Hazard. Mater. 283, 806–814. Marcacci, S., Raveton, M., Ravanel, P., Schwitzguebel, J.P., 2006. Conjugation of atrazine in vetiver (Chrysopogon zizanioides Nash) grown in hydroponics. Environ. Exp. Bot. 56, 205–215. Merini, L.J., Bobillo, C., Cuadrado, V., Corach, D., Giulietti, A.M., 2009. Phytoremediation potential of the novel atrazine tolerant Lolium multiflorum and studies on the mechanisms involved. Environ. Pollut. 157, 3059–3063. Mizutani, M., Ohta, D., 2010. Diversification of P450 genes during land plant evolution. Annu. Rev. Plant Biol. 61, 291–315. Moore, M.T., Kröger, R., 2010. Effect of three insecticides and two herbicides on rice (Oryza sativa) seedling germination and growth. Arch. Environ. Contam. Toxicol. 59, 574–581. Nelson, D.R., Schuler, M.A., Paquette, S.M., Werck-Reichhart, D., Bak, S., 2004. Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot. Plant Physiol. 135, 756–772. Panuwet, P., Nguyen, J.V., Kuklenyik, P., Udunka, S.O., Needham, L.L., Barr, D.B., 2008. Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction-high-performance liquid chromatography–tandem mass spectrometry. Anal. Bioanal. Chem. 391, 1931–1939. Powles, S.B., Yu, Q., 2010. Evolution in action: plants resistant to herbicides. Annu. Rev. Plant Biol. 61, 317–347. Qiu, X., Li, W., Tian, Y., Leng, X.L., 2003. Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera, Noctuidae): tissue difference and induction. J. Econ. Entomol. 96, 1283–1289. Ramel, F., Sulmon, C., Cabello-Hurtado, F., Taconnat, L., Martin-Magniette, M., Renou, J., El Amrani1, A., Couée, I., Gouesbet., G., 2007. Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose-induced tolerance. BMC Genomics 8, 450. Ro, D.K., Ehlting, J., Douglas, C.J., 2002. Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar. Plant Physiol. 130, 1837–1851. Sathiakumar, N., MacLennan, P.A., Mandel, J., Delzell, E.A., 2011. Review of epidemiologic studies of triazine herbicides and cancer. Crit. Rev. Toxicol. 41, 1–34. Shimabukuro, R.H., 1968. Atrazine metabolism in resistant corn and sorghum. Plant Physiol. 43, 1925–1930. Shirakura, S., Ito, K., Aizawa, H., 1995. Effect of cytochrome P450 monooxygenase inhibitors on the rice tolerance of azimsulfuron and bensulfuronmethyl. Weed Res. 40, 218–220. Siminszky, B., 2006. Plant cytochrome P450-mediated herbicide metabolism. Phytochem. Rev. 5, 445–458. Singh, S., Kirkwood, R.C., Marshall, G., 1998. Effect of the monooxygenase inhibitor piperonyl butoxide on the herbicidal activity and metabolism of isoproturon in herbicide resistant and susceptible biotypes of Phalaris minor and wheat. Pestic. Biochem. Physiol. 59, 143–153. Su, Y.H., Zhu, Y.G., Lin, A.J., Zhang, X.H., 2005. Interaction between cadmium and atrazine during uptake by rice seedlings (Oryza sativa L.). Chemosphere 60, 802–809. Topal, A., Adams, N., Hodgson, E., Kelly, S.L., 1996. In vitro metabolism of atrazine by tulip cytochrome P450. Chemosphere 32, 1445–1451. Varsano, R., Rabinowitch, H.D., Rubin, B., 1992. Mode of action of piperonyl butoxide as herbicide synergist of atrazine and terbutryn in maize. Pestic. Biochem. Physiol. 44, 174–182. Wang, H.C., Li, J., Lv., B., Lou, Y.L., Dong, L.Y., 2013. The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.). Pestic. Biochem. Physiol. 107, 334–342. Wang, Q.Q., Liu, F., Chen, X.S., Ma, X.J., Zeng, H.Q., Yang, Z.M., 2010. Transcriptome profiling of early developing cotton fibre by deep-sequencing reveals significantly differential expression of genes in a fuzzless/lintless mutant. Genomics 96, 369–376. Werck-Reichhart, D., Hehn, A., Didierjean, L., 2000. Cytochromes P450 for engineering herbicide tolerance. Trends Plant Sci. 5, 116–123. Yang, Y., Wu, Y., Chen, S., Devine, G.J., Denholm, I., Jewess, P., Moores, G.D., 2004. The involvement of microsomal oxidases in pyrethroid resistance in Helicoverpa armigera from Asia. Insect Biochem. Mol. Biol. 34, 763–773. Yang, Z.M., Wang, J., Wang, S.W., Xu, L.L., 2003. Salicylic acid-induced aluminum tolerance by modulation of citrate efflux from roots of Cassia tora L. Planta 217, 168–174. Zhang, J.J., Lu, Y.C., Zhang, J.J., Tan, L.R., Yang, H., 2014. Accumulation and toxicological response of atrazine in rice crops. Ecotoxicol. Environ. Saf. 102, 104–115. Zhang, J.J., Zhou, Z.S., Song, J.B., Liu, Z.P., Yang, H., 2012. Molecular dissection of atrazine responsive transcriptome and gene networks in rice by high-throughput sequencing. J. Hazard. Mater. 219, 57–68. Zhou, Z.S., Yang, S.N., Li, H., Zhu, C.C., Liu, Z.P., Yang, Z.M., 2013. Molecular dissection of mercury-responsive transcriptome and sense/antisense genes in Medicago truncatula. J. Hazard. Mater. 252, 123–131. Zhou, Z.S., Zeng, H.Q., Liu, Z.P., Yang, Z.M., 2012. Genome-wide identification of Medicago truncatula microRNAs and their targets reveals their differential regulation by heavy metal. Plant Cell Environ. 35, 86–99.