Article pubs.acs.org/JAFC
Rice (Oryza sativa) Laccases Involved in Modification and Detoxification of Herbicides Atrazine and Isoproturon Residues in Plants Meng Tian Huang,†,∥ Yi Chen Lu,†,Δ,∥ Shuang Zhang,§ Fang Luo,† and Hong Yang*,†,‡,∥ †
Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, China § State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China Δ College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211800, China ‡
S Supporting Information *
ABSTRACT: Atrazine (ATR) and isoproturon (IPU) as herbicides have become serious environmental contaminants due to their overuse in crop production. Although ATR and IPU in soils are easily absorbed by many crops, the mechanisms for their degradation or detoxification in plants are poorly understood. This study identified a group of novel genes encoding laccases (EC 1.10.3.2) that are possibly involved in catabolism or detoxification of ATR and IPU residues in rice. Transcriptome profiling shows at least 22 differentially expressed laccase genes in ATR/IPU-exposed rice. Some of the laccase genes were validated by RT-PCR analysis. The biochemical properties of the laccases were analyzed, and their activities in rice were induced under ATR/ IPU exposure. To investigate the roles of laccases in degrading or detoxifying ATR/IPU in rice, transgenic yeast cells (Pichia pastoris X-33) expressing two rice laccase genes (LOC_Os01g63180 and LOC_Os12g15680) were generated. Both transformants were found to accumulate less ATR/IPU compared to the control. The ATR/IPU-degraded products in the transformed yeast cells using UPLC-TOF-MS/MS were further characterized. Two metabolites, hydroxy-dehydrogenated atrazine (HDHA) and 2-OH-isopropyl-IPU, catalyzed by laccases were detected in the eukaryotic cells. These results indicate that the laccase-coding genes identified here could confer degradation or detoxification of the herbicides and suggest that the laccases could be one of the important enzymatic pathways responsible for ATR/IPU degradation/detoxification in rice. KEYWORDS: laccase, detoxification, atrazine, isoproturon, rice (Oryza sativa)
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human health.13,14 Natural degradation by soil microorganisms may eliminate the toxicity of the compounds. However, a complete removal of the contaminants often takes a long time, particularly for persistent pesticides. Due to the nonspecific target, most pesticides (or residues) are readily absorbed by crops. Once taken up in plants, these toxicants are subject to biodegradation through multiple metabolic and transformation pathways.15 In higher plants, several processes have been proposed for pesticide metabolism and detoxification.16 These processes can be divided into three major steps including chemical modification or activation (phase I), conjugation (phase II), and transport/compartmentalization (phase III). Several metabolic enzymes, such as cytochrome P450 in phase I and glutathione-S-transferase in phase II, are well-known to regulate degradation of pesticide.15,17,18 Laccases (EC 1.10.3.2) as multicopper oxidoreductases are able to polymerize lignin monomers in vitro, but recent studies have shown that some laccase genes could be also novel regulators for degrading aromatic or other toxic compounds.5,19 However, reports of
INTRODUCTION Laccases (EC 1.10.3.2) constitute a family of copper-containing oxidase enzymes that use molecular oxygen to catalyze the monoelectronic oxidation of natural metabolites or artificial substrates (e.g., phenolic and aromatic substrates) via a mechanism involving redicals.1,2 Laccases exist in many fungi and plants, and more than a hundred compounds have been identified as substrates for fungal laccases.2−4 Typical substrates of laccases include monophenols, polyamines, aminophenol, aryl diamine, and some inorganic ions (e.g., Cu). The major biological functions of laccase are involved in lignin biodegradation,5,6 cell wall lignification,7 and detoxification of some toxic compounds such as polyaromatic hydrocarbons (PAHs),8,9 organophosphorus pesticides, or azodyes.10,11 It is apparent that molecular modification and detoxification could be one of the major functions for laccases in organisms. To date, most studies have concerned functional laccases on xenobiotics (e.g., pesticides or herbicides) in fungi and bacteria, but very few have described laccase working against toxic compounds in higher plants.9,12 The presence of pesticides in the environment is a matter of particular concern for the conservation of ecosystems. It is also the major problem for crop production because soil contamination with such compounds risks food safety and © 2016 American Chemical Society
Received: Revised: Accepted: Published: 6397
May 14, 2016 July 22, 2016 August 7, 2016 August 8, 2016 DOI: 10.1021/acs.jafc.6b02187 J. Agric. Food Chem. 2016, 64, 6397−6406
Article
Journal of Agricultural and Food Chemistry
The crude homogenized tissues were centrifuged at 12000g at 4 °C for 30 min. The supernatant was collected and used to determine the protein content and laccase activity. Laccase activities were determined using the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate.33 An Alpha-1506 spectral photometer (Puyuan, Shanghai) that monitors the time-increase absorbance at 420 nm (ε = 18460 M−1 cm−1) gave rise to the oxidation of ABTS to the cation radical ABTS•+. The assay mixture contained 20 mM sodium acetate buffer (pH 3.6), 5 mM ABTS, and the appropriate amount of diluted extract fluid. Laccase activity was expressed in units defined as μmol of ABTS•+ formed from ABTS min−1 (U) and g−1 of crude protein (U g−1 protein) at 25 °C. The protein of sample extract was quantified according to the method of Bradford.34 RNA Isolation. Six samples were collected including shoot with/ without atrazine or isoproturon and root with/without atrazine or isoproturon, which were generated from the RNA library received from tissues treated with 0 mg L−1 (control) and 0.4 mg L−1 atrazine or 2.0 mg L−1 isoproturon for 2, 4, and 6 days (samples from each time point were pooled), respectively. Fresh leaves and roots were collected with sterilized aluminum foil after removal of the residues and immediately stored in liquid nitrogen for the following experiments. Total RNA was extracted from sample using Trizol (Invitrogen, Carlsbad, CA, USA). One microgram of RNA sample was incubated at 37 °C with 1 unit of RNase-free DNaseI (Takara) for 30 min to remove the genomic DNA and incubated with 1 μL of 50 mM EDTA for DNaseI inactivation at 65 °C for 10 min. The integrity of RNA was checked by running 1% agarose gel stained by ethidium bromide. 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 on the basis of 28S/18S rRNA band intensity (2:1) and A260 nm/A280 nm ratios between 1.8 and 2.0, A260 nm/A230 nm ratios >1.5.17 The high-throughput transcriptome analysis was described previously.35 Transcript Analysis by Quantitative RT-PCR. A 20 μL reverse transcription reaction mixture contained 1 mg of RNA, 1 mL of 100 mM oligo (dT) primers, 1 μL of 10 mM deoxyribonucleotide triphosphated (NTP) mixture, 0.5 μL of 200 U/μL M-MLV reverse transcriptase (TaKaRa Biochemical), and 4 μL of 5× M-MLV buffer. The mixture was incubated at 42 °C for 30 min and heated at 85 °C for 5 min to inactivate the reverse transcriptase. The cDNA was synthesized from 20 mL of the mRNA solution using the identical reaction conditions indicated above. The resultant cDNA was diluted 5-fold with sterile water and stored at −20 °C for subsequent quantitative RT-PCR (qRT-PCR) analysis. Quantitative RT-PCR was performed using a My-IQ Single Color Real time PCR system (Bio-Rad) and IQ5 software (Bio-Rad).36 Each reaction was prepared with SYBR Green PCR Core Reagents (TaKaRa Biochemical) and performed in a final volume of 25 μL containing 2 μL of diluted template cDNA, 12.5 μL of the 2× TransStart Top Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.), and 200 nM primers (Data S1). The conditions for PCR reaction were as follows: initial denaturing step of 1 cycle of 94 °C for 30 s, followed by 36 cycles of 94 °C for 5 s, and 60 °C for 30 s for annealing and extension. Each qRT-PCR analysis was performed in triplicate. All reactions monitoring the dissociation curve to detect and eliminate the possible primer−dimer and nonspecific amplifications were run in triplicate. Gene expression was presented using a modification described previously.37 The ubiquitin gene was used as an internal standard for normalization. Cloning of Two Laccase Genes and Transformation in Yeast. Two laccase genes encoding LOC_Os01g63180 (from exposure to IPU) and LOC_Os12g15680 (from exposure to ATR) were cloned and amplified by successive RT-PCR. The PCR reaction system was run with NEB Phusion high-fidelity DNA polymerase under the following conditions: initial denaturation at 98 °C, 30 s; denaturation at 98 °C, 10 s; annealing at 61 °C (LOC_Os01g63180) or 62 °C (LOC_Os12g15680), 30 s; extension at 72 °C, 1 min 50 s with 36 cycles; and final extension at 10 °C to ensure complete extension. The primers are summarized in Data S1.
laccase involvement in pesticide detoxification are still rare. The detailed biochemical and molecular processes are largely unknown. Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5triazine, ATR] and isoproturon [3-(4-isopropylphenyl)-1,1dimethylurea, IPU] are herbicides that represent a wide spectrum of broadleaf weeds in the field of crops such as corn, sorghum, wheat, sugar cane, lupins, and rice.20−22 Whereas ATR belongs to the triazine family, IPU is a representative member of the phenylurea herbicide family with a similar function.23 Both were originally designed to block photosystem II through the electron transport necessary for photosynthesis in weeds.24 Over the past decades, the largescale application of ATR/IPU during crop rotation led to widespread residues in soils, ground waters, and crops.24−27 ATR is persistent in the deep or near soil layer,28,29 whereas IPU is less persistent due to its moderately hydrophobic nature and weak soil absorption. Such chemophysical properties are conducive to the uptake of ATR/IPU by monocotyledonous crops such as rice and wheat from wheat−rice rotation soils.30 However, the metabolism of the two herbicides in plants remains poorly understood.14 To figure out the molecular mechanism underlying the process of ATR/IPU degradation, we screened genome-wide transcripts of laccase isoforms responsive to ATR/IPU and identified the laccases responsible for ATR/IPU transformation and degradation in rice (Oryza sativa). The identified laccase genes in response to atrazine or isoproturon were validated by quantitative RT-PCR (qRT-PCR). The total activity of laccase was found to be increased in the ATR/IPU-exposed plants. We further developed recombinant proteins in yeast to prove the roles of two laccase genes (LOC_Os12g15680 and LOC_Os01g63180) in degrading ATR and IPU in eukaryotic cells. Furthermore, UPLC-MS was used to detect several ATR/ IPU-degraded metabolites in yeast transformants. Thus, the goal of the study was to identify the laccase genes in response to ATR/IPU in rice and functionally prove that some of the laccase genes were responsible for the degradation of ATR and IPU in rice. These data may elucidate the mechanism for rice active degradation of ATR/IPU residues in the environment.
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MATERIALS AND METHODS
Plant Materials and Treatment. Seeds of rice (O. sativa, Japonica) were disinfected with 3% H2O2 solution, washed with distilled water, and germinated in an incubator under darkness at 30 °C for 48 h. The sprouted seedlings were transferred in a black polyvinyl chloride plate floating on the half-strength Hoagland nutrient solution.31 Each pot contained 20 seedlings growing in a growth chamber (PGX-350D, SAFE Co.) Rice seedlings at three-leaf stage were treated with 0.4 mg L−1 atrazine or 2.0 mg L−1 isoproturon in the nutrient solution for 2, 4, and 6 days. The pH of the nutrient solution was adjusted to 5.8. The solution was renewed every 2 days. Each pot contained 20 seedlings under the condition of 25/20 °C (day/night), 300 μmol m−2 s−1 artificial illumination with 14/10 h period (light/ dark), and 80% humidity in the growth chamber. Each treatment was biologically repeated in triplicate. Atrazine and isoproturon as technical materials were obtained from the Institute of Pesticide Science, Academy of Agricultural Sciences in Jiangsu, Nanjing, China, with a purity of 98.0% and 96.9%, respectively. Assay of Laccase Activity. Specific activities of laccases (EC 1.10.3.2) were assayed according to the method of Johannes and Majcherczyk.32 Fresh rice tissues (1.0 g of shoot and 0.5 g of root) were ground and extracted with 50 mM Tris-HCl (pH 7.8) buffer containing 50 mM NaCl, 1 mM EDTA, and 1.5% (w/v) polyvinylpolypyrrolidone under the condition of an ice-bath in mortar. 6398
DOI: 10.1021/acs.jafc.6b02187 J. Agric. Food Chem. 2016, 64, 6397−6406
Article
Journal of Agricultural and Food Chemistry
Figure 1. Summary of differentially expressed genes of rice laccases exposed to ATR and IPU: heat maps of expression patterns of laccase genes from roots and shoots of rice (Oryza sativa) treated with atrazine (A) and with isoproturon (B). The color (representing fold change values) shows up or down regulation of laccase genes, with >2-fold changes and with p value 2 or
