Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa

Sep 21, 2017 - focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environ...
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Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago sativa) Jing Jing Zhang,†,‡ Jiang Yan Xu,† Feng Fan Lu,† She Feng Jin,† and Hong Yang*,† †

Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural University, Nanjing 210095, China College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China



S Supporting Information *

ABSTRACT: Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich nonprotein compounds and play primary and multiple roles in cellular redox homeostasis, enzyme activities, and xenobiotics detoxification. This study focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environment. Using high-throughput RNAsequencing, a set of ATZ-responsive thiols-related protein genes highly up-regulated and differentially expressed in alfalfa was identified. Most of the differentially expressed genes (DEGs) were involved in regulation of biotic and abiotic stress responses. By analyzing the genes involved in thiols-mediated redox homeostasis, we found that many of them were thiolssynthetic enzymes such as γ-glutamylcysteine synthase (γECS), homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using liquid chromatography−mass spectrometry/ mass spectrometry (LC−MS/MS), we further characterized a group of ATZ-thiols conjugates, which are the detoxified forms of ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite detected by MS. Several other ATZconjugates were also examined as ATZ-detoxified metabolites. Such results were validated by characterizing their analogs in rice. Our data showed that some conjugates under ATZ stress were detected in both plants, indicating that some detoxified mechanisms and pathways can be shared by the two plant species. Overall, these results indicate that LMW thiols play critical roles in detoxification of ATZ in the plants.

1. INTRODUCTION Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine, ATZ) is a herbicide widely used for controlling weeds or grasses in fields for crop production.1 In China, 10−15 million kg of ATZ is input to the farmland yearly, leading to the widespread contamination of its residues in environments.2 Since ATZ is environmentally persistent with a half-life being about 57 weeks,3 the bulk soil bearing the ATZ residues largely exceeds its natural degradation capacity.4 It has been estimated that the concentration of ATZ residues in waters near the ATZtreated fields is up to 1 mg L−1.5 As a consequence, the soil residues of ATZ not only negatively affect crop production, but also wildlife and human health through food chains.6,7 Alfalfa is an important forage grass planted in the way of rotation with rice or corn in many parts of the world.8 The residues of ATZ in the field of former-planted cereal crops usually impact the growth of alfalfa through the mechanism by interference of photosystem II.9 Our recent studies have shown that ATZ is easily absorbed by alfalfa, which thereby triggers toxic symptoms such as stunt growth, leaf chlorosis, oxidative stress, and many other phenotypes of dysfunction.10 On the other hand, rice is a staple food crop providing the major calories for human diet in Asia and many other areas of the © 2017 American Chemical Society

world. As a model plant, rice is often used to investigate its adaptability to various environmental stresses.11,12 Recently, genome-wide identification of transcriptome and DNA methylation profiled a large number of specific loci for ATZ detoxification and degradation,13,14 and these resistant (tolerant) genes encoding enzymes such as glycosyltransferases, glutathione transferases, ABC transporters, and laccases have been identified in plants.13,15 Plant thiols, with mercapto groups, are endogenous substances abundantly existing in vegetable kingdom, among which LMW thiols plays unique roles in secondary metabolisms and redox homeostasis when plants are exposed to unfavorable environmental stress because thiolate anion is one of the strongest biological nucleophiles in the cell.16 Of these, cysteine (Cys) and glutathione (GSH) are proposed to be the major components.16 Both molecules can separately or coordinately participate in the process of plant defense to invasive toxicants.17,18 For example, toxicants or their metabolites with electrophilic properties may damage macromolecules in cells, but the active electrophilic agents can be drastically detoxified Received: June 15, 2017 Published: September 21, 2017 1835

DOI: 10.1021/acs.chemrestox.7b00166 Chem. Res. Toxicol. 2017, 30, 1835−1846

Article

Chemical Research in Toxicology by thiols-containing molecules in plants.16 There is another subgroup of thiols-compounds called GSH analogues (or derivatives) such as homoglutathione (hGSH) and hydroxymethyl glutathione (hmGSH). Basically, these compounds have the functions similar to GSH. In plants, the majority of pesticides tend to trigger oxidative stress by overgenerating reactive oxygen species (ROS).15,19,20 Plants have evolved enzymatic and nonenzymatic antioxidant systems to lower the concentration of ROS and its damage to cells. 15,16,20 Maintenance of cellular redox balance by interconversion of mercaptan/disulfide bonds of thiols is a crucial strategy for the antioxidant system in plants.16 Several enzymes involved in oxidation and reduction of thiols such as glutaredoxin (Grx), thioredoxin (Trx), glutathione peroxidase (GPX), and glutathione reductase (GR) play crucial roles in scavenging ROS.15−17,21 Although these enzymes and those involved in glutathione-mediated antioxidation to toxicants have been well documented in mammals and plants,10,14,22 there are few such studies in alfalfa, particularly with comparatively analysis on removing toxicants such as pesticides. In this study, we attempted to identify transcripts of oxidoreductases (Grx, Trx, GPX, GHL, etc.), which are closely associated with synthesis of thiols in the glutathione metabolic pathway in ATZ-exposed plants. The ATZ-responsive transcripts and metabolites were characterized in alfalfa. Thus, the goal of the study is to highlight the role of thiols-compounds in mediating ATZ detoxification and to figure out the detoxified mechanisms of ATZ in this legume.

cut into 5 mm length, submerged in 10 mL of deionized water, and maintained at 32 °C for 2 h. The electrical conductivity of the medium (EC1) was read using an electrical conductivity meters (METTLER TOLEDO FE30-FiveEasy). The samples were autoclaved at 121 °C for 20 min to release all electrolytes completely and cooled to the room temperature. The electrical conductivity of killed tissues (EC2) was measured. The electrolyte leakage (EL) was calculated using the following formula: EL = EC1/EC2 × 100.14 2.4. RNA Sequencing and Data Processing. Total RNA was extracted from roots and shoots using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Four libraries were constructed including Root-ATZ (root control, ATZ-free), Root+ATZ (ATZ-treated), Shoot-ATZ (shoot control), and Shoot+ATZ. mRNA (6 μg) was enriched using oligo (dT) magnetic beads and fragmented into short fragments using fragmentation buffer (Invitrogen, USA). The first strand and double strand cDNA were synthesized subsequently. The quality of PCR products was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). The high quality samples were sequenced using the Illumina HiSeq 2500 system. The transcripts of two technologies (454 and Illumina technologies) were combined to get longer and more accurate transcripts. To get a set of Unigenes, the transcripts of two projects studying Medicago sativa were downloaded and clustered with the Gene Indices Clustering Tools (software) to remove redundancy.23 The level of gene expression was calculated by the numbers of genes uniquely mapped to Unigenes using fragments per thousand bases per million reads (FPKM) (eXpress, software).24 Fold changes for differentially expressed genes (DEGs) were calculated and p-value was used to determine the threshold (p ≤ 0.05 and fold change ≥2) for judging the significance of gene expression. Cluster analysis of gene expression patterns was performed using pheatmap package in R (software). The settings for the calculations were described as follows: similarity was measured by euclidean, and clustering method was complete linkage. Each column represents an experimental sample, while each row represents a DEG. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the pathways for genes from the alfalfa data sets. A criterion with e-value threshold 1e−5 was set. The statistical significance of the terms analyzed was calculated with twosided enrichment/depletion hypergeometric test and Bonferroni pvalue correction. 2.5. Measurement of ATZ Accumulation in Alfalfa and Rice. Fresh tissues were harvested and ground using liquid nitrogen. The ground powders (3.0 g) were dissolved in 25 mL of acetone-distilled water (3:1, V:V) and subjected to ultrasonic extraction for 30 min. After centrifuged, the supernatant was collected. This step was repeated in triplicate. The pooled supernatant was vaporized at 42 °C to remove acetone using a rotary vacuum evaporator. The residual water was liquid−liquid extraction by petroleum ether for three times. The extract was concentrated up to dryness. Methanol (0.5 mL) was used to dissolve the residue and 20 mL of distilled water was added to dilute it. The mixture was purified using LC-18 solid phase extraction (SPE) column. Methanol (2 mL) was used to wash the ATZ sample. The content of ATZ was quantified by the external standard method using high performance liquid chromatography (HPLC) (Waters 515; Waters Technologies Co. Ltd., USA) with a UV detector. The performance of HPLC was conducted under the following condition: Hypersil reversedphase C18 column (Thermo, 250 mm × 4.6 mm i.d.); mobile phase, methanol/HPLC grade water (65:35, V/V); wavelength, 225 nm; flow rate, 0.6 mL/min.10 The standard curve ranging from 0.1−10 mg/L of ATZ (R2 = 0.9999) for the quantification of HPLC was shown in Figure S1. The spiked recoveries and relative standard deviation (RSD) of the method for the two plants were 85.32−110.52% and