| Pesticides,as essential materials in agricultural production,play an important role in the prevention of diseases,insects and weed,and production increase of agricultural and sideline products.However,in the process of intensive agricultural production,the long-term,large or non-standard use of pesticides results in pesticide residues in the environment.Atrazine is a widely used herbicide worldwide.Although it has been banned by the European Union,but many developing countries still use it.Due to the migration and diffusion of atrazine,not only water,soil and atmospheric pollution occur,but also human health is threatened through the food chain enrichment.Residues of pesticides in the environment are excessively absorbed by the crop,and cause the metabolic disorders and damage to crop.Therefore,the study of metabolism and detoxification of atrazine in plants is particularly important.In this paper,the toxicity and accumulation of atrazine in alfalfa were studied.The degradation products of atrazine in alfalfa were detected by Liquid Chromatography-Mass spectrometry/Mass spectrometry(LC-MS/MS).The molecular mechanism of regulation and detoxification of atrazine in alfalfa was studied by high throughput sequencing.Glycosyltransferase(GT)is one of the most important detoxifying enzymes in plants.The rice and Arabidopsis thaliana of overexpressing atrazine-responsive GT gene were obtained by molecular cloning technology.It was found that this gene can alleviate the toxicity of atrazine to plants and accelerate the degradation of atrazine in plants.Based on the analysis of the degradation products of atrazine in alfalfa and rice,it was shown that thiol compounds played an important role in the degradation of atrazine.Combined with the data of transcriptome and MS,it was indicated that gene expression of thiol metabolism related enzymes was changed under atrazine stress,and thiols played the different role in rice and alfalfa metabolic pathways.Specific content was as follows:1.In order to investigate the damage of atrazine residues to alfalfa,a series of atrazine concentrations(0,0.02,0.04,0.06,0.08 and 0.10 mg/L)were used to treat alfalfa seedlings.Atrazine inhibited the elongation,dry weight and chlorophyll content of alfalfa.Atrazine affected the normal growth of alfalfa.The content of malondialdehyde in alfalfa first increased and then decreased with the increase of atrazine concentration,and reached the highest value at the treatment concentration of 0.08 mg/L,suggesting the greatest membrane lipid damage of alfalfa.The accumulation of atrazine in alfalfa was detected by pretreatment of acetone-water ultrasonic extraction and purification of C18-SPE column.It was found that the accumulation of atrazine in alfalfa gradually increased with the increase of atrazine concentration and the time of treatment.The data of bioconcentration factor(BCF)and translocation factor(TF)showed that alfalfa had a strong accumulation and conduction effect on atrazine(TF>1).The degradation products of atrazine in alfalfa were identified using UPLC-LTQ-Orbitrap XL-MS/MS.A total of 10 metabolites and 9 conjugates were found.The results showed that atrazine absorbed in alfalfa can be chemically modified into the corresponding derivatives,such as DIA,DHA and MEA and so on.The different functional groups in atrazine and some derivatives reacted with sugars,glutathione and amino acids to form conjugates such as HIA+Glc-H2O,HEA+ Glc-H2O,ATZ-HCl+Cys,ATZ-HCl+hGSH,DHA+hGSH and MEA-HCl+hGSH.Among them,HIA+Glc-H2O,HEA+Glc-H2O,ATZ-HCl+Cys,DHA+Cys,MEA-HCl+hGSH and MEA-HCl+Cys were first found in plants.These results indicated that atrazine can be degraded in alfalfa through different detoxification pathways.Under the treatment of atrazine,glycosyltransferase and glutathione S-transferase activity in alfalfa increased,suggesting that they may play an important role in the detoxification of atrazine.The results revealed the enrichment and detoxification mechanism of atrazine in alfalfa.2.To study the molecular mechanism of atrazine stress in alfalfa,high-throughput sequencing(RNA-Seq)was used to perform large-scale sequencing of alfalfa transcriptome.Four libraries were constructed,including Root-ATZ(root control,without atrazine),Shoot-ATZ(Shoot control),Root+ATZ(root treatment,plus atrazine)and Shoot+ATZ(Shoot treatment).They were used to study the transcriptional changes of different tissues of alfalfa under atrazine stress.A total of 70370 transcripts were obtained.A large number of differentially expressed genes(DEGs)were found by comparison between each library.All DEGs were classified by cluster analysis.Furthermore,Gene Ontology(GO)annotation analysis and Pathway enrichment analysis of DEGs were carried out.It was found that the biological function of many differentially expressed genes was involved in stress response,exogenous degradation and metabolic response.In addition,gene expression in the primary metabolites and secondary metabolites synthetic pathway changed.The differentially expressed genes related to atrazine detoxification(such as GSTs,GTs and ABC transporters)and stress responses(such as redox reactions,conjugations and hydrolysis reactions)were analyzed.And it was found that the related genes in alfalfa were significantly induced.The real-time quantitative PCR and semi-quantitative PCR of eight genes related to degradation of xenobiotics(cytochrome P450,glycosyltransferase and glutathione transferase)were carried out to confirm the accuracy of the sequencing results.The expression pattern of the selected gene was consistent with that of RNA-Seq sequencing.The above results provide a reference value for the study of stress responsive and detoxification genes.3.Plants have evolved complex detoxification pathways to cope with the stress of atrazine in the environment,in which glycosyltransferase is one of the most important detoxifying enzymes.Rice is an important food crop in China.At present,the whole genome sequencing of rice has been completed.Therefore,we selected an atrazine responsive glycosyltransferase gene(OsARGT1)as the research object.First,three rice and three Arabidopsis thaliana OsARGT1 homozygous overexpression lines were obtained,respectively.Compared with the wild type,the elongation,dry weight and chlorophyll content of overexpression rice were significantly increased and the damage of membrane was decreased after 6 d treatment of 0.2,0.4 and 0.8 mg/L atrazine.More importantly,OsARGT1 promotes the degradation of atrazine in overexpression rice.At the same time,the removal of atrazine in the nutrient solution of overexpression rice was higher than that of the wild type.In order to further explore the effect of OsARGT1 on the degradation of atrazine in the environment,we selected 0.01 mg/L atrazine as the treatment concentration,which was close to the environmental pollution concentration.After rice was exposed to this concentration for a long time,the amount of atrazine in rice was assayed.It was found that the contents of atrazine in the old leaves,mature leaves,new leaves and roots of the overexpression rice were lower than those of the wild type after 30 and 60 d exposure.After 100 d exposure,the content of atrazine in the flag leaf of overexpression rice was lower than that of wild type.After 120 d of exposure,the accumulation of atrazine in OsOX-1 grain was lower than that in wild type,which was reduced by 39.84%.In order to further explore the detoxification mechanism of OsARGT1 for atrazine,the degradation of atrazine in the line OsOX-1 of overexpression rice and wild-type rice was detected by UPLC-AB SCIEX Triple TOF 5600 mass spectrometry.Eight metabolites and ten conjugates of atrazine were detected,in which the glucose and hydroxymethyl glutathione conjugates of atrazine DIHA+Glc-H2O,HA+Glc-H2O and ATZ-HCl+hmGSH were detected for the first time in plants.The contents of 5 metabolites and 7 conjugates in shoots of OsOX-1 were higher than those in wild type,and the contents of 2 metabolites and 4 conjugates in roots were higher than those in wild type.OsARGTl is an exogenous gene for Arabidopsis thaliana,and the gene is transferred into Arabidopsis to further verify its detoxification function.The results showed that under atrazine stress,the dry weight and chlorophyll content of overexpression Arabidopsis were significantly higher than that of wild type.In addition,the content of atrazine in overexpression Arabidopsis was significantly lower than that of wild type,and the content of atrazine in the nutrient solution of overexpression lines was lower than that of wild type,indicating that OsARGT1 promoted the degradation of atrazine in Arabidopsis thaliana as well.Above these results showed that the glycosyltransferase can regulate the detoxification and degradation of pesticides in plants.4.It was found that the conjugation of atrazine with mercapto thiols was anothor main pathway in plants.Therefore,combined with transcriptome and MS data,the role of thiol in atrazine response and detoxification in plants was studied.Firstly,the content of reactive oxygen species(H2O2)in leaves of alfalfa was assayed by tissue staining.It was found that atrazine induced the production of H2O2 and produced oxidative stress in alfalfa.Thiols has many biological roles,including the defense of reactive oxygen species and free radicals,maintaining cell redox status and defensive active electrophilic reagents.Therefore,the gene expression of thiol redox-associated enzymes(Grx,GPX,GL and GHL,etc.)was further studied.Under the atrazine stress,many of the relevant genes were differentially expressed.Five genes encoding Grx were significantly up-regulated.Two genes encoding GPX were significantly up-regulated in shoot.One GSNOR gene was significantly down-regulated by atrazine.By analyzing the differential expression gene in the glutathione metabolic pathway,it was found that most of the glutathione synthetases(?ECS,hGSHS and GSHS)were up-regulated and the decomposition enzyme GGT was down-regulated.APN was up-regulated in shoot and down-regulated in root.The thiols in glutathione metabolic pathway form the S-conjugate with atrazine in plants.Cysteine S-conjugate ATZ-HCl+Cys was detected the highest intensity by mass spectrometry.Thus,the expression of genes in cysteine synthesis pathway was analyzed under atrazine stress in alfalfa.Electrophilic atrazine can cause damage to plants cells.Thiols can hinder the activity of these unwanted electrophilic agents and play an important role in the defense of xenobiotic.Therefore,we summarized the thiol S-conjugates of atrazine in alfalfa and rice.The metabolic pathways were drawn.The results showed that the cysteine S-conjugate was the most important metabolite in this two plants.The hGSH S-conjugate was only detected in alfalfa and the hmGSH S-conjugate was only detected in rice.Furthermore,the relative content of other S-conjugates was different in these two plants,indicating that the thiols have differences in detoxification of pesticide in different plants species. |
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