Research On Detoxification And Metabolism Mechanism Of Atrazine In Rice By DNA Demethylation

Pesticide is important input for ensuring a stable increase of crop yield in agricultural production.At the same time,the non-standard and excessive use of pesticides has damaged the stability of agricultural ecological environment.Pesticide residues are one of the environmental pollutants seriously affected crop production,food safety and human health.Herbicide atrazine(ATZ)has been used in China and other countries of the world for many years.Due to long half-life and poor absorption,ATZ concentration in surface water and groundwater is over limits,resulting in soil pollution,and even has negative effect on the growth of succeeding crops.ATZ residue in the environment is readily absorbed and accumulated by crops thus threatening the ecological environment and human health through the food chain.Thus,it is of great significance to study degradation mechanism of ATZ in crops and reduce the risk of ATZ to human.The mechanism of CG DNA demethylation alleviating the toxicity of ATZ to rice and accelerating the degradation of ATZ in rice was studied in this dissertation.In order to find out the molecular mechanism of DNA demethylation involved in ATZ degradation in rice,the relationship between DNA methylation and transcription mechanism in rice under ATZ stress was established by high-throughput sequencing(Bisulfite sequencing,BS-Seq and RNA sequencing,RNA-Seq),Through a cross analysis of DNA methylome and transcriptome,three combined genes were screened.It was found that the activation of demethylated genes promoted the degradation of ATZ by heterologous expression of Pichia.In addition,the genes that are related to jasmonate acid synthesis or signal transduction were analyzed.Among them,the up-regulated hypomethylated gene was screened and then it was revealed that the gene was involved in detoxification and metabolism.Main content was as follows:To figure out the effect of DNA methylation on ATZ detoxification and metabolism in rice,mutants Osmet1 defective in CG DNA methylation and wild types were tested.Compared with wild type,the elongation,dry weight and chlorophyll content of mutants were increased while the electrolyte permeability was decreased after treatment with 0.2 mg/L ATZ for 6 d.The ATZ concentration in rice and growth medium was determined by high-performance liquid chromatography(HPLC).It was found that the accumulation of ATZ in mutants was less than wild types while the removal of ATZ in growth medium by mutants was higher.To simulate the actual ATZ concentration in environment,the rice was treated with 0.01 mg/L ATZ for 120 days and during the entire developmental stage,and the ATZ content in different tissues were measured.The accumulation of ATZ in different leaves(old leaves,new leaves and mature leaves)and roots of the mutant were lower than that of the wild type.After 120 days of treatment,the ATZ concentration in the grains of the mutant was only 67.1%and 82.7%of the wild-type.In order to further investigate the degradation of ATZ in the mutant and wild-type,we characterized ATZ-metabolites by HRLC-Q-TOF-MS/MS.A total of eight degradation products and eight conjugates were identified.Of these,one degradation product(DMHA)and two conjugates(ATZ-HCl+Cys/HAc and ATZ-NGlcA)were first identified in plants.By the relative quantitative analysis of ATZ and its metabolites,it was found that the content of the parent ATZ in the mutant was lower than that of the wild type.Furthermore,sixteen metabolites content in the mutant were higher than that of the wild type,indicating that CG DNA demethylation accelerated the degradation of ATZ in rice.To study the molecular mechanism of DNA demethylation regulating the ATZ degradation in rice,genome-wide methylome analysis by BS-seq was performed and the difference of DNA methylation levels between two biotypes of rice under the stress of ATZ was compared.It was found that ATZ exposure resulted in a significantly lower methylation level both in two biotypes.In addition,the methylation level of wild-type was higher compared with the mutant and it was obviously hypomethylated at CG site in the presence of ATZ.Through KEGG enrichment and GO analysis of differentially methylated genes(DMGs,≥2 fold change,p<0.05),it was found that several differently methylated genes encode ABC transporters and plant hormones.Through the analysis of transcriptome sequencing,a total of 205 differently expressed genes(DEGs,≥1.5 fold change,p<0.05)were found in the comparative library of Osmet1-1/WT1 under ATZ stress.Besides,135 of those were induced and 70 repressed under ATZ stress.Among the up-regulated genes,13 growth-responsive genes(chlorophyll,cytochrome P450 and late embryogenesis abundant protein)and 20 genes closely related to metabolism(laccase and glycosyltransferase)were summarized.It was suggested that the activation of stress resistance genes played a role in the ATZ degradation in rice.To get insights into the relationship between DNA methylation and gene expression,we performed a correlation analysis of DMGs and DEGs and found a total of 159 genes had differential methylation along with altered transcription levels.Through the functional annotation of combined genes,several genes with biological functions of xenobiotics catabolism were identified and mainly concentrated in the database of demethylation and gene up-regulation,indicating that DNA hypomethylation could activate gene expression.It also provided a theoretical basis for the mechanism of DNA demethylation regulating atrazine degradation in rice.To identify the function of combined genes by high-throughput sequencing on ATZ degradation,three demethylated and up-regulated genes(OsGTF,OsHPL1 and OsGLH)which were analyzed.Using McrBC-based DNA methylation assay,chromatin immunoprecipitation and qRT-PCR,histone and DNA methylation level in different regions of combined genes were determined.It was found that the genes were hypomethylated in the exon region(OsGTF),promoter region(OsHPL1)and 5’UTR region(OsGLH),with reduced H3K9me2 marks.By identifying a set of mutant of DNA methylation modulators,DNA methylation,histone methylation,and transcription mechanism in differently methylated regions(DMGs)were analyzed.The results showed that histone and DNA demethylation of the genes were regulated by histone H3K9 methyltransferase,DNA methyltransferase OsMET1 and DNA demethyltransferase,and upregulation of the combined genes were mediated by demethylation.To investigate the functional roles of the genes in regulating ATZ detoxification and degradation,we constructed vectors carrying these loci which were transformed into the eukaryotic yeast cells(Pichia pastoris X-33).In the absence of atrazine,the transformed cells showed no difference of growth compared to the untransformed cells(empty-vector).However,in the presence of atrazine,the transformed cells grew stronger.Besides,compared to the empty-vector cells,the degradation of ATZ in the medium were increased by the transformed cells.These data indicated that these loci were able to enhance elimination of atrazine.To get insights into the mechanism underlying the degradation of atrazine by the cells,the metabolites including atrazine degraded products and conjugations in the medium were characterized by HRLC-Q-TOF-MS/MS.Nine metabolites and seven conjugates were characterized.Compared with the control,the relative content of most of the products was significantly higher in the medium of transformed cells,suggesting the target genes were involved in the metabolism of ATZ in cell growth media.To assess the genes related to the biosynthesis and signal transduction of natural products of jasmonic acid,it was studied that the epigenetic and transcriptional mechanism of the genes related to jasmonic acid in the presence of ATZ,and the DNA methylation and expression levels of genes associated with jasmonic acid synthesis or signal transduction were analyzed in rice.Through the analysis of bisulfite sequencing and transcriptome of the control group(-ATZ)and experimental group(+ATZ),24 jasmonate-related DMGs and 15 DEGs were found.Of these,13 genes belonged to both DMGs and DEGs.Combined with bioinformatics,chromosomal localization,phylogenetic tree and promoter cis acting elements of combined genes were performed.The multiple cis-elements were related to environmental stress,which provided scientific basic information for further research of jasmonic acid involved in plant stress resistance.To further explore the mechanism of DNA methylation regulating jasmonate signal transduction gene involved in detoxification and metabolism of atrazine in rice,the regulation mechanism and function of the gene OsCOI1a were examined,encoding the jasmonic acid receptor protein.DNA methylation,histone methylation and transcription level were detected by McrBC and chromatin immunoprecipitation,combining with qRT-PCR.It was found that the DNA and histone demethylation at the promoter regions associated with the upregulated OsCOI1a were mediated by DNA methyltransferase OsMETl and histone methyltransferase OsSDG714.Verification of the function of the gene was processed by expression in eukaryotic cells.The results demonstrated that transformed cells enhanced the tolerance and promoted the degradation of ATZ.To further investigate the effect of the gene on the degradation of ATZ in rice,the overexpression lines,mutants(experimental group)and wild-types(control)were treated with 0.1,0.2 and 0.4 mg/L ATZ for 6 d.Compared with wild types,the biomass and chlorophyll content of the overexpression lines were significantly increased under ATZ exposure,while the electrolyte leakage and ATZ content in overexpression lines and growth medium were reduced.However,mutants were more sensitive to atrazine,with the opposite phenotype and more ATZ content in plants.To assay the function of OsCOI1a involved in ATZ degradation in the environment,the seedlings were treated with 0.01 mg/L ATZ for 120 days.The concentration of ATZ in different tissues were measured at tillering stage(30 d),panicle initiation(60 d),flowering(90 d)and grain mature(120 d).It was found that similar to the short-term experiment,the accumulation of atrazine in the overexpression lines were lower than that of the wild type,while atrazine content in mutant rice were higher than that of the wild type in the entire developmental stage,indicating that OsCOI1a accelerated degradation of atrazine in rice.The ATZ metabolism in wild types,overexpression line OsOE-1 and mutant oscoila-1 were detected by high resolution liquid chromatography-mass/mass spectrometry.Nine degradation products and nine conjugates were identified.Among them,the degradation products(DA)and conjugates(DIA+NGlcN)were first found in rice.Based on the structure of the metabolites,proposed metabolic pathway of ATZ was speculated in rice,mainly with alkylation reaction,hydrolysis reaction,glycosylation reaction,and combination with thiols.Through the relative quantitative analysis,the content of metabolites in different biotypes of rice were over expression line>wild type>mutant.Above results showed that DNA demethylation gene OsCOI1a played a role in the detoxification and metabolism of atrazine in rice.