| PFOS and PFOA are the two most widely used compounds in the industry and are also two emerging artificial compounds.The C-F bonds make them extremely stable,they were widely detected in the human body,animals and plants,and the environment.The ecotoxicity of PFOA and PFOS are complex and have been reported in animals(including fish and mice),but little research has been done on the effects on plants,especially the mechanism of toxicity.Omics technology can be used as a powerful tool to study the toxic effects of contaminants on organism.In the present study,using HPLC-QTOF based omics technology,the analytical methods of metabolites in A.thaliana leaves were optimized,then qualitatively and quantitatively analyze the endogenous metabolites of A.thaliana and analyze the metabolic pathways.Finally,using indoor controlled pot experiments,trace PFOS and PFOA were exposed to A.thaliana.Samples with different concentrations were subjected to metabolomics analysis,and chromatogram and mass spectrum collection was carried out.Many statistical methods(PCA,PLS-DA,OPLS-DA,Cluster Analysis,T test,Correlation Analysis,etc.)were used to obtain the corresponding biomarkers,then metabolic pathway analysis and metabolic mechanism analysis were carried out to obtain toxic effects on the metabolic level.This article comprehensively discussed the research trend of perfluoroalkyl acids.The metabolomics technology platform and related data analysis and processing methods were introduced,the differential metabolites involved metabolic pathways were summarized,to provide a theoretical basis for further studies related to PFOS and PFOA trace exposure in plant leaves.The purpose of this experiment was timely and comprehensively to reflect the phytotoxic effects of two perfluoroalkyl acids and to supplement ecotoxicological data.It is conducive to discover microscopic toxic effects by other environmental pollutants and provides a reference for the study of pollution toxicology of other environmental pollutants.The main findings were as follows:(1)HPLC-QTOF analysis technology was used to compare the effects of different extraction methods on the extraction results of A.thaliana leaves metabolites,an analysis method of A.thaliana leaves metabolites with T3 and Amide column was established.The number of detected peaks and their stability were used as evaluation indicators.Through the T3 chromatography system,different extraction methods based on methanol-water(including ultrasonic and vortex,dry and fresh leaves extraction)were discussed.The effect of extraction and separation of metabolites in A.thaliana leaves were compared.The results showed that the combination of vortex and dried leaves samples had the higher coverage of A.thaliana leaves metabolites.(2)Sterile control experiment of A.thaliana with PFOS exposure was prepared,high-throughput non-targeted metabolomics methods for comprehensive evaluation were selected to study the metabolic characteristics of A.thaliana leaves exposed to different concentrations of PFOS throughout the growth period(30 days).PFOS concentration gradient treatments of 0.01,and 1.00 mg/kg and control group were set,and 30 parallel samples were treated in each group.The toxic effects of different PFOS concentrations on the metabolites of A.thaliana leaves were studied.All metabolites were analyzed by PCA and OPLS-DA methods,using the cutoff of VIP and p value,53 biomarkers were found and significantly regulated.All amino acids except glutamic acid were inhibited maybe because of PFOS binding with related protein.In addition,auxin,phytohormone and cytokinin were significantly down-regulated.As a response mechanism to PFOS oxidative stress,the phenylpropane pathway was fully activated to form a variety of polyphenols,and several flavonoids were further up-regulated as the main defense pathway,thereby resisting reactive oxygen species(ROS),in addition,ascorbic acid,trehalose and nicotinamide were also activated,to reduce the damage of oxidative stress.These results provided new insights into the potential mechanism of phytotoxicity.(3)PFOA linear chain(PnFOA: Perfluoro-n-octanoic acid)and branched chain(P3MHpA: Perfluoro-3-methylheptanoic acid)isomers were exposed to A.thaliana in agar hydroponic experiments.High-throughput non-targeted metabolomics methods were used to conduct a comprehensive evaluation to study the differences in the metabolic characteristics of A.thaliana leaves after exposure to different concentrations and structures of PFOA throughout the growth period(30 days).PFOA concentration gradient treatments of 0.01,and 0.50 mg/kg and control group were designed,with 30 parallel samples in each treatment group,and the toxicity effects of different exposure concentrations and structures of PFOA on metabolite of A.thaliana leaves were studied.PCA,PLS-DA,OPLS-DA methods were used to analyze the regulation differences of all metabolites,and the biomarkers were screened by VIP and p cutoff value.PnFOA exposure and P3 MHpA exposure were both compared with the control group and the comparison between the two exposure group.65 different metabolites were screened out,differential metabolic pathways were identified,and their toxicity differences and toxicity mechanisms were analyzed.A large number of up-regulations of metabolites of the tryptophan pathway indicated that the defense mechanism of the tryptophan pathway was activated during the stress resistance of plants;sugars,nucleic acids,and flavonoids all showed down-regulation mechanisms,indicated that PFOA exposure enhanceed energy conversion and antioxidant effect,genotoxicity was also found;it was found that the difference in the mechanism of the two monomers on amino acids,the straight chain mostly showed up-regulation phenomenon,while the branch chain showed down-regulation effect,and the four metabolites found with the opposite regulation phenomenon were all mustard oleosides;two exposure groups were simultaneously down-regulated during the urea cycle of detoxification of ammonia in plants,indicating that PFOA caused plants to reduce stress resistance;compared with PFOS exposure,sugars,flavonoids and plant hormones were found the opposite regulation.Proteomics was used for verification.All proteins were analyzed by PCA-DA and differential proteins were screened by FC and p value.The differences in protein regulation of A.thaliana leaves caused by different PFOA structural exposures were discussed,and the expression of the differential proteins was analyzed.By analyzing the correlation between protein expression and metabolite response regulation through P3 exposure,it was found that many plant metabolites were regulated by protein during amino acid metabolism. |
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