Detoxification And Metabolism Of Mesotrione And Fomesafen In Rice

Pesticides have been an important part of agricultural production,playing a decisive role in protecting crops from reduced production.The application of pesticides is still considered to be the most effective means to protect crops from diseases,pests and weeds.But only a few of the pesticides applied can reach the control target in agricultural production,and the rest access to the soil,water and air,and finally enter into our food chain,posing a great threat to the ecological environment and human health.As broad-spectrum herbicides with low toxicity and high efficiency,mesotrione and fomesafen have been widely used in weed control of various crops in recent years.Due to long-term and large-scale application,many incidents of mesotrione and fomesafen causing phytotoxicity to subsequent crops have been found in the past two decades.It is noteworthy that two herbicides have been detected in groundwater all over the world for many times.It is urgent to solve the threat of mesotrione and fomesafen to crops and the environment.Acetyltransferases(ACEs)widely exist in mammals,plants,fungi,algae and bacteria and participated in secondary metabolism involved a variety of abiotic and biotic stress responses.GNAT acetyltransferases,which was isolated from bacteria have been reported to detoxify and metabolize herbicides(glyphosate and glufosinate)through catalytic acetylation reaction.However,the detoxification and metabolism mechanism of rice acetyltransferase to pesticides in rice is not clear.Therefore,the study the mechanism of acetyltransferase involved in the detoxification and metabolism of mesotrione and fomesafen in rice,which can provide a new idea for reducing the risk of pesticides on crops and ensuring the safety of agricultural products.In this paper,the toxic effects of mesotrione and fomesafen on rice and their accumulation and transmission in rice were studied.High-throughput sequencing technique was used to study the molecular regulation mechanism of metabolic detoxification of mesotrione and fomesafen in rice.The different expression acetyltransferase genes induced by mesotrione and fomesafen were screened by the association analysis of transcriptome.The overexpression and CRISPR/Cas9 of OsACE1 rice lines were obtained by molecular cloning technique.The effects of OsACE1 on alleviating the toxicity of mesotrione and fomesafen on rice and promoting the degradation and metabolism of two pesticides in rice were studied.The specific contents are as follows:In order to study the toxic effects of mesotrione and fomesafen on rice,wild type rice was treated with different concentrations of mesotrione and fomesafen(0,0.05,0.1,0.15,0.2 and 0.25 mgL-1).The results displayed that mesotrione and fomesafen affected the growth of rice,and the malondialdehyde content and electrical conductivity in rice tissue increased significantly.The content of malondialdehyde reached the maximum when treated with 0.1 mg L-1 mesotrione and fomesafen,while the electrical conductivity reached the peak value under treated with 0.25 mg L-1 mesotrione and fomesafen.It suggested the damage to the cell membrane of rice has been caused by two pesticides.It was also found that the activities of antioxidant enzymes(SOD,POD,APX and GR)and detoxification enzyme(GST)increased significantly in rice tissues under the stress of two pesticides.Under the treatment of 0.1 mg L-1 mesotrione and fomesafen,the activities of antioxidant enzymes and detoxification enzymes in shoots and roots reached the maximum at 6th and 4th days,respectively.The pre-treatment method of oscillating extraction with acetonitrile-water(pH=3)and purification of graphitization carbon black(GCB)+C18 was used.The accumulation of mesotrione and fomesafen in rice was determined by high performance liquid chromatography(HPLC).The results showed that the accumulation of mesotrione and fomesafen in rice tissue gradually increased with the increase of treatment time and concentration.Through the analysis of bioconcentration factor(BCF)and translocation factors(TF),it was found that rice has strong ability to uptake and transmite mesotrione and fomesafen.Rice could continuously enrich and transmit mesotrione and fomesafen from the growth medium,and lead to the inhibition of the chlorophyll synthesis and causing damage to cell membrane of rice.In order to study the molecular mechanism of stress response and detoxification metabolism of rice to mesotrione,rice transcriptome was sequenced by high-throughput sequencing.Four libraries including Root-mesotrione(root control,without mesotrione),Shoot-mesotrione(shoot control),Root+mesotrione(root treated with mesotrione)and Shoot+mesotrione(shoot treated with mesotrione)were established to study the changes of rice transcriptome under the stress of mesotrione.It was found that 326 differentially expressed genes(DEGs)were up-regulated in shoots and 1995 up-regulated DEGs in roots.Further Gene Ontology(GO)annotation analysis and Pathway enrichment analysis of these DEGs were carried out.It was found that many DEGs in rice were involved in stress response,enzyme activity regulation and xenobiotics detoxification and metabolism response.In addition,the expression of many genes in the synthetic pathway of secondary metabolites has also changed.The DEGs related to the detoxification and metabolism of mesotrione(cytochrome P450s,glycosyltransferase(GTs),glutathione S-transferase(GSTs),methyltransferase(MTs)and acetyltransferase(ACEs))and stress responses(oxidoreductase,hydrolase and transcription factors)were analyzed.It was found that the expression of related genes in rice was significantly induced.In order to verify the accuracy of RNA-Seq,nine genes encoding enzymes(P450s,GTs,GSTs and ACEs)were selected for quantitative real-time PCR(qRT-PCR).The expression patterns of the selected genes induced by mesotrione were consistent with the results of RNA-Seq,indicating that the results of RNA-Seq were true and reliable.The results can provide ideas for plants to alleviate the stress of mesotrione,and detoxify and metabolize mesotrione.To study the molecular mechanism of stress response and detoxification metabolism of rice to fomesafen,rice transcriptome was sequenced by RNA-Seq.Four libraries including Root-fomesafen(root control,without fomesafen),Shoot-fomesafen(shoot control),Root+fomesafen(root treated with fomesafen)and Shoot+fomesafen(shoot treated with fomesafen)were established to study the changes of rice transcriptome under the stress of fomesafen.It was found that 550 DEGs were up-regulated in shoots and there were 499 up-regulated DEGs in rice roots.GO annotation and Pathway enrichment analysis of these DEGs were carried out.It was found that many DEGs in rice were involved in stress response,enzyme activity regulation and xenobiotics detoxification and metabolism response.The expression of many genes in the synthetic pathway of secondary metabolites has also changed.The DEGs related to the detoxification of fomesafen(P450s,GSTs and ACEs)and stress responses(oxidoreductase,hydrolase and transcription factors)were analyzed.It was found that the expression of related genes in rice was significantly stimulated.For verifying the accuracy of RNA-Seq,nine genes encoding enzymes(P450s,GSTs and ACEs)were selected for qRT-PCR.The expression patterns of the selected genes stimulated by fomesafen were consistent with the results of RNA-Seq,indicating that the results of RNA-Seq were true and reliable.The results can provide ideas for plants to alleviate the toxicity of fomesafen and metabolize it.Through the correlation analysis of transcriptome,86 rice acetyltransferase genes responsive to mesotrione stress were screened,of which 13 genes were differentially expressed in rice roots.Eighty three rice acetyltransferase genes responsive to fomesafen stress were screened,and 2 and 5 acetyltransferase genes were differentially expressed in rice roots and shoots,respectively.It showed that the expression of acetyltransferase gene in rice has changed under the induction of two pesticides.The chromosome location,evolutionary tree,promoter cis-acting elements and domains of these DEGs exposed to mesotrione and fomesafen were analyzed.It was found that most acetyltransferase genes contain specific domains or motifs that may interact with other components to cope with various environmental stresses.Among them,8 and 7 differentially expressed acetyltransferase genes under mesotrione and fomesafen stress were verified by qRT-PCR,respectively.The expression patterns of these DEGs were consistent with the results of RNA-Seq sequencing.The activity of rice acetyltransferase was determined under the stress of mesotrione and fomesafen.It was found that rice acetyltransferase was induced and enhanced.These results suggested that acetyltransferase coding genes can respond to mesotrione and fomesafen stress by increasing the level of transcription and enhancing the activity of specific acetyltransferases in rice tissues.In order to study the regulation of acetyltransferases(ACEs)on the detoxification and metabolism of mesotrione and fomesafen in rice,a rice ACE gene(LOC_Os10g01920,namely OsACE1)was screened.The T3 rice lines of OsACEl overexpression and CRISPR/Cas9 were successfully obtained by genetic transformation and identification of molecular cloning.Three lines of rice OsACE1 homozygous overexpression(OE)and Cas9 were selected respectively,with wild-type rice(WT)as the control.The toxic effects of two pesticides on three different biotypes rice these were analyzed.After rice seedlings were treated with 0.1mg L-1 mesotrione and fomesafen for 6 d,the elongation,dry weight and chlorophyll concentration of OEs rice were significantly higher than those of WT,and the damage of cell membrane was also reduced.The opposite results were obtained in Cas9 lines.Meanwhile,overexpression of OsACEl in rice promoted the degradation of mesotrione and fomesafen in rice.The accumulation of mesotrione and fomesafen in OE lines was significantly lower than that in WT,and the removal of mesotrione and fomesafen in growth medium was also significantly higher than that in WT.The opposite results were obtained in cas9 lines.To confirm whether OsACE1 can promote the degradation of mesotrione and fomesafen in the environment,WT,OE and Cas9 rice lines were exposed to 0.01 mg L-1 mesotrione and fomesafen for whole growth period,respectively.It was found that the accumulation of mesotrione and fomesafen in leaves and stem,leaf Ⅲ,roots,leaf Ⅱ,leaf Ⅰ and roots of OE lines rice was lower than that in WT,while the accumulation of two pesticides in Cas9 lines were higher than that in WT after the exposure of mesotrione and fomesafen for 45,90 and 135 d,with the concentration ranking lower leaves and stem>leaf Ⅲ>roots>leaf Ⅱ>leaf Ⅰ>grains>panicle.What’s more,these two pesticides were also detected on rice panicles and grains,and the content of mesotrione and fomesafen in panicles and grains of OE lines rice was significantly lower than that in wild type,which only 69.6%-92.7%and 32.3%-82.0%of WT,respectively.However,the content of mesotrione and fomesafen in panicle and grain of Cas9 lines was 1.01-1.65 and 1.03-1.50 times higher than WT,respectively.In order to further explore the detoxification and metabolism mechanism of OsACE1 on mesotrione and fomesafen,the metabolites and conjugates of mesotrione and fomesafen in rice OE-3,Cas9-1 and WT were detected by LC-Q-TOF-HRMS/MS.Totally,6 metabolites and 6 conjugates of mesotrione were identified,two of which were O-acetylated conjugates(#1-2).The six conjugates were first found in plant.For fomesafen,8 metabolites and 20 conjugates were identified,including three N-acetylated conjugates(#7-9)and seven O-acetylated conjugates(#10-16).The 7 metabolites and 20 conjugates were first time characterized in plant.The possible degradation and metabolism pathways of the two pesticides in rice were speculated.Two pesticides were detoxified and metabolized in rice through oxidation,hydrolysis,reduction,methylation,acetylation,glycosylation,malonylation and bind to glutathione and amino acids.The relative quantitative analysis of metabolites and conjugates of mesotrione and fomesafen exhibited that the content of metabolites and conjugates of two pesticides in roots,shoots and grains of OE-3 line was significantly higher than that in WT,especially acetylated conjugates.However,the content of metabolites and conjugates in Cas9-1 line was significantly lower than that in WT.These results further illustrated that OsACE1 overexpression can promote the metabolic pathways of Phase Ⅰ(oxidation,reduction and hydrolysis)and Phase Ⅱ(methylation,glycosylation,malonylation,binding glutathione and amino acids)of two pesticides in rice by regulating the metabolic pathway of acetylation to reduce the accumulation of two pesticides,and enhancing the degradation and metabolism of two pesticides in rice.