Roles Of TGA2 Transcription Factor In The Metabolism And Degradation Of Chlorothalonil In Solanum Lycopersicum And The Regulatory Mechanism

The rapid development of greenhouse horticulture has solved the long-term problem of insufficient vegetable supply in China.However,the sustainable development of greenhouse horticulture is hindered by major problems,such as high incidence of pests and diseases due to specific enclosed environments and continuous cropping,and high pesticide residues resulting from excessive pesticide application.Therefore,an environment-friendly and efficient technology with potential wide application is urgently needed to reduce pesticide residues in crops.Brassinosteroids(BRs)are a new type of phytohormones which are involved in the regulation of plant growth and development,and biotic/abiotic stress tolerance.In addition,BRs are shown to promote the degradation of xenobiotics by enhancing the detoxification system.However,the underlying regulatory mechanism remains unclear.TGAs is a group of transcription factor in the basic region-leucine zippers(bZIPs)transcription factor family.TGAs were named after their binding motif in gene promoter(TGACG).TGAs participate in many physiological processes such as systemic resistance to pathogens,plant development and detoxification of toxic metabolites.However,their role in degradation of pesticide residue is still unclear.Tomato is the vegetable with the largest planting area in greenhouse horticulture.In this study,tomato was used as experiment material.Using plant physiology and molecular biology approaches,in combination with transgenic technology,the roles of TGA transcription factors in BR-induced degradation of pesticide chlorothalonil(CHT)residue,and the effects of redox modification of TGA on the regulation of detoxification genes and pesticide degradation were studied.The results are as follows:1.The Class-? TGA factors comprising TGA2,TGA2.2 and TGA2.3 are involved in the degradation of CHT residues in tomato.TGA2 can directly bind to the promoter of detoxification-related gene GST3,and regulate its transcription.By blasting in the SGN network using TGA protein sequences of Arabidopsis thaliana,we identified 10 TGA transcription factors in tomato.Among Class ? TGAs.the roles in pesticide degradation are different.Silencing of TGA2.TGA2.2,and TG.A2.3 suppressed the degradation of CHT residues by inhibiting the inductions of transcript level of detoxification genes.the activity of detoxification enzymes.and the content of glutathione.Overexpression of TGA2 enhanced the plant ability of degrading CHT residues.The transcript level of the detoxification gene GST3 was reduced in the TGA2-silenced plants as compared with the pTRV-TRV plants,while the overexpression of TGA2 significantly enhanced the transcript level of GST3 as compared with the wild type.EMSA and ChIP-qPCR assays showed that TGA2 protein could bind to the promoter of GST3 gene and induce its transcriptional expression.Silencing of GST3 inhibited the detoxification capacity of plants and significantly increased the pesticide residues in plants.2.BR-induced apoplastic H2O2 activates the glutaredoxin genes GRXS13 and GRXS25,and promotes their protein interaction with TGA2 transcription factor and their participation in the degradation of CHT residues.BR treatment enhanced the detoxification capacity of tomato plants by activating the detoxification system.Silencing of TGA2 inhibited the detoxification pathway and abolished BR-induced pesticide degradation.By comparing the GRXs gene expression of RBOH1(Respiratory Burst Oxidase Homologue 1)silenced plants with pTRV-TRV plants.we identified glutaredoxin encoding genes GRXS13 and GRXS25 which were induced by BR in an apoplastic H2O2-dependent manner.GRXS13 and GRXS25 acted downstream of RBOH1 and participated in the BR-promoted metabolism of CHT residues.Yeast two-hybrid and BiFC assays showed that GRXS 13 and GRXS25 could interact with TGA2 protein.indicating that TGA2 protein might be regulated by GRX-dependent redox modification.3.CHT and BR treatment induce the accumulation of S-nitrosothiols(SNO)in plants.TGA2 protein can be modified by S-nitrosylation,which enhances its transcriptional activity for regulating the expression of detoxification gene GST3 and degradation of CHT residues.CHT and BR treatment increased the content of SNO in leaves by inducing the transcript level of NR gene encoding nitrate reductase.S-nitrosoglutathione reductase(GSNOR)can reduce the level of SNO by inhibiting the accumulation of GSNO.Silencing of GSNOR increased the transcript level of GSTs,and the activity of GST,thus increasing the plant ability of detoxification and degradation of CHT.In vitro experiments showed that TGA2 protein could be modified by S-nitrosylation after incubation w ith GSNO,which enhanced the binding activity of TGA2 protein to the promoter of detoxification gene GST3.These results suggest that S-nitrosylation of TGA2 protein plays an important role in regulating its transcriptional activity and function in pesticide metabolism.