Mechanisms Of Glutathione-dependent Detoxification In Brassinosteroids-regulated Chlororthalonil Degradation In Tomato

Pesticides are widely used in agriculture for its high efficacy in pathogens control, however, the excessive use of pesticide results in pollution to environment and agricultural products, which directly endangers human health. Therefore, it has become a serious problem to be solved in order to reduce pesticide residue following safety standards. Until now, extensive researches have focused on bioremediation of organic pollutants using enzymes produced from the microorganisms or plants. By contrast, there are only few studies on the metabolism or biotransformation of pesticides in plants, especially on the degradation of fungicides. Recent study shows that brassinosteroids (BRs) can reduce pesticides residue and promote pesticides detoxification in plant. However, the role and mechanism of BR promoting pesticide degradation is still not clear. Therefore, the present study was conducted to investigate the mechanism of BR promoting biotransformation and degradation of CHT in plant.In this study, we investigated the mechanism of chlorothalonil (CHT) degradation in tomato (Solanum lycopersicum Mill.) plants involved in glutathione biosynthesis, regeneration and metabolism by VIGS. We also investigated the role and mechanism of brassinosteroids (BRs) alleviating pesticides phytotoxicity and promoting pesticide metabolism in glutathione-dependent gene silencing plants to understand the role of glutathione-dependent detoxification in BRs promoting pesticides degradation in plant. Meanwhile, we have investigated the role of H2O2in BRs promoting pesticides degradation to further clarify the mechanism of BRs promoting pesticides metabolism. The results are as follows:1. In this study, tobacco rattle virus (TRV) and tomato plants(Solanum lycopersicum Mill. cv. Zheza205) were used to construct the gene silencing vector with the fragment of SlGSH1、SlGSH2、SlGR and seven SIGST genes. VIGS transform system of SIPDS reference gene and other genes were established. Quantitative RT-PCR analysis revealed that transcript levels of genes in silenced plants were decreased significantly, compared with TRV plants, which suggested an effective inhibition of target genes by VIGS. 2. To determine the role of glutathione biosynthesis and regeneration in the CHT metabolism, GSH1, GSH2and GR genes were silenced with VIGS. Silencing of GSH1, GSH2and GR1all resulted in decreased glutathione synthesis and ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), but increased CHT residues in plant tissues, while the GSH1-silenced plants and GR-silenced plants showed the lowest GSH levels and lowest ratio of GSH to GSSG, respectively. CHT exposure study indicated that glutathione-dependent pathway was involved in CHT detoxification and glutathione not only reacted as a substrate to conjugate with CHT, but also induced activities of detoxification enzymes, as well as expression of detoxification genes directly or indirectly. These observations provided evidences for the involvement of the glutathione-dependent pathway in the in vivo metabolism of pesticide in tomato plants.3. Studies were conducted to understand the role of glutathione-dependent detoxification pathway in BRs promoting pesticides degradation in plant. The results demonstrated that BRs could increase glutathione synthesis and NPT, TT levels, and induced the detoxification metabolism reacts, in which GSH conjugated with CHT as a substrate. Meanwhile, BRs induced activities of detoxification enzymes, as well as the expression of detoxification genes directly or indirectly. Consequently, BRs could promote the CHT metabolism processes and finally reduces CHT residue in plant via glutathione-dependent detoxification metabolism.4. The role of H2O2in BRs promoting pesticides degradation was also investigated to further clarify the mechanism of BRs promoting pesticides metabolism. We inhibited ROS burst in plant with the application of chemical inhibitors, and also regulated gene expression of BRI1and RBOH genes by VIGS approach to investigate the signal role of H2O2in BRs promoting pesticides metabolism. The studies confirmed that H2O2signal could be in response to EBR treatment, and induced glutathione-dependent detoxification pathway in BRs promoting pesticides degradation in plant. BRs induced the gene expression of RBOH, resulted in the production of H2O2signal by NADPH oxidase to regulate glutathione-dependent detoxification pathway to promote pesticide metabolism in plant. H2O2played a critical role in BR-induced glutathione-dependent detoxification to pesticide in plant.5. The effects of GST in BRs promoting pesticides metabolism in plant were investigated. GST1、GST2、GST3、GST4、GST5、GST6、GST7genes that encode glutathione S-transferase in tomato, were silenced respectively in tomato plants by a virus induced gene silencing (VIGS) approach. The studies revealed that gene transcripts of seven GST genes were all significantly inhibited by VIGS, however, the inhibition in GST activities were different. GST1and GST7genes silencing inhibited both GST activity and CHT degradation metabolism in plants significantly, which demonstrated the participation of GST encoded by GST1and GST7genes in the detoxification of CHT in plant. However, GST1gene silenced plants could even be induced by EBR treatment significantly, while GST7gene silenced plants showed no response to EBR treatment, which demonstrated that BRs could induce the expression of some GST encoding genes, such as GST7, to increase GST activities to promote pesticides degradation metabolism.