Physiological And Molecular Mechanisms Of Brassinosteroids-regulated Pesticide Degradation In Tomato

Pesticides are important strategies for pest management, improvement of crop yield and quality, plant growth regulation as well as realization of agricultural mechanization. Presently, due to extensive use of chemical pesticides, especially in protected agriculture which involved frequent pesticides application, crops often suffers the pesticide injury and high pesticide residues, which not only directly harm human health and our living environment, but also cause international trade issues. For the development of sustainable agriculture, it is of great importance to study the mechanisms of plant adaptation to environmental stresses and explore the effective strategy to enhance the tolerance of vegetable crops and improve the productivity, quality and safety of agricultural produces. In this study, we have investigated the differences of plant in response to chlorothalonil (CHT), carbendazim (CAR), chlorpyrifos (CPF) and bifenthrin (BIF) by using tomato(Lycopersicon esculentum Mill.) as model plant. After that, the role and mechanism of brassinosteroids (BRs) alleviating pesticides phytotoxicity and promoting pesticide metabolism were investigated in wild type tomato with normal BRs level or mutant lack of BRs. Meanwhile, we have investigated the role of H2O2 in BRs promoting pesticides metabolism. In addition, Affymetrix GeneChip(?) Tomato Genome Array was utilized to further clarify the mechanism of BRs promoting pesticides metabolism. The results are as follows:1. We have investigated the effects of chlorothalonil (CHT), carbendazim (CAR), chlorpyrifos (CPF) and bifenthrin (BIF) on tomato plant growth and physiology as well as detoxification-related parameters. All pesticides used with double recommended dose inhibited photosynthesis and chlorophyll fluorescence parameters of tomato plant by different degrees. Both CHT and CPF treatments increased the content of reduced glutathione (GSH), activities of glutathione S-transferase (GST), glutathione reductase (GR) and peroxidase (POD), as well as transcriptional levels of detoxification-related genes (GSH, GST1, GST2, GST3, GR and GPX). However, in contrast, GSH content, enzyme activity and gene expression were not influenced significantly by CAR and BIF as compared to that of control. In addition, GST, GR, POD activities and most gene expression were induced by CHT and CPF in a concentration-and time-dependent manner.2. The effects of BRs on alleviation of pesticides injury, detoxification-related enzyme activities and gene expression were investigated in tomato wild type (WT) and mutant (M). The inhibition effects of CHT and CPF on parameters of photosynthesis and chlorophyll fluorescence were alleviated by pretreatment with EBR. Moreover, WT plants exhibited higher tolerance to pesticides than M plants. BRs increased GSH content, GST and GR activities and transcriptional levels of CYP724B2, GSH1, GSH2, GST1, GST2, GST3, GR and ABC.3. We have investigated the effects of BRs on pesticide residue and the mechanism of its promoting pesticide degradation. All the concentrations of EBR set in this experiment increased CHT degradation rate. It is more effective to use lower EBR concentration. The pesticide residue of WT was lower than that of M. Furthermore, BRs had shortened half-life of pesticide residue. In addition, the degradation rate of pesticide residue was slowed down by inhibition of GSH synthesis.4. The signal role of H2O2 in that of BRs promoting pesticide degradation was investigated. The different concentrations of EBR significantly increased the endogenous H2O2 content, which exhibited a concentration-dependent manner. WT plants had a higher H2O2 content than that of M plants. The content of H2O2 in WT or M plants was also increased by pretreatment with EBR. The H2O2 content was closely related to the expression of gene RBOH. The content of H2O2 was inhibited by using NADPH oxidase inhibitor (DPI), H2O2 eliminator (DMTU) and NADPH inhibitor (6-AN). After inhibiting H2O2 burst, we observed that the CHT residue degradation rate was slowed down markedly with the decline in GSH content, detoxification-related enzyme activities and gene expression. In contrast, pretreatment with exogenous EBR and H2O2 significantly increased GSH content, detoxification-related enzyme activities and gene expression, resulting in lower pesticide residue as compared with that of control.5. Affymetrix GeneChip(?) Tomato Genome Array was utilized to further clarify the mechanism of BRs promoting pesticides metabolism. There are total of 1545 different expression genes by CHT treatment,670 of up-regulated genes and 875 down-regulated genes. EBR treatment induced 1584 different expression genes,544 of up-regulated genes and 1040 down-regulated genes. EBR&CHT treatment induced 1725 different expression genes,700 of up-regulated genes and 1025 down-regulated genes. Among these different expression genes, most genes were co-regulated by both BRs and CHT. Analyzing by molecule annotation system, we found that these genes involved many molecular functions and the percentage of gene function was dominated by catalytic category. Moreover, many co-regulated genes were involved in the function of detoxification-related process such as P450, transferases and transporters. Meanwhile, there were also some ethylene (ET) receptor and response genes among the co-regulated genes, which suggested that there may be crosstalk between BRs and ET in process of BRs promoting pesticide degradation.