Studies On The Stereoselectivity Degradation And Its Mechanism Of Chiral Fungicide Tebuconazole In Arabidopsis Thaliana

The use of pesticide can ensure the quality and quantity of agricultural products to some extent. However, pesticide is a kind of exogenous compounds. The plant will defense the damage of pesticide exposure through the internal detoxification mechanisms. The defense mechanism can help to switch or decomposition the exogenous substances, such as pesticide residue on crop. Triazole fungicide is one of the most widely used in the agricultural crops, and 80% of them consist one or two pairs of enantiomers because of the stereogenic centers. The established data showed that the obvious enantioselective metabolic of triazole fungicide enantiomers were investigated in plant under the field condition. And the stereoselective degradation varies in the plant species, the compound structure and the uptake suits of plant. Thus, in order to obtain a more accurate evaluation about the metabolic of chiral pesticide in plant, the researches of four typical chiral triazole fungicides at the level of enantiomers and from the point of plant metabolic under aseptic conditions were carried out in this paper. The main results are listed as following:1. The enantioseparation of myclobutanil, tebuconazole, epoxiconazole and diniconazole was carried out using ultra performance liquid chromatography coupled with tandem mass spectrometry combined with reversed chiral columns. Moreover, a chiral residue analytical method was established in the Arabidopsis thaliana. The sample was extracted with acetonitrile, then purify with the sorbent of GCB and PSA. The matrix-matched calibration curve was used for quantification of sample. The linearity, accuracy, precision and sensitivity of the method were also investigated. The results showed that excellent linearity of the method with all the R2 > 0.999. And the average recoveries ranged from 72.5% to 91.3% with all RSDs below 13.1%, which meet the demand for pesticide residue analyses. The developed method was proven to be suitable for residue determination in the Arabidopsis thaliana.2. Based on the established chiral analytical methods, enantioselective degradation of four triazole fungicide enantiomers in Arabidopsis thaliana was assayed in this paper. Resulted indicated that under Sterile culture conditions, the significant enanselectivity was observed in the degradation process when plant suffer the exposure of racemic tebuconazole under three different concentration. The EF value of tebuconazole enatimers increase from 0.48 to 0.70, which showed the(-)-R-tebuconazole was preferentially degraded and(+)-S-tebuconazole was more persistent. However, no obvious enantioselectivity was observed for myclobutanil and diniconazole in Arabidopsis thaliana. As for epoxiconazole, it exhibited the difference between enantiomers in the degradation process under low concentration exposure, but on the contrary, there is no enantioselective degradation happened when the concentration of enantiomer is relatively high.3. The verification test was conducted to confirm the degradation behavior of pure enantiomer of tebuconazole in Arabidopsis thaliana. The results proved that it is in accordance with the behavior of racemic tebuconazole with the(-)-R-tebuconazole preferentially degraded and the relative enrichment for(+)-S-tebuconazole.4. Genome-wide transcriptional profiling of Arabidopsis thaliana from plants subjected at pure enantiomer of tebuconazole exposure was carried out in order to figure out the molecular mechanism for the plant response to the stress of different compounds. Through the comparison between Genome-wide transcriptional profiling of(-)-R-tebuconazole and(+)-S-tebuconazole, we found there are a large scale for relevant changes, and 4353 genes in 0h and 1958 genes in 6d were differentially expressed. Moreover, 1222 genes were both found differentially expressed in 0h and 6d. GO and KEGG analyses of differentially expressed genes(DEGs) showed modified biological functions and metabolic pathways associated with the oxidoreduction, signal transduction, regulation and molecular metabolism. Besides, DEGs encoding proteins involved in xenobiotic metabolism, detoxification, transporters, and transcription factors were comprehensively investigated, for example, the gene AT4G15440 and AT2G29440 encoding cytochrome P450, glutathione S-transferase, and three ABC transporter genes all were up-regulated expression. The results suggeste that these difference may led to the enantioselective degradation of tebuconazole enantiomers in plant.