Research On The Biotoxicity And Degradation Mechanism Of Isoproturon And Atrazine For Wheat And Rice

Rice (Oryza sativa) and wheat (Triticum aestivum) are two main planted crops in China,whose production safety even concerns national security. As the residue of herbicides has been a rising problem among the issues of environmental and food safety, effective ways to enhance the ability of self-detoxification as well as prompt the herbicide-degradation in planted soil are among essential issue for crop protection and agro-ecology improvement.Thus it is an important scientific problem to investigate these methods. Isoproturon (IPU)and atrazine (ATR) were widely used to prevent pre- and post-emergence weeds in crop.Field and laboratory research have shown their harmful effects on freshwater algae, soil microbial communities and higher plants, as well as the threat to human health through the food chain. As a result, IPU and ATR have been classified as possible carcinogens in human beings by the annex X of the Water Framework Directive,and have been forbidden by European Union. Unexpectedly, IPU and ATR are still applied in many developing countries, including China.The dissertation firstly focused on the biotoxity and degradation mechanism of two herbicides (IPU and ATR) in wheat and rice. In addition, the effect of exogenous salicylic acid (SA) on herbicide IPU degradation and detoxification in wheat and its planted soil was investigated. Finally, based on the cutting-edge knowledge of epigenetics, our work explored the association between DNA methylation level and gene expression under ATR exposure, and further illustrated the regulatory mechanism of DNA methylation on herbicide-induced transcript. Main original conclusions are shown as follows:1. To investigate the toxic effect of isoproturon on crop rice, the rice seedlings were treated with 0?8 mg L-1. IPU significantly prompted the content of MDA in IPU-treated rice seedlings,compared to the control,which the maximum was at 2 mg L-1 isoproturon.The result indicated that rice seedlings were damaged by oxidative stress. Meanwhile, the physiological experiment confirmed that IPU inhibited the growth and chlorophyll content of rice. To further investigate the biochemical responses to isoproturon, superoxide dismutase (SOD), laccase, peroxidase (POD) and ascorbate peroxidase (APX) were assayed.The result suggested that IPU stress stimulated the activities of antioxidant enzymes which were able to be resistance to oxidative stress from isoprotuorn.2. In order to figure out the molecular mechanisms for plant tolerance and degradation to the herbicide, rice (Oryza sativa) was selected as experimental object, due to a wealth of knowledge about its genetics, molecular biology, genomic sequence, utilized the RNA-Seq to get hints on a larger scale for relevant changes in rice transcriptome, and constructed four RNA libraries (Shoot+IPU, Shoot-IPU, Root+IPU and Root-IPU) which were taken from rice shoot and root at 0 and 2 mg L-1 IPU, respectively. The result showed that 10,879,252?14,519,285 clean tags were generated. Mapping the clean reads to rice genomic databases generated 31,009?32,118 annotated genes for single library. Moreover,11,680 genes in root and 11,927 genes in shoots were differentially expressed under IPU stress. Gene Ontology (GO) and Kyoto Encylopeida of Genes and Genomes (KEGG)analyses of differentially expressed genes (DEGs) showed modified biological functions and metabolic pathways associated with the resistance to environmental stress, degradation of xenobiotics and molecular metabolism. Validation of gene expressions by qRT-PCR confirmed the RNA-Seq results. DEGs encoding proteins involved in xenobiotic metabolism, detoxification, transporters, and transcription factors were comprehensively investigated. The activities of several enzymes closely related to xenobiotic metabolism were determined, such as cytochrome P450, glycosyltransferase and methyltransferase.Notably, the specific cis-elements of degradation-associated DEGs were predicted, and included many phytohormone-responsive elements. To evidence the IPU-metabolism in rice, 19 degradations and 5 conjugates were chemically characterized using UPLC/Q-LTQ-MS2. Of these, 11 metabolites have been reported in plant for the first time.Base on the structures of metabolites, we inferred the proposed pathway of enzyme-regulated IPU metabolism in rice.3. The prediction of cis-elements above demonstrated that phytohormone possibly induced the expression of genes involving in herbicide-metabolism, as a result of the enhancement of detoxified ability in rice. In order to confirm the hypothesis, we investigated the effect of phytohormone on herbicide metabolism in crop. Because IPU was usually applied in wheat field, wheat plants were selected as the treated object. Salicylic acid (SA) is an important phytohormone in plant, which was well-reported to enhance the resistance to many abiotic stresses, such as cold, salinity and heavy metals. Firstly, the conductivity and phenotype under the IPU treatment were determined in the present of salicylic acid. The result indicated that salicylic acid (5 mg L-1) alleviated toxicity of wheat growth under IPU exposure. Then, 15 IPU-metabolites in wheat tissues were characterized using UPLC/Q-TOF-MS2. Of these, 4 metabolites which were different with those in rice were found in wheat for the first time. Interestingly,most detected IPU-derivatives were sugar-conjugated. Degradation and glycosylation of IPU-derivatives could be enhanced by the provision of salicylic acid. It suggested salicylic acid treatment accelerated the rate of IPU-metabolism in wheat, especially phase II metabolism. In addition, the activities of glycosyltransferase and their encoding genes expression under IPU exposure were tested in the presence of salicylic acid. The result showed that IPU remarkably induced the expression of four glycosyltransferase genes. Notably, gene CD876318 was up-regulated by salicylic acid. The observations may let us to illustrate the reason that the reduced biotoxicity of IPU could be attributed to S A-accelerated degradation with the high level of IPU-degradations and conjugations in wheat.4. Meanwhile, the herbicide isoproturon residues was determined in soil, where wheat was cultivated and sprayed with salicylic acid. Provision of salicylic acid led to a lower level of IPU residues in planted soil compared to IPU treatment alone, especially in rhizosphere soil. In order to explore the reason about the SA-accelerated IPU-degradation in soil, we investigated the effects of SA on low-molecular-weight organic acid (LMWOAs)from root exudation and soil microbes which played an essential role in xenobiotic metabolism. Three LMWOAs (tartaric acid, malic acid and oxalic acid) were enhanced in rhizosphere with SA-treated wheat. Due to the close association between root exudation and soil microbe biomass, we further probed the effects of SA on soil microbial biomass and population in IPU-treated soil. Soil microbial biomass carbon (SMBC),soil microbial biomass nitrogen (SMBN) and phospholipid fatty acids (PLFAs) were determined in soils(rhizosphere, bulk and non-rhizosphere soil). The results showed that in soils much higher SMBC, SMBN and total PLFAs contents which represent soil microbe biomass, were examined in the presence of SA compared to the control, especially in rhizosphere soil.Additionally, the decline of soil stress indicator and the enhancement of several specific microbial PLFAs (Gram+/- bacteria, fungi and AM fungi) could be found in the presence of SA. It was indicated that the SA-induced LMWOAs enriched soil microbe biomass, and subsequently lower the IPU content, as a result of releasing IPU-stress in soil. Besides,three soil enzyme activities were assayed, including catalase (CAT), dehydrogenase (DHA),phenol oxidase (PO),suggesting that the inhibitions of DHA and PO activities under IPU exposure were recovered in the presence of SA. We further assessed the correlation matrix and principal component to figure out the positive correlation between IPU degradation and the following factors: LMWOAs, soil microbes and soil enzymes. Finally, six IPU degraded products in rhizosphere soil were characterized using UPLC/Q-TOF-MS . A relatively higher level of IPU derivatives was identified in soil with SA-treated wheat than those in soil without SA-treated wheat plant. In a nutshell, provision of SA stimulated exudation of organic acids in rhizosphere soil. The increased organic acids enriched the soil microbial commnities, modified the soil enzyme activities, and consequently accelerated the degradation of IPU.5. Currently, the epigenetic mechanisms such as DNA methylation that control gene expression have been described. DNA methylation may be an important mechanism involved in the regulation of plant response to abiotic stress. This study presents the first genome-wide single-base-resolution maps of DNA methylation in ATR-exposed rice.Widespread differences were identified in CG and non-CG methylation marks between the ATR-exposed and ATR-free (control) rice. Most of DNA methyltransferases, histone methyltransferases and DNA demethylase were differentially regulated by ATR. We found more genes hypermethylated than those hypomethylated in the regions of upstream,genebody and downstream under ATR exposure. A stringent group of 674 genes (p< 0.05,two-fold change) with a strong preference of differential expression in ATR-exposed rice was identified. Some of the genes were identified in a subset of loss of function mutants defective in DNA methylation/demethylation. Provision of 5-azacytidine (AZA, inhibitor of DNA methylation) promoted the rice growth and reduced ATR content. By UPLC/Q-TOF-MS/MS,8 degraded products and 9 conjugates of ATR in AZA-treated rice were characterized. Two of them are newly identified in this study. Furthermore, more ATR-metabolites were detected in the presence of AZA, including cytochrome P450-reuglated degradations (DIA, HA) and most GST-regulated conjugations. Our data provide evidence that ATR-inducible DNA methylation/demethylation is an epigenetic mechanism associated with activation of metabolism-based genes involving in ATR degradation and detoxification.