Methyltransferases Involved In Detoxification And Degradation Of Atrazine Residues In Rice

Rice(Oryza sativa),as one of the economical crops,nourishes nearly half the world’s population.Importantly,rice is one of the best studied model plants with resourceful knowledge about its genetics,molecular biology and genetic transformation.Atrazine is a member of the triazine herbicide,which is predominantly used to control certain annual broadleaf weeds such as corn,sugarcane sorghum and rice.Moreover,our previous studies showed that atrazine could lead to the ROS-mediated toxicity in rice and alfalfa.Methyltransferase(MTs;EC 2.1.x.y),as a metabolic enzyme,play important roles in response to various abiotic and biotic stresses.Methyltransferases(MTs)catalyze transfer of methyl-groups from donors such as S-adenosine methionine(SAM)to certain groups of their substrates,thus modifying many functional molecules in organisms for development and environmental stress response.In this study,we demonstrated 166 differentially expressed MT genes retrieved from the genome wide sequencing database from atrazine-exposed rice.Additionally,the expression of MT genes was investigated by DNA microarray,which 45 genes were validated to be agreement with those from RNA sequence.With bioinformation analysis,we presented phylogenetic assay of these 45 MT genes,cis-elements prediction,chromosome location and domain analysis.The 15 MT genes were randomly selected and validated to their expression pattern of RNA-Seq using quantitative real-time PCR.The activity of total O-methyltransferase was induced by atrazine exposure in both rice shoots and roots with HPLC assay.In order to study the effect of O-MT on degradation of atrazine,we successfully generated transgenic yeast cells(Pichia pastoris X-33)expressing two rice MTs genes(LOC_Os04g09604 and LOC_Os11g15040).The two tranformatants were found to accumulate less atrazine and produced more ATR degraded products comparied with blank vectors.The ATR degradation rate increased by 1.7 and 2.3 times for LOC_Os04g09604-and LOC_Os11g15040-transformants,respectively.The ATR-degraded products in the rice and transformed yeast cells were characterized using ultra performance liquid chromatography mass spectrometer(UPLC/MS/MS).Seven ATR-derivatives were detected in the rice,including hydroxyisopropylatrazine(HIA),hydroxyethylatrazine(HEA),atraton,demethylated atrazine(DMA),hydroxyatrazine(HA),desethylatrazine(DEA),desisopropylatrazine(DIA).Atraton and hydroxyatrazine(HA)were detected in the transformed yeast cells.The topological interaction between enzyme LOC_Os04g09604 and its substrate was specially analyzed by homology modeling programs and well confirmed by the molecular docking with ATR and its metabolites.The significance of the study understood the degradation of the remaining ATR in the rice mediated by O-MT,and it has important significance on understanding the mechanism of degradation toxic herbicide in O-MT crop.