Brassinosteroids Confer Resistance to Isoproturon through OsBZR4-Mediated Degradation Genes in Rice (Oryza sativa L.).

Plants have complex detoxification and metabolic systems that enable them to deal with environmental pollutants. We report accumulation of the pesticide isoproturon (IPU) in a BR signaling pathway for mutant bzr4-3/5 rice to be significantly higher than in wild-type (WT) rice controls and for exogenous 24-epibrassinolide to reverse toxic symptoms in WT rice but not in mutants. A genome-wide RNA sequencing study of WT/bzr4 rice is performed to identify transcriptomic changes and metabolic mechanisms under IPU exposure. Three differentially expressed genes in yeast cells increase the degradation rate of IPU in a growth medium by factors of 1.61, 1.51, and 1.29 after 72 h. Using UPLC/Q-TOF-MS/MS, five phase I metabolites and five phase II conjugates are characterized in rice grains, with concentrations generally decreasing in bzr4 rice grains. OsBZR4, a regulator of IPU degradation in rice, may eliminate IPU from edible parts of food crops by regulating downstream metabolic genes.

Cytochrome P450 CYP90D5 Enhances Degradation of the Herbicides Isoproturon and Acetochlor in Rice Plants and Grains.

The rice cytochrome P450 gene has been comprehensively studied in the present study. This gene encodes CYP90D5 in promoting the degradation of isoproturon (IPU) and acetochlor (ACT) in rice tissues and grains. It has here been found that CYP90D5 improved the resistance of the plant to IPU and ACT, which was reflected in the improvement of the growth of the overexpression (OE) lines. CYP90D5 also reduced the levels of IPU and ACT accumulation in rice, and the CRISPR-Cas9 (Cas9) lines displayed the opposite effects. This function of CYP90D5 for pesticide degradation was also confirmed by the transformation of CYP90D5 in Pichia pastoris. Compared with the control yeast, it grew better and could degrade more pesticides. In addition, the relative contents of the IPU and ACT derivatives increased in the OE rice, while they decreased in the Cas9 rice. This suggested that CYP90D5 plays a pivotal role in the pesticide detoxification and degradation.

Chlorpyrifos and chlorfenapyr resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) relies on UDP-glucuronosyltransferases.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), has become an important agricultural pest worldwide. S. frugiperda is mainly controlled by the chemical insecticides, whereas the frequent application of insecticides would result in the resistance development. Insect uridine diphosphate-glucuronosyltransferases (UGTs), as phase II metabolism enzymes, play vital roles in the breakdown of endobiotic and xenobiotics. In this study, 42 UGT genes were identified by RNA-seq, including 29 UGT genes were elevated compared to the susceptible population, and the transcript levels of 3 UGTs (UGT40F20, UGT40R18, and UGT40D17) were increased by more than 2.0-fold in the field populations. Expression pattern analysis revealed that S. frugiperda UGT40F20, UGT40R18, and UGT40D17 were increased by 6.34-, 4.26-, and 8.28-fold, compared the susceptible populations, respectively. The expression of UGT40D17, UGT40F20, and UGT40R18 was affected after exposure to phenobarbital, chlorpyrifos, chlorfenapyr, sulfinpyrazone, and 5-nitrouracil. The induced expression of UGT genes may have improved UGT enzymatic activity, while the inhibition of UGTs genes expression may decreased UGT enzymatic activity. Sulfinpyrazone, and 5-nitrouracil, significantly increased the toxicity of chlorpyrifos and chlorfenapyr, as well as phenobarbital significantly reduced the toxicity of chlorpyrifos and chlorfenapyr against the susceptible populations and field populations of S. frugiperda. The suppression of UGTs (UGT40D17, UGT40F20, and UGT40R18) significantly increased the insensitivity of the field populations to chlorpyrifos and chlorfenapyr. These findings strongly supported our viewpoint that UGTs may play a critical role in insecticide detoxification. This study provides a scientific basis for the management of S. frugiperda.

Biodegrading Two Pesticide Residues in Paddy Plants and the Environment by a Genetically Engineered Approach.

Accumulating pesticide (and herbicide) residues in soils have become a serious environmental problem. This study focused on identifying the removal of two widely used pesticides, isoproturon (IPU) and acetochlor (ACT), by a genetically developed paddy (or rice) plant overexpressing an uncharacterized glycosyltransferase (IRGT1). IRGT1 conferred plant resistance to isoproturon-acetochlor, which was manifested by attenuated cellular injury and alleviated toxicity of rice under isoproturon-acetochlor stress. A short-term study showed that IRGT1-transformed lines removed 33.3-48.3% of isoproturon and 39.8-53.5% of acetochlor from the growth medium, with only 59.5-72.1 and 58.9-70.4% of the isoproturon and acetochlor remaining in the plants compared with the levels in untransformed rice. This phenotype was confirmed by IRGT1-expression in yeast ( Pichia pastoris) which grew better and contained less isoproturon-acetochlor than the control cells. A long-term study showed that isoproturon-acetochlor concentrations at all developmental stages were significantly lower in the transformed rice, which contain only 59.3-69.2% (isoproturon) and 51.7-57.4% (acetochlor) of the levels in wild type. In contrast, UPLC-Q-TOF-MS/MS analysis revealed that more isoproturon-acetochlor metabolites were detected in the transformed rice. Sixteen metabolites of isoproturon and 19 metabolites of acetochlor were characterized in rice for Phase I reactions, and 9 isoproturon and 13 acetochlor conjugates were characterized for Phase II reactions in rice; of these, 7 isoproturon and 6 acetochlor metabolites and conjugates were reported in plants for the first time.

Isoproturon-Induced Salicylic Acid Confers Arabidopsis Resistance to Isoproturon Phytotoxicity and Degradation in Plants.

This study identified the effect of salicylic acid on degradation of isoproturon in Arabidopsis. Three T-DNA insertion mutant lines pal1- 1, pal1- 2, and eps1- 1 defective in salicylic acid synthesis were tested, which showed higher isoproturon accumulation and a toxic symptom in the mutants. When treated with 5 mg/L salicylic acid, these lines displayed a lower level of isoproturon and showed an attenuated toxic symptom. An RNA-sequencing study identified 2651 (1421 up and 1230 down) differentially expressed genes (DEGs) in eps1- 1 and 2211 (1556 up and 655 down) in pal1- 2 mutant plants (>2.0 fold change, p < 0.05). Some of the DEGs covered Phase I-III reaction components, like glycosyltransferases (GTs) and ATP-binding cassette transporters (ABCs). Using ultra performance liquid chromatography-time-of-flight-tandem-mass spectrometer/mass spectrometer (UPLC/Q-TOF-MS/MS), 13 Phase I and four Phase II metabolites were characterized. Of these, two metabolites 1-OH-isopropyl-benzene-O-glucoside and 4-isopropylphenol-S-2-methylbutanoyl-serine, have been identified and reported for the first time.

Degrading and Phytoextracting Atrazine Residues in Rice (Oryza sativa) and Growth Media Intensified by a Phase II Mechanism Modulator.

Atrazine (ATZ) residue in farmland is one of the environmental contaminants seriously affecting crop production and food safety. Understanding the regulatory mechanism for ATZ metabolism and degradation in plants is important to help reduce ATZ potential toxicity to both plants and human health. Here, we report our newly developed engineered rice overexpressing a novel Phase II metabolic enzyme glycosyltransfearse1 (ARGT1) responsible for transformation of ATZ residues in rice. Our results showed that transformed lines, when exposed to environmentally realistic ATZ concentration (0.2-0.8 mg/L), displayed significantly high tolerance, with 8-27% biomass and 36-56% chlorophyll content higher, but 37-69% plasma membrane injury lower than untransformed lines. Such results were well confirmed by ARGT1 expression in Arabidopsis. ARGT1-transformed rice took up 1.6-2.7 fold ATZ from its growth medium compared to its wild type (WT) and accumulated ATZ 10%-43% less than that of WT. A long-term study also showed that ATZ in the grains of ARGT1-transformed rice was reduced by 30-40% compared to WT. The ATZ-degraded products were characterized by UPLC/Q-TOF-MS/MS. More ATZ metabolites and conjugates accumulated in ARGT1-transformed rice than in WT. Eight ATZ metabolites for Phase I reaction and 10 conjugates for Phase II reaction in rice were identified, with three ATZ-glycosylated conjugates that have never been reported before. These results indicate that ARGT1 expression can facilitate uptake of ATZ from environment and metabolism in rice plants.