24-epibrassinolide as a multidimensional regulator of rice (Oryza sativa) physiological and molecular responses under isoproturon stress.

Brassinosteroids (BRs) can regulate various processes in plant development and defense against environmental stress. In this study, the contribution of BRs in the degradation of isoproturon (IPU) in rice has been established. IPU has a significant effect on rice growth, chlorophyll content, and membrane permeability. When treated with 1.0 µmol/L 24-epibrassinolide (EBR), a BR analogue, the associated symptoms of rice poisoning were alleviated as the IPU levels in the rice and growth media were decreased. In the presence of EBR, the activities of several IPU-related detoxification enzymes were enhanced to cope with the stress due to IPU. An RNA-sequencing (RNA-Seq) has been performed to determine the variation of transcriptomes and metabolic mechanisms in rice treated with EBR, IPU, or IPU+EBR. Some of the differentially expressed genes (DEGs) were Phase I-III reaction components of plants, such as cytochrome P450 (CYP450), glutathione S-transferase (GST), glycosyltransferases (GTs), and the ATP-binding cassette transporter (ABC transporter). The expression of some signal transduction genes was significantly up-regulated. The relative content of low-toxicity IPU metabolites increased due to the presence of EBR as determined by UPLC/Q-TOF-MS/MS. The IPU metabolic pathways include enzyme-catalyzed demethylation, hydroxylation, hydrolysis, glycosylation, and amino acid conjugation processes. The results suggest that EBR plays a key role in the degradation and detoxification of IPU. This study has provided evidence that BRs regulate the metabolism and detoxification of IPU in rice, and offers a new approach to ensuring cleaner crops by eliminating pesticide residues in the environment.

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China.

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Phytoremediation of isoproturon-contaminated sites by transgenic soybean.

The widespread application of isoproturon (IPU) can cause serious pollution to the environment and threaten ecological functions. In this study, the IPU bacterial N-demethylase gene pdmAB was transferred and expressed in the chloroplast of soybean (Glycine max L. 'Zhonghuang13'). The transgenic soybeans exhibited significant tolerance to IPU and demethylated IPU to a less phytotoxic metabolite 3-(4-isopropylphenyl)-1-methylurea (MDIPU) in vivo. The transgenic soybeans removed 98% and 84% IPU from water and soil within 5 and 14 days, respectively, while accumulating less IPU in plant tissues compared with the wild-type (WT). Under IPU stress, transgenic soybeans showed a higher symbiotic nitrogen fixation performance (with higher total nodule biomass and nitrogenase activity) and a more stable rhizosphere bacterial community than the WT. This study developed a transgenic (TS) soybean capable of efficiently removing IPU from its growing environment and recovering a high-symbiotic nitrogen fixation capacity under IPU stress, and provides new insights into the interactions between rhizosphere microorganisms and TS legumes under herbicide stress.

The Importance of Humic Substances in Transporting "Chemicals of Emerging Concern" in Water and Sewage Environments.

In this study, we examined the sorption of selected "chemicals of emerging concern" (CEC) on humic substances commonly found in water and municipal wastewater. These were ibuprofen, diclofenac, caffeine, carbamazepine, estrone, triclosan, bisphenol A, and isoproturon. The humic substances (HSs) were synthetic and not contaminated by the tested organic substances. The elemental composition and content of mineral micropollutants, gravimetric curves, and the IR spectrum of HSs were determined. We determined a relationship between the process efficiency and the characteristics of a sorbent and sorbate using the properties of organic substances sorbed on HSs. This relationship was confirmed by sorption tests on the HS complex, i.e., the HS-organic micropollutant. It has been shown that the given complexes have a greater affinity for hydrophobic surfaces than hydrophilic surfaces. To confirm the nature of the sorbent surfaces, we determined their zeta potential dependence on the pH of the solution. Studies have shown that HSs are carriers of both mineral substances and CEC in water and sewage environments.

Degradation of isoproturon in vitro by a mix of bacterial strains isolated from arable soil.

Isoproturon (IPU) is widely used to control annual grasses and broad leaf weeds in cereal crops. In this study, four IPU-degrading bacterial strains, i.e., Sphingomonas sp. ISP1, Arthrobacter sp. ISP2, Acinetobacter baumannii 4IA, and Pseudomonas sp. ISP3, were isolated from agricultural soil. The mixed culture of four isolates completely degraded the herbicide at 100 mg/L within 10 days. During IPU degradation, several transient accumulations of the metabolites, including 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, 4-isopropylaniline, and 4-toluidine, were also identified. Moreover, the inoculation of the isolated mixed culture into the soil from a mountain with no previous herbicide application increased the degradation rate by 51% of the herbicide on average. Furthermore, bioaugmentation with isolated bacteria in the soil resulted in short-term variations in bacterial structure compared to the unaugmented soil. The findings of this study were instrumental in understanding the mechanisms of pesticide breakdown and bioremediation in liquid media and soil.

Insights into the Function and Horizontal Transfer of Isoproturon Degradation Genes (pdmAB) in a Biobed System.

Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.IMPORTANCE Our study provides novel insights into the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes, and verifies that on-farm biobed systems are hot spots for the evolution of pesticide catabolic traits.

Sensitive simultaneous voltammetric determination of the herbicides diuron and isoproturon at a platinum/chitosan bio-based sensing platform.

Phenylurea herbicides are persistent contaminants, which leads their transport to the surface and ground waters, affecting human and aquatic organisms. Different analytical methods have been reported for the detection of phenylureas; however, several of them are expensive, time-consuming, and require complex pretreatment steps. Here, we show a simple method for the simultaneous electrochemical determination of two phenylurea herbicides by differential pulse adsorptive stripping voltammetry (DPAdSV) using a modified platinum/chitosan electrode. The one-step synthesized platinum/chitosan PtNPs/CS was successfully characterized by TEM, XRPD, and FT-IR, and applied through the sensing platform designated as PtNPs/CS/GCE. This bio-based modified electrode is proposed for the first time for the individual and/or simultaneous electrochemical detection of the phenylurea herbicides diuron and isoproturon compounds extensively used worldwide that present a very similar chemical structure. Electrochemical and interfacial characteristics of the modified electrode were evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). It was found that the oxidation mechanism of diuron and isoproturon occurs in two different pathways, with a peak-to-peak definition of ca. 0.15 V. Under differential pulse adsorptive stripping voltammetry (DPAdSV) optimized conditions, the limit of detection (LOD) was estimated as 7 µg L-1 for isoproturon and 20 µg L-1 for diuron (Ed = +0.8 V; td = 100 s). The proposed method was successfully applied to the determination of both analytes in river water samples, at three different levels, with a recovery range of 90-110%. The employment of the bio-based sensing platform PtNPs/CS/GCE allows a novel and easy analytical method to the multi-component phenylurea herbicides detection.

Effect of interspecific competition on species sensitivity distribution models: Analysis of plant responses to chemical stress.

Species Sensitivity Distributions (SSD) are widely used in environmental risk assessment to predict the concentration of a contaminant that is hazardous for 5% of species (HC5). They are based on monospecific bioassays conducted in the laboratory and thus do not directly take into account ecological interactions. This point, among others, is accounted for in environmental risk assessment through an assessment factor (AF) that is applied to compensate for the lack of environmental representativity. In this study, we aimed to assess the effects of interspecific competition on the responses towards isoproturon of plant species representative of a vegetated filter strip community, and to assess its impact on the derived SSD and HC5 values. To do so, we realized bioassays confronting six herbaceous species to a gradient of isoproturon exposure in presence and absence of a competitor. Several modelling approaches were applied to see how they affected the results, using different critical effect concentrations and investigating different ways to handle multiple endpoints in SSD. At the species level, there was a strong trend toward organisms being more sensitive to isoproturon in presence of a competitor than in its absence. At the community level, this trend was also observed in the SSDs and HC5 values were always lower in presence of a competitor (1.12-11.13 times lower, depending on the modelling approach). Our discussion questions the relevance of SSD and AF as currently applied in environmental risk assessment.

Growth inhibition and recovery patterns of common duckweed Lemna minor L. after repeated exposure to isoproturon.

Aquatic non-targeted organisms are more likely to be exposed to herbicides in multiple pulse events then long continuous exposure. The potential of an organism to recover between exposures has an important role in the overall effects of the toxicant. Common duckweeds show high potential for recovery after a single exposure to isoproturon. To evaluate the growth patterns and recovery potential between multiple exposures, L. minor plants were exposed to isoproturon in three repetitive 7-day treatment cycles in three time-variable exposure scenarios with equivalent time-weighted average concentrations. The growth was significantly inhibited during each exposure phase with significant cumulative effects in every subsequent treatment cycle resulting in a cumulative decrease in biomass production. However, inhibitory effects were reversible upon transferring plants to a herbicide-free nutrient solution. These results indicate that L. minor plants have a high recovery potential even after multiple exposures to isoproturon. Observed cumulative decrease in biomass production, as well as the potential for fast and efficient recovery from repeated herbicide exposure, might affect the competitiveness of L. minor in surface water communities. The observations made during each exposure period, recovery patterns, and the resulting cumulative effects over time may contribute to further development, calibration and validation of mechanistic toxicokinetic/toxicodynamic models for simulating the effects of pesticides on aquatic plants populations in the laboratory and environmental conditions.

Kinetics of inhibition of isoproturon to glutathione-associated enzymes in wheat.

The present study aimed at investigating the kinetic of inhibition of isoproturon to the GSH-associated enzymes [γ-glutamyl-cysteine synthetase (γ-GCS), glutathione synthetase (GS), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione peroxidase (GPX)] in wheat. Isoproturon, applied to 10-day-old seedlings for the following 12 days, provoked significant reductions in shoot fresh and dry weights, protein, thiols and glutathione (GSH); however, oxidized glutathione (GSSG) was elevated while GSH/GSSG ratio was declined with concomitant significant inhibitions in the activities of γ-GCS, GS, GR, GST and GPX; the effect was time dependent. IC50 and Ki values of isoproturon were lowest for GPX, highest for both GST and GR, and moderate for both γ-GCS and GS. The herbicide markedly decreased Vmax of γ-GCS, GS and GPX but unchanged that of GST and GR; however, Km of γ-GCS, GS, GST and GR increased but unchanged for GPX. The pattern of response of changing Vmax, Km, Vmax/Km, kcat and kcat/Km for in vivo and in vitro tests of each enzyme seemed most likely similar. These results indicate that a malfunction to defense system was induced in wheat by isoproturon resulting in inhibitions in GSH-associated enzymes, the magnitude of inhibition was most pronounced in GPX followed by γ-GCS, GS, GST, and GR. These findings could conclude that isoproturon competitively inhibited GST and GR; however, the inhibition was noncompetitive for GPX but mixed for both γ-GCS and GS.

Pesticide contact dermatitis in agricultural workers of Himachal Pradesh (India).

BACKGROUND: Common pesticides used in the region by agricultural workers may cause contact allergy. METHODS: Thirty agricultural workers with a history of pesticide exposure and dermatitis involving the face, neck, trunk or extremities, and 20 controls comprising 2 groups of 10 subjects each, group 1 with dermatitis and no exposure to pesticides, and group 2 with neither exposure to pesticides nor dermatitis, were patch tested with 10 pesticides commonly used in the region by use of the Finn Chamber method. RESULTS: The 30 patients, 20 of whom were male, aged 30-77 years, had dermatitis for 1 month to 18 years, with relapses and remissions. Seasonal exacerbation was present in 18 patients. Six patients attributed aggravation of their dermatitis to pesticide exposure, and 2 of these reacted positively to propiconazole. Positive patch test reactions to pesticides occurred in 10 patients, but not in controls. Thiuram was the commonest sensitizer (4 patients). Three patients were sensitized to propiconazole, and 2 patients reacted positively to metaldehyde. Formaldehyde, mercaptobenzothiazole, cypermethrin and isoproturon gave positive reactions in 1 patient each. CONCLUSION: The sensitizing potential of pesticides remains a concern. Apparently, pesticide contact dermatitis is more common than expected, but remains under-reported, as the implicated pesticides vary across regions and according to the crop patterns.

Inoculation of soil with an Isoproturon degrading microbial community reduced the pool of "real non-extractable" Isoproturon residues.

During pesticides degradation, biogenic non-extractable residues ("apparent NER") may not share the same environmental fate and risks with the "real NER" that are bound to soil matrix. It is not clear how microbial community (MC) inoculation for pesticides degradation would influence the NER composition. To investigate degradation efficiency of pesticides Isoproturon (IPU) and NER composition following MC inoculation, clay particles harboring MC that contains the IPU degrading strain, Sphingomonas sp., were inoculated into soil receiving 14C-labeled IPU addition. Mineralization of IPU was greatly enhanced with MC inoculation that averagely 55.9% of the applied 14C-IPU was consumed up into 14CO2 during 46 days soil incubation. Isoproturon degradation was more thorough with MC than that in the control: much less amount of metabolic products (4.6% of applied IPU) and NER (35.4%) formed in MC treatment, while the percentages were respectively 30.3% for metabolites and 49.8% for NER in the control. Composition of NER shifted with MC inoculation, that relatively larger amount of IPU was incorporated into the biogenic "apparent NER" in comparison with "real NER". Besides its well-recognized role on enhancing mineralization, MC inoculation with clay particles benefits soil pesticides remediation in term of reducing "real NER" formation, which has been previously underestimated.

Modelling the effect of fluctuating herbicide concentrations on algae growth.

Herbicide concentrations fluctuate widely in watercourses after crop applications and rain events. The level of concentrations in pulses can exceed the water chronic quality criteria. In the present study, we proposed modelling the effects of successive pulse exposure on algae. The deterministic model proposed is based on two parameters: (i) the typical growth rate of the algae, obtained by monitoring growth rates of several successive batch cultures in growth media, characterizing both the growth of the control and during the recovery periods; (ii) the growth rate of the algae exposed to pulses, determined from a dose-response curve obtained with a standard toxicity test. We focused on the herbicide isoproturon and on the freshwater alga Scenedesmus vacuolatus, and we validated the model prediction based on effect measured during five sequential pulse exposures in laboratory. The comparison between the laboratory and the modelled effects illustrated that the results yielded were consistent, making the model suitable for effect prediction of the herbicide photosystem II inhibitor isoproturon on the alga S. vacuolatus. More generally, modelling showed that both pulse duration and level of concentration play a crucial role. The application of the model to a real case demonstrated that both the highest peaks and the low peaks with a long duration affect principally the cell density inhibition of the alga S. vacuolatus. It is therefore essential to detect these characteristic pulses when monitoring of herbicide concentrations are conducted in rivers.

Alleviation of isoproturon toxicity to wheat by exogenous application of glutathione.

Treatment with the recommended field dose of isoproturon to 7-d-old wheat seedlings significantly decreased shoot height, fresh and dry weights during the subsequent 15days. Meanwhile contents of carotenoids, chlorophylls and anthocyanin as well as activities of δ-aminolevulinate dehydratase (ALA-D), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were significantly inhibited. On the other hand, the herbicide significantly increased malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and H2O2, while it significantly decreased the contents of glutathione (GSH) and ascorbic acid (AsA) and reduced the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). These findings indicate an induction of a stress status in wheat seedlings following isoproturon treatment. However, exogenous GSH appeared to limit the toxic effects of isoproturon and seemed to overcome this stress status. Most likely, contents of pigment and activities of enzymes were raised to approximate control levels. Moreover, antioxidants were elevated and the oxidative stress indices seemed to be alleviated by GSH application. These results indicate that exogenous GSH enhances enzymatic and nonenzymatic antioxidants to alleviate the effects of isoproturon.

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.

Remediation of isoproturon-contaminated soil by Sphingobium sp. strain YBL2: Bioaugmentation, detoxification and community structure.

The widely used phenylurea herbicide isoproturon (IPU) and its residues can inhibit the growth of subsequently planted crops. However, reports on bioremediation of IPU-contaminated soil are scarce. In this study, Sphingobium sp. strain YBL2-gfp (a derivative of the IPU-degrading Sphingobium sp. strain YBL2 isolated by our lab) was constructed to bioremediate IPU-contaminated soil. In pot experiments, strain YBL2-gfp colonized the roots of wheat and eliminated IPU residues in the soil within 21 d, effectively alleviating its toxicity and restoring wheat growth. IPU treatment reduced the richness and diversity of soil bacteria, while inoculation YBL2-gfp mainly affected richness with less impact on diversity. The high concentrations of IPU and inoculation of YBL2-gfp alone reduced the soil microbial community connections, while bioaugmentation treatment enhanced the soil microbial community connections. Additionally, strain YBL2-gfp stimulated the metabolic capacity of the indigenous microbes, promoting the degradation of IPU and reducing the negative impact of high concentrations of IPU on microbial community. Taken together, this study offers relatively comprehensive insights into the practical application of bioaugmentation, demonstrating that strain YBL2 has the potential to remediate IPU-contaminated soils.

Engineering multi-degrading bacterial communities to bioremediate soils contaminated with pesticides residues.

Parallel to the important use of pesticides in conventional agriculture there is a growing interest for green technologies to clear contaminated soil from pesticides and their degradation products. Bioaugmentation i. e. the inoculation of degrading micro-organisms in polluted soil, is a promising method still in needs of further developments. Specifically, improvements in the understanding of how degrading microorganisms must overcome abiotic filters and interact with the autochthonous microbial communities are needed in order to efficiently design bioremediation strategies. Here we designed a protocol aiming at studying the degradation of two herbicides, glyphosate (GLY) and isoproturon (IPU), via experimental modifications of two source bacterial communities. We used statistical methods stemming from genomic prediction to link community composition to herbicides degradation potentials. Our approach proved to be efficient with correlation estimates over 0.8 - between model predictions and measured pesticide degradation values. Multi-degrading bacterial communities were obtained by coalescing bacterial communities with high GLY or IPU degradation ability based on their community-level properties. Finally, we evaluated the efficiency of constructed multi-degrading communities to remove pesticide contamination in a different soil. While results are less clear in the case of GLY, we showed an efficient transfer of degrading capacities towards the receiving soil even at relatively low inoculation levels in the case of IPU. Altogether, we developed an innovative protocol for building multi-degrading simplified bacterial communities with the help of genomic prediction tools and coalescence, and proved their efficiency in a contaminated soil.

Photo-transformation of isoproturon under UV-A irradiation: The synergy of nitrite and natural organic matter.

Isoproturon (IPU), a widely utilized phenylurea herbicide, is recognized as an emerging contaminant. Previous studies have predominantly attributed the degradation of IPU in natural waters to indirect photolysis by natural organic matter (NOM). Here, we demonstrate that nitrite (NO2-) also serves as an important photosensitizer that induces the photo-degradation of IPU. Through radical quenching tests, we identify hydroxyl radicals (•OH) and nitrogen dioxide radicals (NO2•) originating from NO2- photolysis as key players in IPU degradation, resulting in the generation of a series of hydroxylated and nitrated byproducts. Moreover, we demonstrate a synergistic effect on the photo-transformation of IPU when both NOM and NO2- are present in the reaction mixture. The observed rate constant (kobs) for IPU removal increases to 0.0179 ± 0.0002 min-1 in the co-presence of NO2- (50 µM) and NOM (2.5 mgC/L), surpassing the sum of those in the presence of each alone (0.0135 ± 0.0004 min-1). NOM exhibits multifaceted roles in the indirect photolysis of IPU. It can be excited by UV and transformed to excited triplet states (3NOM*) which oxidize IPU to IPU•+ that undergoes further degradation. Simultaneously, NOM can mitigate the reaction by reducing the IPU•+ intermediate back to the parent IPU. However, the presence of NO2- alters this dynamic, as IPU•+ rapidly couples with NO2•, accelerating IPU degradation and augmenting the formation of mono-nitrated IPU. These findings provide in-depth understandings on the photochemical transformation of environmental contaminants, especially phenylurea herbicides, in natural waters where NOM and NO2- coexist.

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.

Identification of a Phase I mechanism gene of rice (OsCYP1) in response to isoproturon.

The long-term use of isoproturon may threaten food security and human health. Cytochrome P450 (CYP or P450) can catalyze the biosynthetic metabolism, and play a crucial role in the modification of plant secondary metabolites. Therefore, it is of great importance to explore the genetic resources for isoproturon degradation. This research focused on a phase I metabolism gene (OsCYP1) with significant differential expression in rice under isoproturon pressure. Specifically, the high-throughput sequencing results of rice seedling transcriptome in response to isoproturon stress were analyzed. The molecular information and tobacco subcellular localization of OsCYP1 were studied. The subcellular localization of OsCYP1 in tobacco was assessed, where it is located in the endoplasmic reticulum. To analyze the expression of OsCYP1 in rice, the wild-type rice was treated with 0-1 mg/L isoproturon for 2 and 6 days, and qRT-PCR assays were conducted to detect the transcription levels. Compared with the control group, the expression of OsCYP1 in shoots was progressively upregulated after exposure to isoproturon, with 6.2-12.7-fold and 2.8-7.9-fold increases in transcription levels, respectively. Moreover, treatment with isoproturon upregulated the expression of OsCYP1 in roots, but the upregulation of transcripts was not significant except for 0.5 and 1 mg/L isoproturon at day 2. To confirm the role of OsCYP1 in enhancing isoproturon degradation, the vectors overexpressing OsCYP1 were transformed into recombinant yeast cells. After exposure to isoproturon, the growth of OsCYP1-transformed cells was better than the control cells, especially at higher stress levels. Furthermore, the dissipation rates of isoproturon were increased by 2.1-, 2.1- and 1.9-fold at 24, 48 and 72 h, respectively. These results further verified that OsCYP1 could enhance the degradation and detoxification of isoproturon. Collectively, our findings imply that OsCYP1 plays vital role in isoproturon degradation. This study provides a fundamental basis for the detoxification and regulatory mechanisms of OsCYP1 in crops via enhancing the degradation and/or metabolism of herbicide residues.