Mutation of the Cytochrome P450 CYP360A8 Gene Increases Sensitivity to Paraquat in Daphnia magna

Abstract

The freshwater crustacean Daphnia magna has traditionally been a model for ecotoxicological studies owing to its sensitivity to many xenobiotics. Because it is used in many toxicity assessments, its detoxification mechanism for xenobiotics is important and requires further study. However, studies related to detoxification genes are limited to transcriptomic profiling, and there are no D. magna mutants for use in the understanding of xenobiotic metabolism in vivo. We report the generation of a D. magna CYP360A8 mutant—the gene is a cytochrome P450 (CYP) clan 3 gene. Based on RNA sequencing of adult D. magna, we found that CYP360A8 has the highest expression level among all CYP genes. At ovarian maturation, its expression level is up‐regulated 6‐fold compared to the juvenile stages and is maintained thereafter. Using the CRISPR/CRISPR‐associated 9 (Cas9) system, we disrupted CYP360A8 by coinjecting CYP360A8‐targeting guide RNA and Cas9 proteins into D. magna eggs and established one monoallelic CYP360A8 mutant line. This CYP360A8 mutant had a higher sensitivity to the herbicide paraquat compared to the wild type. We confirmed the up‐regulation of CYP360A8 by paraquat. The results demonstrate the role of CYP360A8 in paraquat detoxification. The present study establishes a CYP mutant of D. magna, and this strategy can be a basic platform to document a range of CYP gene–xenobiotic relationships in this species. Environ Toxicol Chem 2021;40:1279–1288. © 2020 SETAC

Degradation of Carbendazim by Molecular Hydrogen on Leaf Models

“…Meanwhile, three detoxification genes, including CYP72A , ABC3 , and ABC4 , were selected to evaluate their involvement of exogenous H 2 control of CAR degradation. Among these, CYP72A encodes one of the members of cytochrome P450s, which are indispensable for hormone synthesis and detoxification in both animals and plants [ 43 ]. ATP-binding cassette (ABC) transporters also participated in the detoxification of environmental pollutants [ 44 , 45 ].…”

“…As shown in Figure 5A-C, HRW strongly stimulated the expression of GR, GSH1 (encoding γ-ECS) [42], and GST2 in the presence of CAR, all of which were matched with the increased activities of GR (Figure 4H), γ-ECS (Figure 4F), and GST (Figure 4G), thus promoting endogenous GSH production in CAR-treated tomato seedling leaves (Figure 4A). Among these, CYP72A encodes one of the members of cytochrome P450s, which are in-dispensable for hormone synthesis and detoxification in both animals and plants [43]. ATP-binding cassette (ABC) transporters also participated in the detoxification of environmental pollutants [44,45].…”

Daphnia as a versatile model system in ecology and evolution

“…Manipulation of gene expression, as well as gene knock-in and knock-out technology are now possible for Daphnia, with microinjection-based RNA interference (RNAi) used routinely [102][103][104]. Clustered regularly interspaced short palindromic repeats/ CRISPR-associated (CRISPR/Cas) system have been used for gene knock-outs [105,106], but also for CRISPR/Casmediated knock-in via non-homologous end-joining, e.g., for reporter-genes [107]. Likewise, Transcription Activator-Like Effector Nucleases (TALENs) are increasingly used as a versatile genomic manipulation tool in D. magna [108][109][110].…”

“…Current experimental approaches mainly focus on genes known from other organisms for diverse biological roles, such as development, sex expression (e.g., producing white eyes, knock-in of Green Fluorescent Protein) [97,106,107,[112][113][114]. So far, genes that have a function in the direct interaction of the Daphnia with their environment, for example genes for local adaptation, phenotypic plasticity, predator defence, parasite resistance, perception of environmental factors, such as water quality, photoperiod, and toxins, have hardly been manipulated [115], partly due to the low number of good candidates for genes with such function.…”

CRISPR-Cas gene editing technology and its application prospect in medicinal plants

“…Their roles can be further studied, and the result may lay a foundation for improving the herbicide resistance of medicinal plants. In addition, herbicide detoxification enzymes introduced into plants with the CRISPR technology reduce damage caused by herbicides to medicinal plants and cause herbicide resistance [164].…”