Role of CYP9E2 and a long non-coding RNA gene in resistance to a spinosad insecticide in the Colorado potato beetle, Leptinotarsa decemlineata.

Spinosads are insecticides used to control insect pests, especially in organic farming where limited tools for pest management exist. However, resistance has developed to spinosads in economically important pests, including Colorado potato beetle (CPB), Leptinotarsa decemlineata. In this study, we used bioassays to determine spinosad sensitivity of two field populations of CPB, one from an organic farm exposed exclusively to spinosad and one from a conventional farm exposed to a variety of insecticides, and a reference insecticide naïve population. We found the field populations exhibited significant levels of resistance compared with the sensitive population. Then, we compared transcriptome profiles between the two field populations to identify genes associated primarily with spinosad resistance and found a cytochrome P450, CYP9E2, and a long non-coding RNA gene, lncRNA-2, were upregulated in the exclusively spinosad-exposed population. Knock-down of these two genes simultaneously in beetles of the spinosad-exposed population using RNA interference (RNAi) resulted in a significant increase in mortality when gene knock-down was followed by spinosad exposure, whereas single knock-downs of each gene produced smaller effects. In addition, knock-down of the lncRNA-2 gene individually resulted in significant reduction in CYP9E2 transcripts. Finally, in silico analysis using an RNA-RNA interaction tool revealed that CYP9E2 mRNA contains multiple binding sites for the lncRNA-2 transcript. Our results imply that CYP9E2 and lncRNA-2 jointly contribute to spinosad resistance in CPB, and lncRNA-2 is involved in regulation of CYP9E2 expression. These results provide evidence that metabolic resistance, driven by overexpression of CYP and lncRNA genes, contributes to spinosad resistance in CPB.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae.

Thymoquinone, extracted from the black seeds of Nigella sativa, is a natural substance with highly beneficial effects against various human diseases. In this study, we aimed to construct a Saccharomyces cerevisiae strain that, produce thymoquinone from thymol, a relatively inexpensive substrate. To achieve this, cytochrome P450 from Origanum vulgare was expressed in S. cerevisiae for the bioconversion of thymol to thymoquinone, with the co-expression of cytochrome P450 reductase (CPR) from Arabidopsis thaliana, ATR1. Additionally, flexible linkers were used to connect these two enzymes. Furthermore, modifications were performed to expand the endoplasmic reticulum (ER) space, leading to increased thymoquinone production. After integrating the genes into the chromosome and optimizing the media components, a significant improvement in the thymol-to-thymoquinone conversion rate and yield were achieved. This study represents a possibility of the production of thymoquinone, a bioactive ingredient of a plant, using an engineered microbial cell.

Genome-wide identification, molecular evolution and gene expression of P450 gene family in Cyrtotrachelus buqueti.

BACKGROUND: Insect Cytochrome P450 monooxygenase (CYPs or P450s) plays an important role in detoxifying insecticides, causing insect populations to develop resistance. However, the molecular functions of P450 gene family in Cyrtotrachelus buqueti genome are still lacking. RESULTS: In this study, 71 CbuP450 genes have been identified. The amino acids length of CbuP450 proteins was between 183 aa ~ 1041 aa. They are proteins with transmembrane domains. The main component of their secondary structure is α-helix and random coils. Phylogenetic analysis showed that C. buqueti and Rhynchophorus ferrugineus were the most closely related. This gene family has 29 high-frequency codons, which tend to use A/T bases and A/T ending codons. Gene expression analysis showed that CbuP450_23 in the female adult may play an important role on high temperature resistance, and CbuP450_17 in the larval may play an important role on low temperature tolerance. CbuP450_10, CbuP450_17, CbuP450_23, CbuP450_10, CbuP450_16, CbuP450_20, CbuP450_23 and CbuP450_ 29 may be related to the regulation of bamboo fiber degradation genes in C. buqueti. Protein interaction analysis indicates that most CbuP450 proteins are mainly divided into three aspects: encoding the biosynthesis of ecdysteroids, participating in the decomposition of synthetic insecticides, metabolizing insect hormones, and participating in the detoxification of compounds. CONCLUSIONS: We systematically analyzed the gene and protein characteristics, gene expression, and protein interactions of CbuP450 gene family, revealing the key genes involved in the stress response of CbuP450 gene family in the resistance of C. buqueti to high or low temperature stress, and identified the key CbuP450 proteins involved in important life activity metabolism. These results provided a reference for further research on the function of P450 gene family in C. buqueti.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

CYP2C19 and CYP2J2 genotypes predict praziquantel plasma exposure among Ethiopian school-aged children.

Metabolism of praziquantel (PZQ), a racemic mixture and the only drug approved to treat S. mansoni infection, is mediated by genetically polymorphic enzymes. Periodic school-based mass drug administration (MDA) with PZQ is the core intervention to control schistosomiasis. However data on the impact of pharmacogenetic variation, nutrition, and infection status on plasma PZQ exposure is scarce. We investigated genetic and non-genetic factors influencing PZQ plasma concentration and its metabolic ratios (trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ). Four hundred forty-six school children aged 7-15 years from four primary schools in southern Ethiopia who received albendazole and PZQ preventive chemotherapy through MDA campaign were enrolled. Genotyping for common functional variants of CYP3A4 (*1B), CYP3A5 (*3, *6), CYP2C19 (*2, *3, *17), CYP2C9 (*2, *3), and CYP2J2*7 was performed. Plasma concentrations of PZQ, trans-4-OH-PZQ, and cis-4-OH-PZQ were quantified using UPLCMS/MS. Carriers of CYP2C19 defective variant alleles (*2 and *3) had significantly higher mean PZQ plasma concentration than CYP2C19*1/*1 or *17 carriers (p = 0.005). CYP2C19*1/*1 and CYP2C19*17 carriers had higher trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ metabolic ratios compared with CYP2C19*2 or *3 carriers (p < 0.001). CYP2J2*7 carriers had lower mean PZQ plasma concentration (p = 0.05) and higher trans-4-OH-PZQ/PZQ and cis-4-OH-PZQ/PZQ metabolic ratios. Male participants had significantly higher PZQ concentration (p = 0.006) and lower metabolic ratios (p = 0.001) than females. There was no significant effect of stunting, wasting, S. mansoni or soil-transmitted helminth infections, CYP3A4, CYP3A5, or CYP2C9 genotypes on plasma PZQ or its metabolic ratios. In conclusion, sex, CYP2C19 and CYP2J2 genotypes significantly predict PZQ plasma exposure among Ethiopian children. The impact of CYP2C19 and CYP2J2 genotypes on praziquantel treatment outcomes requires further investigation.

St. John's wort extract with a high hyperforin content does not induce P-glycoprotein activity at the human blood-brain barrier.

St. John's wort (SJW) extract, a herbal medicine with antidepressant effects, is a potent inducer of intestinal and/or hepatic cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), which can cause clinically relevant drug interactions. It is currently not known whether SJW can also induce P-gp activity at the human blood-brain barrier (BBB), which may potentially lead to decreased brain exposure and efficacy of certain central nervous system (CNS)-targeted P-gp substrate drugs. In this study, we used a combination of positron emission tomography (PET) imaging and cocktail phenotyping to gain a comprehensive picture on the effect of SJW on central and peripheral P-gp and CYP activities. Before and after treatment of healthy volunteers (n = 10) with SJW extract with a high hyperforin content (3-6%) for 12-19 days (1800 mg/day), the activity of P-gp at the BBB was assessed by means of PET imaging with the P-gp substrate [11C]metoclopramide and the activity of peripheral P-gp and CYPs was assessed by administering a low-dose phenotyping cocktail (caffeine, omeprazole, dextromethorphan, and midazolam or fexofenadine). SJW significantly increased peripheral P-gp, CYP3A, and CYP2C19 activity. Conversely, no significant changes in the peripheral metabolism, brain distribution, and P-gp-mediated efflux of [11C]metoclopramide across the BBB were observed following the treatment with SJW extract. Our data suggest that SJW does not lead to significant P-gp induction at the human BBB despite its ability to induce peripheral P-gp and CYPs. Simultaneous intake of SJW with CNS-targeted P-gp substrate drugs is not expected to lead to P-gp-mediated drug interactions at the BBB.

Bioinspired axial S-coordinated single-atom cobalt catalyst to efficient activate peroxymonosulfate for selective high-valent Co-Oxo species generation.

The efficient activation and selective high-valent metal-oxo (HVMO) species generation remain challenging for peroxymonosulfate (PMS)-based advanced oxidation processes (PMS-AOPs) in water purification. The underlying mechanism of the activation pathway is ambiguous, leading to a massive dilemma in the control and regulation of HVMO species generation. Herein, bioinspired by the bio-oxidase structure of cytochrome P450, the axial coordination strategy was adopted to tailor a single-atom cobalt catalyst (CoN4S-CB) with an axial S coordination. CoN4S-CB high-selectively generated high-valent Co-Oxo species (Co(IV)=O) via PMS activation. Co(IV)=O demonstrated an ingenious oxygen atom transfer (OAT) reaction to achieve the efficient degradation of sulfamethoxazole (SMX), and this allowed robust operation in various complex environments. The axial S coordination modulated the 3d orbital electron distribution of the Co atom. Density functional theory (DFT) calculation revealed that the axial S coordination decreased the energy barrier for PMS desorption and lowered the free energy change (ΔG) for Co(IV)=O generation. CoN4S-PMS* had a narrow d-band close to the Fermi level, which enhanced charge transfer to accelerate the cleavage of O-O and O-H bonds in PMS. This work provides a broader perspective on the activator design with natural enzyme structure-like active sites to efficient activate PMS for selective HVMO species generation.

Transformation of 6:6 PFPiA in the gut of Xenopus laevis: Synergistic effects of CYP450 enzymes and gut microflora.

As a frequently detected per- and polyfluoroalkyl substance in the environment, 6:6 perfluoroalkylhypophosphinic acid (6:6 PFPiA) is vulnerable to transformation in the liver of organisms, but the transformation in gut is still unclear. This study investigates the molecular mechanisms of 6:6 PFPiA transformation in the gut of Xenopus laevis upon a 28-day exposure in water. Before Day 16, a notable correlation (p = 0.03) was observed between the transformation product (PFHxPA) and cytochrome P450 (CYP450) enzyme concentration in gut. This suggests that CYP450 enzymes played an important role in the transformation of 6:6 PFPiA in the gut, which was verified by an in vitro incubation with gut tissues, and supported by the molecular docking results of 6:6 PFPiA binding with CYP450 enzymes. From the day 16, the CYP450 concentration in gut decreased by 31.3 % due to the damage caused by 6:6 PFPiA, leading to a decrease in the transformation capacity in gut, but the transformation rate was stronger than in liver. This was in contrast with the in vitro experiment, where transformation was stronger in liver. In the mean time, the abundance of Bacteroidota in gut increased, which released hydrolytic enzyme and then could participate in the transformation as well. This study reveals the potential of the gut in metabolizing environmental pollutants, and provides profound insights into the potential health risks caused by 6:6 PFPiA in organisms.

Cytochrome P450 SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11.

Accumulating salinity in soil critically affected growth, development, and yield in plant. However, the mechanisms of plant against salt stress largely remain unknown. Herein, we identified a gene named SmCYP78A7a, which encoded a cytochrome P450 monooxygenase and belonged to the CYP78A sub-family, and its transcript level was significantly up-regulated by salt stress and down-regulated by dehydration stress. SmCYP78A7a located in the endoplasmic reticulum. Silencing of SmCYP78A7a enhanced susceptibility of eggplant to salt stress, and significantly down-regulated the transcript levels of salt stress defense related genes SmGSTU10 and SmWRKY11 as well as increased hydrogen peroxide (H2O2) content and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) enzyme activities. In addition, SmCYP78A7a transient expression enhanced eggplant tolerance to salt stress. By chromatin immunoprecipitation PCR (ChIP-PCR), luciferase reporter assay, and electrophoretic mobility shift assay (EMSA), SmWRKY11 activated SmCYP78A7a expression by directly binding to the W-box 6-8 (W-box 6, W-box 7, and W-box 8) within SmCYP78A7a promoter to confer eggplant tolerance to salt stress. In summary, our finds reveal that SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11, and provide a new insight into regulatory mechanisms of eggplant to salt stress.

Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay.

The continuous emergence of new psychoactive substances (NPS) attracted a great deal of attention within recent years. Lately, the two hallucinogenic NPS 1cP-LSD and 4-AcO-DET have appeared on the global market. Knowledge about their metabolism to identify potential metabolic targets for analysis and their cytotoxic properties is lacking. The aim of this work was thus to study their in vitro and in vivo metabolism in pooled human liver S9 fraction (pHLS9) and in zebrafish larvae (ZL) by means of liquid chromatography-high-resolution tandem mass spectrometry. Monooxygenases involved in the initial metabolic steps were elucidated using recombinant human isozymes. Investigations on their cytotoxicity were performed on the human hepatoma cell line HepG2 using a multiparametric, fluorescence-based high-content screening assay. This included measurement of CYP-enzyme mediated effects by means of the unspecific CYP inhibitor 1-aminbenzotriazole (ABT). Several phase I metabolites of both compounds and two phase II metabolites of 4-AcO-DET were produced in vitro and in vivo. After microinjection of 1cP-LSD into the caudal vein of ZL, three out of seven metabolites formed in pHLS9 were also detected in ZL. Twelve 4-AcO-DET metabolites were identified in ZL after exposure via immersion bath and five of them were found in pHLS9 incubations. Notably, unique metabolites of 4-AcO-DET were only produced by ZL, whereas 1cP-LSD specific metabolites were found both in ZL and in pHLS9. No toxic effects were observed for 1cP-LSD and 4-AcO-DET in HepG2 cells, however, two parameters were altered in incubations containing 4-AcO-DET together with ABT compared with incubations without ABT but in concentrations far above expected in vivo concentration. Further investigations should be done with other hepatic cell lines expressing higher levels of CYP enzymes.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Cytochrome GmGLY1 is Involved in the Biosynthesis of Glycitein in Soybean.

Isoflavones, the major secondary metabolites of interest due to their benefits to both human and plant health, are exclusively produced by legumes. In this study, we profiled the isoflavone content in dry seeds from 211 soybean [Glycine max (L.) Merr.] accessions grown across five environments. Broad and discernible phenotypic variations were observed among accessions, regions, and years of growth. Twenty-six single-nucleotide polymorphisms (SNPs) associated with the sum of glycitein (GLE), glycitin (GL), 6″-O-acetylglycitin (AGL), and 6″-O-malonylglycitin (MGL) contents were detected in multiple environments via a genome-wide association study (GWAS). These SNPs were located on chromosome 11 (8,148,438 bp to 8,296,956 bp, renamed qGly11-01). Glyma.11g108300 (GmGLY1), a gene that encodes a P450 family protein, was identified via sequence variation analysis, functional annotation, weighted gene coexpression network analysis (WGCNA), and expression profile analysis of candidate gene, and hairy roots transformation in soybean. Overexpression of GmGLY1 increased the glycitein content (GLC) in soybean hairy roots and transgenic seeds, while CRISPR/Cas9-generated mutants exhibited decreased GLC and increased daidzein content (DAC). Haplotype analysis revealed that GmGLY1 allelic variations significantly affect the GLC accumulation. These findings enhance our understanding of genes influencing GLC in soybean and may guide breeding for lines with high and stable GLC.

Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

Phytomediated stress modulates antioxidant status, induces overexpression of CYP6M2, Hsp70, α-esterase, and suppresses the ABC transporter in Anopheles gambiae (sensu stricto) exposed to Ocimum tenuiflorum extracts.

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-ß-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds.

SlCYP94B18 and SlCYP94B19 monooxygenases for the catabolic turnover of jasmonates in tomato leaves.

(3R,7S)-Jasmonoyl-L-isoleucine (JA-Ile) is a plant hormone that regulates plant defense responses and other physiological functions. The mechanism of attenuation of JA-Ile signaling in the plant body is essential because prolonged JA-Ile signaling can be detrimental to plant survival. In Arabidopsis thaliana, the cytochrome P450 monooxygenases, CYP94B1/B3/C1, inactivate JA-Ile by converting it into 12-hydroxy-jasmonoyl-L-isoleucine (12-OH-JA-Ile), and CYP94C1 converts 12-OH-JA-Ile into 12-carboxy-jasmonoyl-L-isoleucine (12-COOH-JA-Ile). In the present study, we aimed to identify the cytochrome P450 monooxygenases involved in the catabolic pathway of JA-Ile in tomato leaves. Based on a gene expression screening of SlCYP94 subfamily monooxygenases using qPCR and the time-course of JA-Ile catabolism, we identified SlCYP94B18 and SlCYP94B19 expressed in tomato leaves as candidate monooxygenases catalyzing the two-step catabolism of JA-Ile. An in vitro enzymatic assay using a yeast expression system revealed that these enzymes efficiently converted JA-Ile to 12-OH-JA-Ile, and then to 12-COOH-JA-Ile. SlCYP94B18 and SlCYP94B19 also catalyzed the oxidative catabolism of several JA-amino acid conjugates (JA-AAs), JA-Leu and JA-Val, in tomatoes. These results suggest that SlCYP94B18 and SlCYP94B19 plays a role in the two-step oxidation of JA-AAs, suggesting their broad involvement in regulating jasmonate signaling in tomatoes. Our results contribute to a deeper understanding of jasmonate signaling in tomatoes and may help to improve tomato cultivation and quality.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.