Search Results (115)

A male sterile mutant, S201, was identified in Brassica napus. Genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, male sterility (MS), which was stably inherited. The results of microscopy showed that the main reason for male sterility was a defect in microspore development, resulting in the absence of typical exine and mature microspores. Bulked segregant analysis (BSA) and genotyping of an F₂ population showed that the MS gene was located in a 1.4 Mb region. Sequence analysis showed that the CYP704B1 gene in this region contained two non-synonymous SNPs, leading to substitutions of two amino acids. A high-throughput KASP marker was characterized to detect the presence of the ms gene in the breeding population. The data presented here indicate that the male sterile mutant S201 can be applied in rapeseed breeding by producing the male sterile line and that the KASP marker developed for male sterility will be useful in marker-assisted selection of male sterile individuals in rapeseed-breeding programs. Full article

Phytoalexins are specialized metabolites that are synthesized by plants in response to pathogens. A paradigm in transcription factor (TF) biology is that conserved TFs have dedicated roles across plant lineages in regulating specific branches of specialized metabolism. However, the Arabidopsis (Arabidopsis thaliana) NAC family TF ANAC042 (a.k.a. JUNGBRUNNEN1 or JUB1) regulates the synthesis of camalexin, a Trp-derived phytoalexin specifically produced by several Brassicaceae species, whereas its homolog in soybean (Glycine max) regulates the synthesis of glyceollins, which are Phe-derived phytoalexins specific to soybean. The question addressed by this research is whether ANAC042 broadly regulates phytoalexin biosynthetic pathways in Arabidopsis. Using a novel matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS) method, we found that the Arabidopsis loss-of-function mutant anac042–1 elicited with bacterial flagellin (Flg22) is deficient in lineage-specific Trp- and conserved Phe-derived phytoalexins—namely camalexin and 4-hydroxyindole-3-carbonyl nitrile (4OH-ICN), and pathogen-inducible monolignols and scopoletin, respectively. Overexpressing ANAC042 in the anac042-1 mutant restored or exceeded wildtype amounts of the metabolites. The expression of phytoalexin biosynthetic genes in mutant and overexpression lines mirrored the accumulation of metabolites. Yeast-one hybrid and promoter-reporter assays in Nicotiana benthamiana found that the ANAC042 protein directly binds and activates the promoters of CYP71B15, CYP71A12, and PAL1 genes for the synthesis of camalexin, 4OH-ICN, and pathogen-inducible monolignol/scopoletin, respectively. Our results demonstrate that ANAC042 regulates conserved and lineage-specific phytoalexin pathways in Arabidopsis. The latter suggests that it is an opportunistic TF that has coopted lineage-specific genes into phytoalexin metabolism, thus providing an exception to the current paradigm. Full article

Aspergillus flavus and its secondary metabolites aflatoxins pose a significant threat to the health of humans, animals, and plants. Therefore, there is an urgent need to control A. flavus contamination. AfverB plays a key role in the aflatoxin gene cluster; however, its function and mechanism in fungal development and virulence remain poorly understood. In this study, we constructed afVerB gene deletion mutants (∆afVerB−1 and ∆afVerB−2) and two CYP domain mutants (afVerB^∆D1 and afVerB^∆D2) through homologous recombination. Phenotype analysis revealed that, via its two CYP domains, AfVerB is deeply involved in fungal morphogenesis and aflatoxin synthesis. Insect and crop colonization models revealed that AfVerB plays a key role in the fungus’s ability to infect hosts, and stress experiments discovered that AfVerB plays a significant role in the response to various environmental stresses, which explains why AfVerB is a key factor in fungal infection to some extent. RT-qPCR analysis demonstrated that AfVerB performs its bio-function through corresponding regulatory factors. We ultimately discovered that AfVerB is deeply involved in cell membrane stress stability, thereby participating in the regulation of fungal drug resistance (sensitive to AMB and resistant to VOR in this study). The CYP domain of AfVerB, particularly its second CYP domain, is crucial for the execution of its biological functions. This study elucidated the regulatory mechanisms by which AfVerB regulates fungal pathogenicity and aflatoxin biosynthesis, providing potential strategies for controlling A. flavus and its aflatoxin contamination. Full article

Heat stress is one of the major factors affecting crop growth and yield. However, the molecular mechanisms underlying rice heat stress tolerance remain largely unclear. In this study, we identified and characterized the rice high temperature sensitive 2 (hts2) mutant, which is highly susceptible to heat stress. Map-based cloning revealed that the HTS2 encodes a cytochrome P450 protein (CYP71P1) involved in serotonin biosynthesis. HTS2 is ubiquitously expressed across plant tissues and shows strong upregulation in response to heat stress. The HTS2 mutation significantly impairs basal serotonin synthesis in rice, and the heat-sensitive phenotype of the hts2 mutant is completely rescued by exogenous serotonin supplementation. Compared to the wild type, the hts2 mutant exhibits reduced antioxidant capacity, leading to excessive reactive oxygen species (ROS) accumulation and severe oxidative damage, ultimately reducing heat stress tolerance. Furthermore, disruption of HTS2 significantly affects the rice heat shock response, with the heat-induced expression of HsfA2s and their downstream target genes, such as HSP18.0 (heat shock protein 18.0) and OsAPX2 (ascorbate peroxidase 2), markedly depressed in hts2 mutant. Our results suggest a pivotal role of HTS2 in modulating serotonin metabolism and maintaining ROS homeostasis during heat stress, offering new perspectives on the mechanisms underlying heat tolerance and potential strategies to enhance rice resilience to heat stress. Full article

Cytochromes P450 are a superfamily of heme-containing monooxygenases involved in a variety of oxidative metabolic reactions, primarily catalyzing the insertion of an oxygen atom into a C-H bond. CYP102 represents the first example of a bacterial P450 that can be classified as a type II (eukaryotic-like) P450 and functions as a catalytically self-sufficient enzyme. These unique features have made CYP102 an attractive system for studying P450 structure and function. However, an overall picture of the specific amino acid residues that are crucial to the functioning of CYP102 and the effect of mutations on the P450 structure and catalysis is yet to be reported. Such an approach will aid protein engineering approaches used to improve this enzyme. To address this research knowledge gap, we have investigated 105 CYP102 crystal structures in this study. We demonstrate that the CYP102 active site is highly dynamic and flexible. Amino acid residues that play critical roles in substrate binding, orientation, and anchoring were identified. Mutational studies highlighted the roles of amino acids and provided possible bioengineering improvement strategies for CYP102. Decoy molecules are a promising agent for deceiving CYP102 and permitting non-native substrates into the active site. Ru(II)-diimine photosensitizers and zinc/cobalt (III) sepulchrate (Co(III)Sep) could be used as alternative electron sources. The present study serves as a reference for understanding the structure–functional analysis of CYP102 family members precisely and of P450 enzymes in general. Significantly, this work contributes to the effort to develop an improved CYP102 enzyme, thereby advancing the field of P450 research and potentially leading to new industrial applications. Full article

Background: β-cypermethrin (β-CYP) exhibits high toxicity to aquatic organisms and poses significant risks to aquatic ecosystems. Tetrahymena thermophila, a protozoa widely distributed in aquatic environments, can tolerate high concentrations of β-cypermethrin. However, the comprehensive detoxification mechanisms remain poorly understood in Tetrahymena. Methods: Untargeted metabolomics was used to explore the detoxification mechanisms of T. thermophila under β-CYP stress. Results: Trehalose, maltose, glycerol, and D-myo-inositol were upregulated under β-CYP exposure in Tetrahymena. Furthermore, the expression level of CYP5011A1 was upregulated under β-CYP treatment. CYP5011A1 knockout mutants resulted in a decreasing proliferation rate of T. thermophila under β-CYP stress. The valine–leucine and isoleucine biosynthesis and glycine–serine and threonine metabolism were significantly affected, with significantly changed amino acids including serine, isoleucine, and valine. Conclusions: These findings confirmed that T. thermophila develops β-CYP tolerance by carbohydrate metabolism reprogramming and Cyp5011A1 improves cellular adaptations by influencing amino acid metabolisms. Understanding these mechanisms can inform practices aimed at reducing the adverse effects of agricultural chemicals on microbial and environmental health. Full article

CYP199A4 is a cytochrome P450 and can catalyze the hydroxylation of 4-propionylbenzoic acid (4-pIBA) to generate α-hydroxyketone with high stereoselectivity. The F182L mutant of CYP199A4 (F182L-CYP199A4) has been shown to support the cleavage of the C–C bond between the carbonyl and hydroxyl groups of α-hydroxyketone, whereas wild-type CYP199A4 cannot. To uncover how the Phe182 regulates substrate reactivity, we conducted classical molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) MD simulations on these systems. The results predicted that the formation of α-hydroxyketone preferentially led to the (S)-enantiomer. Moreover, the findings revealed that the F182L-CYP199A4 facilitated the formation of a hydrogen bond between the α-hydroxyketone and the reactive peroxoanion (POA) species. This interaction stabilized the α-hydroxyketone near POA and promoted the subsequent C–C bond cleavage. The mechanism of α-hydroxyketone formation and the subsequent C–C bond cleavage were elucidated by employing the hybrid density functional theory (DFT). The α-hydroxyketone formation mechanism involved C–H hydroxylation of 4-pIBA with a rate-limiting energy barrier of 17.1 kcal/mol. The C–C bond cleavage of α-hydroxyketone catalyzed by F182L-CYP199A4 occurred via a radical attack mechanism. Full article

The trade-off between growth and defense is common in plants. We previously demonstrated that BnaA03.WRKY28 weakened resistance strength but promoted shoot branching in Brassica napus (rapeseed). However, the molecular mechanism by which WRKY28 promotes branching formation is still obscure. In this study, we found that BnaA01.BRC1, BnaC01.BRC1, and BnaC03.BRC1 are mainly expressed in the leaf axils and contained W-box cis-acting elements in the promoter regions. BnaA03.WRKY28 directly bound to the promoter regions of these three copies and inhibited their expression. The brc1 mutants, the BnaA01.BRC1, BnaC01.BRC1 BnaA03.BRC1 and BnaC03.BRC1 were simultaneously knocked out, mediated by CRISPR/Cas9, and exhibited excessive branching. The expression level of the ABA biosynthesis encoding gene NCED3 was significantly reduced in the mutant compared to that in the WT. Instead, the expression level of the ABA catabolism encoding gene CYP707A3 was significantly higher than that in WT. These results suggest that the excessive branching of the brc1 mutant may be caused by the release of ABA-mediated bud dormancy. This study provides direct evidence for the potential mechanism of the WRKY28-BRC1 transcription factor module contributing to shoot branching in rapeseed. Full article

Aspergillus fumigatus is a common fungus which has gained attention due to its resistance to azole compounds, substances used in both medical and agricultural settings. One of the genetic alterations responsible for this resistance is the mutation TR₃₄/L98H in the cyp51A gene. The aim of this study was to understand the impact of azoles and non-azoles on Aspergillus fumigatus. By examining clinical samples, soil samples, and compost material, this research aims to provide insights into the susceptibility of these strains to antifungal substances. To deepen our understanding of the factors potentially involved in antifungal resistance, we combined in vitro studies of sixteen compounds against Aspergillus fumigatus with results from the sequencing of the cyp51 gene. We observed that compounds generally displayed a similar pattern activity against wild-type Aspergillus fumigatus. Non-azoles, except Pyrisoxazole and Amisulbrom, did not show any activity against Aspergillus fumigatus, while azole compounds displayed differential activity against the fungus, except for Tetraconazole. For the mutant strains, a generally similar activity was observed in both clinical and environmental samples, likely due to the same mutation in all the isolates. The implications of these findings may be relevant for better understanding the relationship between Aspergillus fumigatus and its ability to develop resistance to antifungal substances. Full article

Onychostoma macrolepis is not only a protected Cyprinid species in the wild but also an emerging commercial aquaculture fish in China. The objective of this research was to genetically modify the genes associated with commercial traits by CRISPR/Cas9 for the protection and utilization of the germplasm resources of O. macrolepis. To that end, one-cell stage embryos were obtained via hormone-induced ovulation and artificial insemination in O. macrolepis. Eight genes related to body color, growth, intermuscular bone, and sex differentiation were mutated in O. macrolepis using the CRISPR/Cas9 system by microinjection of gRNA/Cas9 mRNA. The optimal dose of gRNA/Cas9 mRNA was determined by injection of different concentrations of tyr (tyrosinase)-gRNA/Cas9 and examination of the mutation rate and hatching rate of embryos. Indels were detected by T7 endonuclease I digestion and Sanger sequencing. F0 mutants with high mutation rates were selected for phenotype analyses. Disruption of body color gene tyr, mpv17 (mitochondrial inner membrane protein MPV17), and csf1ra (colony-stimulating factor 1 receptor, a) resulted in obvious phenotype with decreased or even absence of melanophores, iridophores, and xanthophores, respectively. Mutation of mstnb (myostatin b) led to improved growth performance. Mutation of mc4r (melanocortin 4 receptor) led to no obvious phenotype. Mutation of runx2b (RUNX family transcription factor 2b) and bmp6 (bone morphogenetic protein 6) resulted in decreased or absence of intermuscular bones, as revealed by alizarin red S staining. Mutation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a) resulted in ovarian degeneration as revealed by gonadal histological examination. Therefore, this study successfully obtained mutants with obvious phenotypes of genes associated with body color, growth, intermuscular bone, and sex differentiation by CRISPR/Cas9 in O. macrolepis. Full article

Acute alcoholic liver injury (AALI) remains a significant global health concern, primarily driven by oxidative stress. This study investigated the protective mechanisms of Weizmannia coagulans BC99 against alcohol-induced oxidative stress using a dual model in rats and Caenorhabditis elegans. In rats, excessive alcohol was predominantly metabolized via the CYP2E1 pathway, leading to severe oxidative stress. However, intervention with BC99 suppressed CYP2E1 expression and enhanced antioxidant enzyme activities through the Nrf2/SKN-1 pathway, thereby alleviating oxidative stress. Additionally, BC99 treatment elevated glutamate and aspartate levels while reducing glycerate and glucose, which collectively increased glutathione levels and mitigated oxidative stress triggered by glucose metabolism disorders. In C. elegans, BC99 reduced excessive ROS by upregulating Nrf2/skn-1, daf-16, and their downstream antioxidant genes, consequently alleviating the biotoxicity associated with alcohol-induced oxidative damage. The protective effects of BC99 were markedly diminished in the skn-1 mutant (GR2245) and daf-16 mutant (CF1038), further confirming the pivotal roles of SKN-1 and DAF-16 pathways in BC99-mediated antioxidant protection. Taken together, these findings reveal that BC99 mitigates alcohol-induced oxidative stress by activating the Nrf2/SKN-1 pathway and regulating liver metabolites to eliminate excess ROS, thereby providing a theoretical basis for the application of probiotics in preventing acute alcoholic liver injury. Full article

3,4-Dimethylaniline (3,4-DMA) is present in cigarette smoke and widely used as an intermediate in dyes, drugs, and pesticides. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human CYP1A2 and N-acetyltransferase 1 (NAT1) alleles: NAT1*4 (reference allele) or NAT1*14B (the most common variant allele) were utilized to assess 3,4-DMA N-acetylation and hypoxanthine phosphoribosyl transferase (HPRT) mutations, double-strand DNA breaks and reactive oxygen species (ROS). CHO cells expressing NAT1*4 exhibited significantly (p < 0.001) higher 3,4-DMA N-acetylation rates than CHO cells expressing NAT1*14B both in vitro and in situ. In CHO cells expressing CYP1A2 and NAT1, 3,4-DMA caused concentration-dependent increases in reactive oxygen species (ROS), double-stranded DNA damage, and HPRT mutations. CHO cells expressing NAT1*4 and NAT1*14B exhibited concentration-dependent increases in ROS following treatment with 3,4-DMA (linear trend p < 0.001 and p < 0.0001 for NAT1*4 and NAT1*14B, respectively) that were lower than in CHO cells expressing CYP1A2 alone. DNA damage and oxidative stress induced by 3,4-DMA did not differ significantly (p >0.05) between CHO cells expressing NAT1*4 and NAT1*14B. CHO cells expressing NAT1*14B showed higher HPRT mutants (p < 0.05) than CHO cells expressing NAT1*4. These findings confirm 3,4-DMA genotoxicity consistent with potential carcinogenicity. Full article

Alternaria brassicicola is a seed-borne pathogen that causes black spot disease in Brassica crops, yet the seed defense mechanisms against this fungus remain poorly understood. Building upon recent reports that highlighted the involvement of indole pathways in seeds infected by Alternaria, this study provides transcriptomic resources to further elucidate the role of these metabolic pathways during the interaction between seeds and fungal pathogens. Using RNA sequencing, we examined the gene expression of glucosinolate-deficient mutant lines (cyp79B2/cyp79B3 and qko) and a camalexin-deficient line (pad3), generating a dataset from 14 samples. These samples were inoculated with Alternaria or water, and collected at 3, 6, and 10 days after sowing to extract total RNA. Sequencing was performed using DNBseq™ technology, followed by bioinformatics analyses with tools such as FastQC (version 0.11.9), multiQC (version 1.13), Venny (version 2.0), Salmon software (version 0.14.1), and R packages DESeq2 (version 1.36.0), ClusterProfiler (version 4.12.6) and ggplot2 (version 3.4.0). By providing this valuable dataset, we aim to contribute to a deeper understanding of seed defense mechanisms against Alternaria, leveraging RNA-seq for various analyses, including differential gene expression and co-expression correlation. This work serves as a foundation for a more comprehensive grasp of the interactions during seed infection and highlights potential targets for enhancing crop protection and management. Full article

Background: Bouquet is a crucial characteristic indicative of wine quality that develops during the aging stage. The cytochrome P450 VvCYP76F14 multi-functionally catalyzes linalool into (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, which are direct precursors for wine bouquet. Wine bouquet was closely related to VvCYP76F14 activities. Method: The VvCYP76F14 genes were cloned from five wine grape varieties using a homologous cloning method. The variation in residues of VvCYP76F14s were assessed by multiple alignment of amino acid sequences. Functional studies were implemented by in vitro enzyme activity and transient over-expression systems. Results: D299T variation was observed in VvCYP76F14s of ‘Yantai 2-2-08’, ‘Yantai 2-2-19’, and ‘Yantai 2-3-37’ offspring lines, which was correlated with the decreased content of wine bouquet precursors of (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, respectively. Notably, the key amino acid residue D299 was located at the phase 0 intron positions of VvCYP76F14 genes isolated from distinct wine grape varieties or offspring lines, respectively. Notably, VvCYP76F14s of the ‘Yantai2-2-08’, ‘Yantai 2-2-19’, and ‘Yantai 2-3-37’ mutant lines exhibited lower in vitro enzymatic activities than those of ‘L35’ and ‘Merlot’. In addition, the transient expression of VvCYP76F14 cloned from ‘L35’ and ‘Merlot’ restored the levels of wine bouquet precursors in berries of three D299T mutant lines, respectively, whereas VvCYP76F14 cloned from D299T mutant lines failed. Conclusions: D299T variation was observed in three offspring lines and D299T mutation in VvCYP76F14 led to the decrease in wine bouquet precursor contents. VvCYP76F14 was implicated in the regulation of wine bouquet precursors in wine grapes. Full article

Background/Objectives: The increase in fungal infections, both systemic and invasive, is a major source of morbidity and mortality, particularly among immunocompromised people such as cancer patients and organ transplant recipients. Because of their strong therapeutic activity and excellent safety profiles, azole antifungals are currently the most extensively used systemic antifungal drugs. Antibacterial properties of various topical antifungals, such as oxiconazole, which features oxime ether functionality, were discovered, indicating an exciting prospect in antimicrobial chemotherapy. Methods: In this study, eleven new oxime ether derivatives with the azole scaffold (5a–k) were synthesized and tested for their antimicrobial effects using the microdilution method to obtain broad-spectrum hits. Results: Although the title compounds showed limited efficacy against Candida species, they proved highly effective against dermatophytes. Compounds 5c and 5h were the most potent derivatives against Trichophyton mentagrophytes and Arthroderma quadrifidum, with minimum inhibitory concentration (MIC) values lower than those of the reference drug, griseofulvin. The MIC of 5c and 5h were 0.491 μg/mL and 0.619 μg/mL against T. mentagrophytes (MIC of griseofulvin: 2.52 μg/mL). The compounds were also tested against Gram-positive and Gram-negative bacteria. Briefly, 5c was the most active against Escherichia coli and Bacillus subtilis, with MIC values much better than that of ciprofloxacin (MIC of 5c = 1.56 μg/mL and 1.23 μg/mL, MIC of ciprofloxacin = 31.49 and 125.99 μg/mL, respectively). Molecular docking suggested a good fit in the active site of fungal lanosterol 14α-demethylase (CYP51) and bacterial FtsZ (Filamenting temperature-sensitive mutant Z) protein. Conclusions: As a result, the title compounds emerged as promising entities with broad antifungal and antibacterial effects, highlighting the utility of oxime ether function in the azole scaffold. Full article