Effective prediction of biosynthetic pathway genes involved in bioactive polyphyllins in Paris polyphylla

The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

Expression Levels of Detoxification Enzyme Genes from Dendroctonus armandi (Coleoptera: Curculionidae) Fed on a Solid Diet Containing Pine Phloem and Terpenoids

Bark beetles overcome the toxic terpenoids produced by pine trees by both detoxifying and converting them into a pheromone system. Detoxification enzymes such as cytochrome P450s, glutathione S-transferases, and carboxylesterases are involved in the ability of Dendroctonus armandi to adapt to its chemical environment. Ten genes from these three major classes of detoxification enzymes were selected to study how these enzymes help D. armandi to respond to the host defenses. The expression profile of these detoxification enzyme genes was observed in adult beetles after feeding on different types of diet. Significant differences were observed between two types of seminatural diet containing the phloem of pines, and a purely artificial diet containing five monoterpenes ((−)-α-pinene, (−)-β-pinene, (+)-3-carene, (±)-limonene, and turpentine oil) also caused differential transcript levels in the detoxification enzyme genes. The results suggest that monoterpenes enter the beetles through different routes (i.e., respiratory and digestive systems) and cause the expression of different genes in response, which might be involved in pheromone metabolism. In addition, the xenobiotic metabolism in bark beetles should be considered as a system comprising multiple detoxifying enzymes.

The Role of Cytochrome P450s in Insect Toxicology and Resistance

Insect cytochrome P450 monooxygenases (P450s) perform a variety of important physiological functions, but it is their role in the detoxification of xenobiotics, such as natural and synthetic insecticides, that is the topic of this review. Recent advances in insect genomics and postgenomic functional approaches have provided an unprecedented opportunity to understand the evolution of insect P450s and their role in insect toxicology. These approaches have also been harnessed to provide new insights into the genomic alterations that lead to insecticide resistance, the mechanisms by which P450s are regulated, and the functional determinants of P450-mediated insecticide resistance. In parallel, an emerging body of work on the role of P450s in defining the sensitivity of beneficial insects to insecticides has been developed. The knowledge gained from these studies has applications for the management of P450-mediated resistance in insect pests and can be leveraged to safeguard the health of important beneficial insects. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.

Comprehensive Investigation of Stereoselective Food Drug Interaction Potential of Resveratrol on Nine P450 and Six UGT Isoforms in Human Liver Microsomes

The stereoselectivity of the food drug inhibition potential of resveratrol on cytochrome P450s and uridine 5′-diphosphoglucuronosyl transferases was investigated in human liver microsomes. Resveratrol enantiomers showed stereoselective inhibition of CYP2C9, CYP3A, and UGT1A1. The inhibitions of CYP1A2, CYP2B6, and CYP2C19 by resveratrol were stereo-nonselective. The estimated Ki values determined for CYP1A2 were 13.8 and 9.2 μM for trans- and cis-resveratrol, respectively. Trans-resveratrol noncompetitively inhibited CYP3A and UGT1A1 activities with Ki values of 23.8 and 27.4 μM, respectively. Trans-resveratrol inhibited CYP1A2, CYP2C19, CYP2E1, and CYP3A in a time-dependent manner with Ki shift values >2.0, while cis-resveratrol time-dependently inhibited CYP2C19 and CYP2E1. The time-dependent inhibition of trans-resveratrol against CYP3A4, CYP2E1, CYP2C19, and CYP1A2 was elucidated using glutathione as a trapping reagent. This information helped the prediction of food drug interaction potentials between resveratrol and co-administered drugs which are mainly metabolized by UGT1A1, CYP1A2, CYP2C19, CYP2E1, and CYP3A.

The birth-and-death evolution of cytochrome P450 genes in bees

The birth-and-death model of multigene family evolution describes how families can expand by duplication and contract by gene deletion and formation of pseudogenes. The phylogenetic stability of a gene is thought to be related to the degree of functional importance. However, it is unclear how much evolution of a gene in a gene family is driven by adaptive versus neutral processes. The cytochrome P450s are one of the most diverse and well-studied multigene families, involved in both physiological and xenobiotic functions. Bees have a high toxin exposure due to their diet of nectar and pollen, as well as the resin-collecting behavior exhibited by some bees. Here, we describe the P450s of the orchid bee Euglossa dilemma. Orchid bees are a neotropical clade in which males form perfume bouquets used in courtship displays by collecting a diverse set of volatile compounds, resulting in high chemical compound exposure. We conducted phylogenetic and selection analyses across ten bee species encompassing three bee families. We do not find a relationship between the ecology of a bee species and its P450 repertoire. Our analyses reveal that P450 clades can be classified into stable and unstable clades, and that genes involved in xenobiotic metabolism are more likely to belong to unstable clades. Furthermore, we find that unstable clades are under more dynamic evolutionary pressures, with signals of adaptive evolution detected, suggesting that both gene duplication and positive selection driving sequence divergence have played a role in the diversification of bee P450s. Our works highlights the complexity of multigene family evolution which does not always follow generalized predictions.

Evaluation of a high-throughput deorphanization strategy to identify cytochrome p450s important for odor degradation in Drosophila

Members of the cytochrome p450 (CYP) enzyme family are abundantly expressed in insect olfactory tissues, where they are thought to act as Odorant Degrading Enzymes (ODEs). However, their contribution to olfactory signaling in vivo is poorly understood. This is due in part to the challenge of identifying which of the dozens of antennal-expressed CYPs might inactivate a given odorant. Here, we tested a high-throughput deorphanization strategy in Drosophila to identify CYPs that are transcriptionally induced by exposure to a plant volatile. We discovered three CYPs selectively upregulated by the odorant using transcriptional profiling. Although these CYPs are shown to be broadly expressed in the antenna in non-neuronal cells, electrophysiological recordings from CYP mutants did not reveal any changes in olfactory neuron responses to the odorant. Neurons were desensitized by pre-exposing flies to the odorant, but this effect was similar in CYP mutants. Together, our data suggest that this transcriptomic approach may not be useful for identifying CYPs that contribute to olfactory signaling. We go on to show that some CYPs have highly restricted expression patterns in the antenna, and suggest that such CYPs may be useful candidates for further studies on olfactory CYP function.