Cytochrome P450 Monooxygenase Genes Research Articles

OsCYP714D1 improves plant growth and salt tolerance through regulating gibberellin and ion homeostasis in transgenic poplar

Cytochrome P450 monooxygenases (CYP450s) play crucial roles in the regulation of plant growth and response to abiotic stress. However, their functions in woody trees are still largely unknown. Previously, we reported that expression of the rice cytochrome P450 monooxygenase gene OsCYP714D1 increased gibberellic acid (GA) accumulation and shoot growth in transgenic poplar. In this work, we demonstrate that expression of OsCYP714D1 improved the salt tolerance of transgenic poplar plants. Compared to wild type, plant height and K+ content were significantly higher, whereas plant growth inhibition and Na+ content were significantly lower, in transgenic plants grown under high salt stress condition. Transcriptomic analyses revealed that OsCYP714D1 expression up-regulated the expressions of GA biosynthesis, signaling and stress responsive genes in transgenic plants under both normal and high salt stress conditions. Further gene ontology (GO) analyses indicated that genes involved in plant hormone and ion metabolic activities were significantly enriched in transgenic plants. Our findings imply that OsCYP714D1 participated in the regulation of both shoot growth and salt resistance through regulating gibberellin and ion homeostasis in transgenic poplar, and it can be used as a candidate gene for the engineering of new tree varieties with improved biomass production and salt stress resistance.

Functional validation of key cytochrome P450 monooxygenase and UDP-glycosyltransferase genes conferring cyantraniliprole resistance in Aphis gossypii Glover

Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are major detoxifying enzymes that metabolize plant toxins and insecticides. In the present study, the synergists of piperonyl butoxide, sulfinpyrazone and 5-nitrouracil significantly increased cyantraniliprole and α-cypermethrin toxicity against the resistant strain. The transcripts of UGT341A4, UGT344B4, UGT344D6, UGT344J2 and UGT344M2 increased significantly in the CyR strain compared with the susceptible strain. Among these upregulated genes (including P450s), CYP6CY7 and UGT344B4 were highly expressed in the midgut. Transgenic expression of the P450 and UGT genes in broad body tissues in Drosophila melanogaster indicated that the expression of CYP380C6, CYP4CJ1, UGT341A4, UGT344B4 and UGT344M2 is sufficient to confer cyantraniliprole resistance, and CYP380C6, CYP6CY7, CYP6CY21, UGT341A4 and UGT344M2 are related to α-cypermethrin cross-resistance. The midgut-specific overexpression of CYP380C6, CYP6CY7, CYP6CY21, CYP4CJ1, UGT341A4, UGT344B4 and UGT344M2 significantly increased insensitivity to cyantraniliprole, and CYP380C6, CYP6CY7, CYP6CY21, UGT344B4 and UGT344M2 confer α-cypermethrin cross-resistance. The expression of CYP380C6, CYP4CJ1, UGT341A4 and UGT344M2 in broad tissues or in midgut has similar effects on insensitivity to insecticides; however, CYP6CY7, CYP6CY21 and UGT344B4 are more effective in the midgut. This result indicates that broad body tissues and midgut tissue are involved in insecticide resistance mediated by the candidate P450s and UGTs examined.

Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Does RNAi-Based Technology Fit within EU Sustainability Goals?

European Union (EU) and global sustainability policies emphasize the need to replace contentious pesticides with safe, efficient, and cost-effective alternatives to ensure sustainable food production. However, R&D for alternatives to contentious pesticides are lagging behind and need to be broadened. Here, we discuss how RNAi-based technology can contribute to pesticide risk reduction.

Chromosome-level genome of the peach fruit moth Carposina sasakii (Lepidoptera: Carposinidae) provides a resource for evolutionary studies on moths.

The peach fruit moth (PFM), Carposina sasakii Matsumura, is a major phytophagous orchard pest widely distributed across Northeast Asia. Here, we report the chromosome-level genome for the PFM, representing the first genome for the family Carposinidae, from the lepidopteran superfamily Copromorphoidea. The genome was assembled into 404.83Mb sequences using PacBio long-read and Illumina short-read sequences, including 275 contigs, with a contig N50 length of 2.62Mb. All contigs were assembled into 31 linkage groups assisted by the Hi-C technique, including 30 autosomes and a Z chromosome. BUSCO analysis showed that 98.3% of genes were complete and 0.4% of genes were fragmented, while 1.3% of genes were missing in the assembled genome. In total, 21,697 protein-coding genes were predicted, of which 84.80% were functionally annotated. Because of the importance of diapause triggered by photoperiod in PFM, five circadian genes in the PFM as well as in the other related species were annotated, and potential genes related to diapause and photoperiodic reaction were also identified from transcriptome sequencing. In addition, manual annotation of detoxification gene families was undertaken and showed a higher number of glutathione S-transferase (GST) gene in PFM than in most other lepidopterans, in contrast to a lower number of uridine diphosphate (UDP)-glycosyltransferase (UGT) gene, carboxyl/cholinesterases (CCE) gene and cytochrome P450 monooxygenase (P450) gene, suggesting different detoxication pathways in this moth. The high-quality genome provides a resource for comparative evolutionary studies of this moth and its relatives within the context of radiations across Lepidoptera.

Genome mining and functional analysis of cytochrome P450 genes involved in insecticide resistance in Leucinodes orbonalis (Lepidoptera: Crambidae).

Genome-wide analysis of cytochrome P450 monooxygenase (CYP) genes from the advanced genome project of the Leucinodes orbonalis and the expression analysis provided significant information about the metabolism-mediated insecticide resistance. A total of 72 putative CYP genes were identified from the genome and transcriptome of L. orbonalis. The genes were classified under 30 families and 46 subfamilies based on the standard nomenclature. In the present study, a novel CYP gene, CYP324F1, was identified and it has not been reported from any other living system so far. Biochemical assays showed enhanced titers (5.81-18.5-fold) of O-demethylase of CYP in five field-collected populations. We selected 34 homologous CYP gene sequences, seemed to be involved in insecticide resistance for primer design and quantitative real-time PCR studies. Among the many overexpressed genes (>10 fold), the expression levels of CYP324F1 and CYP306A1 were prominent across all the field populations as compared with the susceptible iso-female line. Oral delivery of ds-CYP324F1 and ds-CYP306A1 directed against CYP324F1 and CYP306A1 to the larvae of one of the insecticide resistance populations caused reduced expression of these two transcripts in a dose-dependent manner (53.4%-85.0%). It appears that the increased titer of O-demethylase is the result of increased transcription level of CYP genes in resistant populations. The data provide insight for identifying the novel resistance management strategies against L. orbonalis.

Cytochrome P450s CYP380C6 and CYP380C9 in green peach aphid facilitate its adaptation to indole glucosinolate-mediated plant defense.

Overexpressing CIRCADIAN CLOCK ASSOCIATED1 in Arabidopsis thaliana (CCA1-ox) increases indole glucosinolate production and resistance to green peach aphid (Myzus persicae). Little is known of how aphids respond to this group of plant defense compounds or of the underlying molecular mechanism. Aphids reared on CCA1-ox for over 40 generations (namely the CCA population) became less susceptible to CCA1-ox than aphids maintained on the wild-type Col-0 (namely the COL population). This elevated tolerance was transgenerational as it remained for at least eight generations after the CCA population was transferred to Col-0. Intriguingly, transcriptome analysis indicated that all differential cytochrome P450 monooxygenase genes (MpCYPs), primarily MpCYP4s, MpCYP380s and MpCYP6s, were more highly expressed in the CCA population. Application of a P450 inhibitor to the CCA population resulted in decreased aphid reproduction on CCA1-ox, which was not observed if aphids were reared on Col-0. When indole glucosinolate biosynthesis in CCA1-ox was blocked using virus-induced gene silencing, the effect of the P450 inhibitor on the CCA population was attenuated, affirming the essential role played by MpCYPs in counteracting the defense mechanism in CCA1-ox that is low or absent in Col-0. Furthermore, we used host-induced gene silencing to identify MpCYP380C6 and MpCYP380C9 that specifically facilitated the CCA population to cope with CCA1-mediated plant defense. Expression profiles revealed their possible contribution to the transgenerational tolerance observed in aphids. MpCYP380C6 and MpCYP380C9 in aphids play a crucial role in mitigating indole glucosinolate-mediated plant defense, and this effect is transgenerational.

Genetic variations in the drug metabolizing enzyme, CYP2E1, among various ethnic populations of Pakistan.

Genetic polymorphism in cytochrome P450 (CYP) monooxygenase genes is an important source of interindividual variability of drug response. CYP enzyme activities may change as a result of such polymorphisms which then, may affect drug metabolism. This would result in a change in the severity and frequency of adverse effects in addition to the non-responder phenomenon. CYP2E1, a member of CYP superfamily, affects the metabolism of several clinically important drugs such as halothane, paracetamol, etc. Genetic variation in CYP2E1 is known to cause significant inter-individual differences in drug response and adverse effects. The degree of genetic variation is found to be different in different populations around the world. The frequencies of two important polymorphisms in the CYP2E1*7C, NC_000010.10:g.135340548A>G (rs2070672) and CYP2E1, NC_000010.10:g.135339244G>C (rs3813865), are not known in the Pakistani population. In the present investigation, 636 healthy human volunteers were screened for these two single nucleotide polymorphism. Our results indicate that about 18% (rs2070672) and 28% (rs3813865) of the Pakistani population has a genotype containing at least one low activity allele. A significant interethnic variation in the frequencies of both the polymorphisms was observed. These results suggest that pharmacogenetics screening for low activity genotypes would be a helpful tool for clinicians when they prescribe medications metabolized by CYP2E1, as a significant fraction of the Pakistani population is expected to have a variable response to these drugs.

Improved 11α-hydroxycanrenone production by modification of cytochrome P450 monooxygenase gene in Aspergillus ochraceus.

Eplerenone is a drug that protects the cardiovascular system. 11α-Hydroxycanrenone is a key intermediate in eplerenone synthesis. We found that although the cytochrome P450 (CYP) enzyme system in Aspergillus ochraceus strain MF018 could catalyse the conversion of canrenone to 11α-hydroxycanrenone, its biocatalytic efficiency is low. To improve the efficiency of 11α-hydroxycanrenone production, the CYP monooxygenase-coding gene of MF018 was predicted and cloned based on whole-genome sequencing results. A recombinant A. ochraceus strain MF010 with the high expression of CYP monooxygenase was then obtained through homologous recombination. The biocatalytic rate of this recombinant strain reached 93 % at 60 h without the addition of organic solvents or surfactants and was 17-18 % higher than that of the MF018 strain. Moreover, the biocatalytic time of the MF010 strain was reduced by more than 30 h compared with that of the MF018 strain. These results show that the recombinant A. ochraceus strain MF010 can overcome the limitation of substrate biocatalytic efficiency and thus holds a high poten tial for application in the industrial production of eplerenone.

Overexpression of a novel cytochrome P450 monooxygenase gene, CYP704B1, from Panax ginseng increase biomass of reproductive tissues in transgenic Arabidopsis.

Cytochrome P450 monooxygenase 704B (CYP704B), a member of the CYP86 clan, was found to be needed in Arabidopsis and rice to biosynthesize precursors of sporopollenin through oxidizing fatty acids. In the present study, we cloned and characterized a CYP704B gene in Panax ginseng, named PgCYP704B1. It shared high sequence identity (98-99%) with CYP704 of Arabidopsis, Theobroma cacao, and Morus notabilis. The phylogenetic comparison of ginseng and higher plants between the members of CYP86 clan revealed that ginseng CYP704 was categorized as a group of CYP704B with dicot plants. The expression of PgCYP704B1 is low in the stem, leaf, and fruit, and high in flower buds, particularly detected in the young gametic cell and tapetum layer of the developing anther. Arabidopsis plants overexpressing PgCYP704B1 improved plant biomass such as plant height, siliques and seed number and size. A cytological observation by transverse and longitudinal semi-thin sections of the siliques cuticles revealed that the cell length increased. Furthermore a chemical analysis showed that PgCYP704B1ox lines increased their cutin monomers contents in the siliques. Our results suggest that PgCYP704B1 has a conserved role during male reproduction for fatty acid biosynthesis and its overexpression increases cutin monomers in siliques that eventually could be used for seed production.

Geographical distribution and molecular insights into abamectin and milbemectin cross-resistance in European field populations of Tetranychus urticae.

Milbemectin and abamectin are frequently used to control the spider mite Tetranychus urticae. The development of abamectin resistance in this major pest has become an increasing problem worldwide, potentially compromising the use of milbemectin. In this study, a large collection of European field populations was screened for milbemectin and abamectin resistance, and both target-site and metabolic (cross-)resistance mechanisms were investigated. High to very high levels of abamectin resistance were found in one third of all populations, while milbemectin resistance levels were low for most populations. The occurrence of well-known target-site resistance mutations in glutamate-gated chloride channels (G314D in GluCl1 and G326E in GluCl3) was documented in the most resistant populations. However, a new mutation, I321T in GluCl3, was also uncovered in three resistant populations, while a V327G and L329F mutation was found in GluCl3 of one resistant population. A differential gene-expression analysis revealed the overexpression of detoxification genes, more specifically cytochrome P450 monooxygenase (P450) and UDP-glycosyltransferase (UGT) genes. Multiple UGTs were functionally expressed, and their capability to glycosylate abamectin and milbemectin, was tested and confirmed. We found a clear correlation between abamectin and milbemectin resistance in European T. urticae populations, but as milbemectin resistance levels were low, the observed cross-resistance is probably not of operational importance. The presence of target-site resistance mutations in GluCl genes was confirmed in most but not all resistant populations. Gene-expression analysis and functional characterization of P450s and UGTs suggests that also metabolic abamectin resistance mechanisms are common in European T. urticae populations. © 2020 Society of Chemical Industry.

CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum.

The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

Multiple down-regulated cytochrome P450 monooxygenase genes contributed to synergistic interaction between chlorpyrifos and imidacloprid against Nilaparvata lugens

Abstract Insecticide mixtures are an effective strategy in pest resistance management. The synergistic chlorpyrifos and imidacloprid mixture could significantly increase toxicity against rice pest, Nilaparvata lugens, despite their high levels of resistance. However, synergism mechanisms to explain this phenomenon remain unknown. Chlorpyrifos and imidacloprid at a 1:0.5 ratio showed significant synergism on N. lugens with a combination index value of 0.18 after topical exposure. We constructed a genetic database of the genes expressed in individual and synergistic chlorpyrifos and imidacloprid treatments of N. lugens using Illumina Hiseq™ X Ten, and 17 co-downregulated genes putatively involved in synergism were detected by comparative transcriptome analyses. Expression patterns of the 17 candidate synergistic genes matched with transcriptome sequencing data by quantitative real-time PCR analyses. Feeding of dsRNAs further reduced the expression levels of 10 of these candidate synergistic genes (from 1.68 to 4.13-fold). Nymphs fed with only dsRNAs of CYP4DE1, CYP6AY1v2, CYP353D1, and CYP439A1 experienced more high mortality rates (81.45–90.34%) to improve synergism between chlorpyrifos and imidacloprid. Multiple reductive expressed P450 genes were potentially associated with synergism of a mixture of chlorpyrifos and imidacloprid, as confirmed by comparative transcriptome analyses and RNAi assays. Our findings suggested that synergistic interactions between chlorpyrifos and imidacloprid might be controlled by P450s.

Candidate detoxification-related genes in brown planthopper, Nilaparvata lugens, in response to β-asarone based on transcriptomic analysis

Nilaparvata lugens(Stål) is a serious pest of rice and has evolved different levels of resistance against most chemical pesticides. β-asarone is the main bioactive insecticidal compound of Acorus calamus L. that shows strong insecticidal activity against pests. In this study, we conducted a bioassay experiment to determine the contact toxicity of β-asarone to N. lugens nymphs. The LD30 sublethal dose was 0.106 μg per nymph, with 95% confidence limits of 0.070–0.140 μg. We applied the LD30 concentration of β-asarone to nymphs for 24 h or 72 h and then performed a transcriptome sequence analysis by referencing the N. lugens genome to characterize the variation. The transcriptomic analysis showed that several GO terms and KEGG pathways presented significant changes. Individually, 126 differentially expressed genes (DEGs), including 72 upregulated and 54 downregulated genes, were identified at 24 h, and 1771 DEGs, including 882 upregulated and 889 downregulated genes, were identified at 72 h. From the DEGs, we identified a total of 40 detoxification-related genes, including eighteen Cytochrome P450 monooxygenase genes (P450s), three Glutathione S-transferase genes, one Carboxylesterase gene, twelve UDP-glucosyltransferases and six ATP-binding cassette genes. We selected the eighteen P450s for subsequent verification by quantitative PCR. These findings indicated that β-asarone presented strong contact toxicity to N. lugens nymphs and induced obvious variation of detoxification-related genes that may be involved in the response to β-asarone.

TaCYP81D5, one member in a wheat cytochrome P450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging.

SummaryAs one of the largest gene families in plants, the cytochrome P450 monooxygenase genes (CYPs) are involved in diverse biological processes including biotic and abiotic stress response. Moreover, P450 genes are prone to expanding due to gene tandem duplication during evolution, resulting in generations of novel alleles with the neo‐function or enhanced function. Here, the bread wheat (Triticum aestivum) gene TaCYP81D5 was found to lie within a cluster of five tandemly arranged CYP81D genes, although only a single such gene (BdCYP81D1) was present in the equivalent genomic region of the wheat relative Brachypodium distachyon. The imposition of salinity stress could up‐regulate TaCYP81D5, but the effect was abolished in plants treated with an inhibitor of reactive oxygen species synthesis. In SR3, a wheat cultivar with an elevated ROS content, the higher expression and the rapider response to salinity of TaCYP81D5 were related to the chromatin modification. Constitutively expressing TaCYP81D5 enhanced the salinity tolerance both at seedling and reproductive stages of wheat via accelerating ROS scavenging. Moreover, an important component of ROS signal transduction, Zat12, was proven crucial in this process. Though knockout of solely TaCYP81D5 showed no effect on salinity tolerance, knockdown of BdCYP81D1 or all TaCYP81D members in the cluster caused the sensitivity to salt stress. Our results provide the direct evidence that TaCYP81D5 confers salinity tolerance in bread wheat and this gene is prospective for crop improvement.

A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma.

Trichothecenes are sesquiterpene toxins produced by diverse fungi, including some species of Trichoderma that are potential plant disease biocontrol agents. Trichoderma arundinaceum produces the trichothecene harzianum A (HA), which consists of the core trichothecene structure (12,13-epoxytrichothec-9-ene, EPT) with a linear polyketide-derived substituent (octa-2,4,6-trienedioyl) esterified to an oxygen at carbon atom 4. The genes required for biosynthesis of EPT and the eight-carbon polyketide precursor of the octa-2,4,6-trienedioyl substituent, as well as for esterification of the substituent to EPT have been described. However, genes required for conversion of the polyketide (octa-2,4,6-trienoic acid) to octa-2,4,6-trienedioyl-CoA, the immediate precursor of the substituent, have not been described. Here, we identified 91 cytochrome P450 monooxygenase genes in the genome sequence of T. arundinaceum, and provided evidence from gene deletion, complementation, cross-culture feeding, and chemical analyses that one of them (tri23) is required for conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioyl-CoA. The gene was detected in other HA-producing Trichoderma species, but not in species of other fungal genera that produce trichothecenes with an octa-2,4,6-trienoic acid-derived substituent. These findings indicate that tri23 is a trichothecene biosynthetic gene unique to Trichoderma species, which in turn suggests that modification of octa-2,4,6-trienoic acid during trichothecene biosynthesis has evolved independently in some fungi.

De novo Analysis of Heortia vitessoides (Lepidoptera: Crambidae) Transcriptome and Identification of Putative Cytochrome P450 Monooxygenase Genes

Abstract We analyzed a transcriptome generated from four developmental stages of Heortia vitessoides Moore (Lepidoptera: Crambidae), a defoliator of the agarwood tree, Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae). A total of 110.53 Mb clean reads were produced, and de novo assembled into 42,946 unigenes of an average length of 1,059 base pairs (bp). Among these unigenes, 22,106 (51.47%) showed significant similarity (E-value <10–5) to known proteins in the National Center for Biotechnology Information (NCBI) nonredundant database. Gene ontology (GO), eukaryotic ortholog groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to classify the functionality of unigenes. Moreover, 46 putative cytochrome P450 (CYP) monooxygenase genes were identified from this dataset. These CYP genes were classified into four clans consisting of 22 families and 34 subfamilies. The expression profiles of genes belonging to CYP4 and CYP6 families and exposed to half the lethal concentrations (LC50) of chlorantraniliprole and beta-cypermethrin were determined by real-time reverse transcription-quantitative PCR (RT-qPCR). The results showed that the transcription levels of eight (CYP4M39, CYP6AB49, CYP6AB53, CYP6AB61, CYP6AE17, CYP6AW1, CYP6BD6, CYP6CV1) and five (CYP6AB10, CYP6AB53, CYP6AE12, CYP6AE17, CYP6BD6) genes significantly increased in the fourth-instar larvae following exposure to the insecticides chlorantraniliprole and beta-cypermethrin, respectively. Therefore, these genes are potential candidates involved in the detoxification of these two insecticides. Further studies utilizing the RNA interference approach are required to enhance our understanding of the functionality of these genes in this forest pest.

Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2

Carbon availability is a major regulatory factor in plant growth and development. Cytokinins, plant hormones that play important roles in various aspects of growth and development, have been implicated in the carbon-dependent regulation of plant growth; however, the details of their involvement remain to be elucidated. Here, we report that sugar-induced cytokinin biosynthesis plays a role in growth enhancement under elevated CO2 in Arabidopsis thaliana. Growing Arabidopsis seedlings under elevated CO2 resulted in an accumulation of cytokinin precursors that preceded growth enhancement. In roots, elevated CO2 induced two genes involved in de novo cytokinin biosynthesis: an adenosine phosphate-isopentenyltransferase gene, AtIPT3, and a cytochrome P450 monooxygenase gene, CYP735A2. The expression of these genes was inhibited by a photosynthesis inhibitor, DCMU, under elevated CO2, and was enhanced by sugar supplements, indicating that photosynthetically generated sugars are responsible for the induction. Consistently, cytokinin precursor accumulation was enhanced by sugar supplements. Cytokinin biosynthetic mutants were impaired in growth enhancement under elevated CO2, demonstrating the involvement of de novo cytokinin biosynthesis for a robust growth response. We propose that plants employ a system to regulate growth in response to elevated CO2 in which photosynthetically generated sugars induce de novo cytokinin biosynthesis for growth regulation.

Transcriptomic analysis of differentially expressed genes in the oriental armyworm Mythimna separata Walker at different temperatures

The oriental armyworm Mythimna separata Walker is a serious migratory and polyphagous pest that damages major crops and some pastures from the family Gramineae. Temperature is a crucial abiotic factor that affects its survival, development and reproduction, but the thermal responses of this moth at the molecular level are largely unknown. In this research, we sequenced the transcriptomes of oriental armyworms that were reared at three temperatures (20 °C, 25 °C and 30 °C) using an Illumina high-throughput RNA-sequencing (RNA-seq) method. We obtained 54.0 Gb of clean reads and 113,396 transcripts. From a total of 46,681 unigenes identified, 22,911 were annotated to the non-redundant (NR) database. We identified 333 downregulated and 1588 upregulated genes in 20 °C versus 25 °C, and 1096 downregulated and 875 upregulated genes at 30 °C versus 25 °C by differential expression of genes (DEGs). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses revealed several functional terms related to carbohydrate metabolism, energy metabolism, and xenobiotics metabolism. DEGs involved in glycolysis, the citrate cycle, oxidative phosphorylation, and the composition of myofilaments were significantly downregulated, while most heat shock protein genes (HSPs) and genes in the ubiquitin-mediated proteasome pathway were upregulated at 30 °C. Many cytochrome P450 monooxygenase genes (CYPs) in clan 3 were upregulated at 20 °C, while two genes involved in ecdysteroid biosynthesis, CYP302A1 and CYP315A1, were upregulated at 30 °C. These data may improve the understanding of the complex molecular mechanisms involved in the thermal responses of M. separata.

Identification of cytochrome P450 monooxygenase genes and their expression in response to high temperature in the alligatorweed flea beetle Agasicles hygrophila (Coleoptera: Chrysomelidae)

Cytochrome P450 monooxygenases (P450s) are a large class of enzymes that play essential roles in metabolic processes such as hormone synthesis and the catabolism of toxins and other chemicals in insects. In the present study, we identified 82 P450 genes using comprehensive RNA sequencing in the flea beetle Agasicles hygrophila, and all of the sequences were validated by cloning and sequencing. Phylogenetic analysis showed that the P450 genes in A. hygrophila fell into the mitochondrial clan, CYP2 clan, CYP3 clan and CYP4 clan and were classified into 20 families and 48 subfamilies. Most A. hygrophila P450 genes had high sequence homology with those from other coleopteran insects. To understand the effects of high temperatures on the metabolic processes of female and male adults, we studied the effects of two temperature regimes (constant temperature of 28 °C for 20 h with a 4-h period of high temperatures of 30 °C and 39 °C) on the expression levels of P450 genes in A. hygrophila using RT-PCR and qRT-PCR. The results showed that there were no differences in expression in 30 P450 genes between the control and high-temperature-treated A. hygrophila adults, while 22 P450 genes showed up-regulated expression and 19 P450 genes were down-regulated in A. hygrophila female adults after high-temperature treatment. For A. hygrophila male adults exposed to high temperatures, we found that 8 P450 genes had higher expression levels and 12 P450 genes had lower expression levels under the same conditions. The P450 genes are candidates that showed significantly different expression levels after high-temperature treatments in A. hygrophila adults, and further studies are needed to determine their possible roles in metabolic processes during the response to elevated temperatures.