Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Quantification of Three Key Enzymes Involved in Artemisinin Biogenesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> by Polyclonal Antisera-Based ELISA

To elucidate the fine-tuned temporal and spatial modulation of artemisinin production in annual wormwood (Artemisia annua), we conducted enzyme-linked immunosorbent assay-based immunoquantification of three key enzymes involved in artemisinin biosynthesis, amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), and cytochrome P450 reductase (CPR), in various tissues and under different growth conditions. The field-grown plants accumulate abundant ADS and CYP71AV1 but a trace amount of CPR in all tested tissues. Furthermore, ADS and CYP71AV1 accumulations in leaves are 16- and eightfold higher than in roots, and ten- and fourfold higher than in stems, respectively, demonstrating a tissue-specific expression pattern. Interestingly, the flowering field plants and cold-acclimated cultural plants produce higher levels of ADS and CYP71AV1 than non-flowering field plants or untreated cultural plants, indicating the environmental and developmental induction on ADS and CYP71AV1 genes and providing possible explanation for the observation that elevation of artemisinin level occurs after flowering.     

Tissue-specific expression of rice CYP72A21 induced by auxins and herbicides

Cytochrome monooxygenase P450s (CYPs) comprise one of the largest enzyme families in plants. Some P450s are involved in xenobiotic metabolism: they confer herbicide tolerance and are induced by chemical treatments. We isolated a novel P450 cDNA, CYP72A21 (accession number, AB237166), from rice (Oryza sativa L. cv. Nipponbare) seedlings treated with a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), chlorotoluron, phenobarbital, salicylic acid, and naphthalic anhydride (each 100 μM). We also isolated the gene’s promoter region. Endogenous CYP72A21 expression in rice seedlings treated with 2,4-D, herbicides esprocarb, or trifluralin was increased in the aerial part of seedlings. An expression plasmid, pI21pg, containing the GUS gene under the control of the CYP72A21 promoter was introduced into rice plants. GUS was expressed constitutively in roots, but this expression was suppressed by 2,4-D treatment. 2,4-D and other auxins induced GUS expression effectively in the stem and leaves. Histological observation revealed that GUS was expressed mainly in the base of the stem. Treatment with the herbicides acetochlor, esprocarb, and propyzamide induced GUS expression in the aerial parts of the seedlings. The CYP72A21 promoter was highly responsive to treatments with various chemicals, and thus might be useful for producing transgenic plants for biomonitoring of environmental chemicals.     

The expression of Spodoptera exigua P450 and UGT genes: tissue specificity and response to insecticides

Cytochrome P450 and UDP-glucosyltransferase (UGT) as phase I and phase II metabolism enzymes, respectively, play vital roles in the breakdown of endobiotics and xenobiotics. Insects can in crease the expression of detoxificatio n enzymes to cope with the stress from xenobiotics including insecticides. However, the molecular mechanisms for insecticide detoxification in Spodoptera exigua remain elusive, and the genes conferring insecticide metabolisms in this species are less well reported. In this study, 68 P450 and 32 UGT genes were identified. Phylogenetic analysis showed gene expansions in CYP3 and CYP4 clans of P450 genes and UGT33 family of this pest. P450 and UGT genes exhibited specific tissue expression patterns. Insecticide treatments in fat body cells of S. exigua revealed that the expression levels of P450 and UGT genes were significantly influenced by challenges of abamectin, lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb. Multiple genes for detoxification were affected in expression levels after insecticide exposures. The results demonstrated that lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb induced similar responses in the expression of P450 and UGT genes in fat body cells;eight P450 genes and four UGT genes were co-up-regulated significantly, and no or only a few CYP/UGT genes were down-regulated significantly by these four insecticides. However, abamectin triggered a distinct response for P450 and UGT gene expression;more P450 and UGT genes were down-regulated by abamectin than by the other four compounds. In con elusion, P450 and UGT genes from S. exigua were identified, and different responses to abamectin suggest a different mechanism for insecticide detoxification.     

Molecular characterization and chromosomal localization of cytochrome P450 genes involved in the biosynthesis of cyclic hydroxamic acids in hexaploid wheat

The cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), are defensive secondary metabolites found in gramineous plants including wheat, maize and rye. cDNAs for five cytochromes P450 (P450s) involved in DIBOA biosynthesis (CYP71C6, CYP71C7v2, CYP71C8v2, CYP71C9v1 and CYP71C9v2) were isolated from seedlings of hexaploid wheat [( Triticum aestivum L. cv. Chinese Spring (2n=6x=42, genomes AABBDD)] by RT-PCR and screening of a cDNA library. CYP71C9v1 and CYP71C9v2 are 97% identical to each other in amino acid and nucleotide sequences. The cloned P450 species showed 76-79% identity at the amino acid level to the corresponding maize P450 species CYP71C1-C4, which are also required for DIBOA biosynthesis. The wheat P450 cDNAs were heterologously expressed in the yeast ( Saccharomyces cerevisiae) strain AH22. Microsome fractions from yeast cells expressing these P450 species catalyzed the same reactions as their maize orthologs. The chromosomes carrying the cyp71C6- C9v1 orthologs were identified by Southern hybridization using aneuploid lines of Chinese Spring wheat. The cyp71C9v1 orthologs were located on the chromosomes of wheat homoeologous group-4. The orthologs of the other P450 genes, cyp71C7v2, cyp71C6 and cyp71C8v2, were located on group-5 chromosomes. The same P450 genes were also present in the three ancestral diploid species of hexaploid wheat, T. monococcum (AA), Aegilops speltoides [BB (approximately SS)] and Ae. squarrosa (DD).     

Chronic Ethanol Exposure Increases Cytochrome P‐450 and Decreases Activated in Blocked Unfolded Protein Response Gene Family Transcripts in Caenorhabditis elegans

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA‐Seq and quantitative real‐time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P‐450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione‐S‐transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes.     

<Emphasis Type="Italic">Helitron</Emphasis> mediated amplification of cytochrome P450 monooxygenase gene in maize

The mass movement of gene sequences by Helitrons has significantly contributed to the lack of gene collinearity reported between different maize inbred lines. However, Helitron captured-genes reported to date represent truncated versions of their progenitor genes. In this report, we provide evidence that maize CYP72A27-Zm gene represents a cytochrome P450 monooxygenase (P450) gene recently captured by a Helitron and transposed into an Opie-2 retroposon. The four exons of the CYP72A27 gene contained within the element contain a putative open reading frame (ORF) for 428 amino acid residues. We provide evidence that Helitron captured CYP72A27-Zm is transcribed. To identify the progenitor gene and the evolutionary time of capture, we searched the plant genome database and discovered other closely related CYP72A27-Zm genes in maize and grasses. Our analysis indicates that CYP72A27-Zm represents an almost complete copy of maize CYP72A26-Zm gene captured by a Helitron about 3.1 million years ago (mya). The Helitron-captured gene then duplicated twice, approximately 1.5-1.6 mya giving rise to CYP72A36-Zm and CYP72A37-Zm. These data provide evidence that Helitrons can capture and mobilize intact genes that are transcribed and potentially encode biologically relevant proteins.     

Muscle force arises by actin filament rotation and torque in the Z-filaments

Cytochrome P450 partial sequences were isolated by PCR using genomic DNA from two hymenopteran insects of agronomical importance, Trichogramma cacoeciae, a parasitoid wasp, and Apis mellifera, the honeybee. Four new P450 genes were identified: one honeybee gene belongs to the CYP4 family and was named CYP4G11; the three other genes were from Trichogramma and belong to the CYP4 family (CYP4G12) and to a novel family, the CYP48 one (CYP48A1 and CYP48A2). The four genes contain a short intron (72-95 bp) at the same position as already described for other insect species. The two genes CYP48A1 and CYP48A2 have a supernumary intron (57-71 bp) upstream the first one. Only the two CYP4 genes were constitutively transcribed, at a high level for CYP4G12 and at a low level for CYP4G11. No expression was observed for CYP48A1 and CYP48A2.     

Biosynthesis of the leucine derived α‐, β‐ and γ‐hydroxynitrile glucosides in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) produces five leucine‐derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non‐cyanogenic HNGs are the β‐HNG epidermin and the γ‐HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP‐glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co‐expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi‐enzyme complex.     

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.     

Genome-Wide Analysis of Mitogen-Activated Protein Kinase Gene Family in Maize

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. As the last component of the MAPK cascade (MAPKKK–MAPKK–MAPK), MAPK plays important roles in linking upstream kinases and downstream substrates. The MAPK proteins belong to a complex gene family in plants, with 20 MAPK genes in the Arabidopsis genome, 17 in the rice genome, and 21 in the poplar genome. Although the maize genome sequencing has been completed, no comprehensive study has been reported thus far for the MAPK gene family in maize. In this study, we identified 19 MAPK genes in maize. These ZmMPK genes belong to four groups (A–D) found in other plants. The phylogeny, chromosomal location, gene structure, and the functional relevancy of ZmMPK genes were analyzed. Moreover, we discuss the evolutionary divergence of MAPK genes in maize. Furthermore, we analyzed the expression profiles of ZmMPKs using the public microarray data and performed expression analyses in maize seedlings and adult plants. The data obtained from our study contribute to a better understanding of the complexity of MAPKs in plants and provide a useful reference for further functional analysis of MAPK genes in maize.     

Microarray-based global differential expression profiling of P450 monooxygenases and regulatory proteins for signal transduction pathways in the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Whole genome sequencing of the model white rot basidiomycete Phanerochaete chrysosporium has revealed the largest P450 contingent known to date in fungi, along with related phase I and phase II metabolic genes and signaling cascade genes. As a part of their functional characterization, genome-wide expression profiling under physiologically distinct conditions, nutrient-limited (ligninolytic) and nutrient-rich (non-ligninolytic), was investigated using a custom-designed 70-mer oligonucleotide microarray developed based on 190 target genes and 23 control genes. All 150 P450 genes were found to be expressible under the test conditions, with 27 genes showing differential expression based on a >twofold arbitrary cut-off limit. Of these, 23 P450 genes were upregulated (twofold to ninefold) in defined high-nitrogen cultures whereas four genes were upregulated (twofold to twentyfold) in defined low-nitrogen cultures. Furthermore, tandem P450 member genes in ten of the 16 P450 genomic clusters showed nonassortative regulation of expression reflecting their functional diversity. Full-length cDNAs for two of the high-nitrogen upregulated genes pc-hn1 (CYP5035A1) and pc-hn2 (CYP5036A1) and partial cDNA for a low-nitrogen upregulated gene pc-ln1 (CYP5037A1) were cloned and characterized. The study provided first molecular evidence for the presence of active components of the cAMP- and MAP kinase-signaling pathways in a white rot fungus; four of these components (cpka and ste-12 of cAMP pathway and two MAP kinases, mps1 and sps1) were significantly upregulated (fourfold to eightfold) under nutrient-limited conditions, implying their likely role in the regulation of gene expression involved in secondary metabolism and biodegradation processes under these conditions.     

Functional evaluation of genetic and environmental regulators of p450 mRNA levels

Variations in the activities of Cytochrome P450s are one of the major factors responsible for inter-individual differences in drug clearance rates, which may cause serious toxicity or inefficacy of therapeutic drugs. Various mRNA level is one of the key factors for different activity of the major P450 genes. Although both genetic and environmental regulators of P450 gene expression have been widely investigated, few studies have evaluated the functional importance of cis- and trans-regulatory factors and environmental factors in the modulation of inter-individual expression variations of the P450 genes. In this study, we measured the mRNA levels of seven major P450 genes (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) in 96 liver biopsy samples from Chinese population. Both trans-acting (mRNA levels and non-synonymous SNPs of putative regulator genes) and cis-acting (gene copy number and functional SNPs) factors were investigated to identify the determinants of the expression variations of these seven P450 genes. We found that expression variations of most P450 genes, regulator genes and housekeeping genes were positively correlated at the mRNA level. After partial correlation analysis using ACTB and GAPDH expression to eliminate the effect of global regulators, a UPGMA (Unweighted Pair Group Method with Arithmetic Mean) tree was constructed to reveal the effects of specific regulation networks potentially masked by global regulators. Combined with the functional analysis of regulators, our results suggested that expression variation at the mRNA level was mediated by several factors in a gene-specific manner. Cis-acting genetic variants might play key roles in the expression variation of CYP2D6 and CYP3A5, environmental inducers might play key roles in CYP1A1 and CYP1A2 variation and global regulators might play key roles in CYP2C9 variation. In addition, the functions of regulators that play less important roles in controlling expression variation for each P450 gene were determined.     

Molecular cloning and expression analysis of a cytochrome P450 gene in tomato

A full-length cytochrome P450 cDNA, CYP71A2, was cloned from tomato (Lycopersicon esculentum Mill.) by RT-PCR and RACE. CYP71A2 (GenBank accession no. GQ370622) encoded a single polypeptide of 495 amino acid residues and shared 46–68% of identity with CYP71A1 which associated with avocado fruit ripening. The polypeptide, which held the conserved domains in all P450s, was classified as CYP71. CYP71A2-GFP fusion protein localised in the endoplasmic reticulum. The expression of CYP71A2 was detected in all the tissues (root, leaf, stem, bud, flower, immature green fruit, mature green fruit, breaker fruit, ripe fruit); however, the CYP71A2 expression was utmost in immature green fruit. During development of fruit, the expression of CYP71A2 reduced rapidly at mature green stage, then gradually increased at breaker and ripening stages. CYP71A2 was regulated by wounding, methyl jasmonate and ethylene. Promoter analysis indicated that CYP71A2 regulatory region had all the specific responding elements to these stresses. This suggested that the role of CYP71A2 is pleiotropic in tomato development and its adaptability to the environment.