Hideo Ohkawa

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

Abstract. 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 (≈SS)] and Ae. squarrosa (DD).

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene.

Abstract.A rat P450 monooxygenase gene (CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene

Abstractu2008u2008A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 µmol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

Molecular and Immunochemical Characteristics of Monoclonal and Recombinant Antibodies Specific to Bisphenol A

Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA). Among these mabs, BBA-2187 was the most reactive towards bisphenol A. The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported. Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells. The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs. The most sensitive assay was achieved with BBA-2187scFv. Its cross-reactivity to the related compounds was similar to that of the parent mab. Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH.

Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a β-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methylcholanthrene (MC), β-naphthoflavone (βNF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5xa0nM MC on solid medium and at 12xa0h after soaking in 25xa0μM MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants.

CYP92B1, A Cytochrome P450, Expressed in Petunia Flower Buds, That Catalyzes Monooxidation of Long-Chain Fatty Acids

In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues. The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development. CYP92B1 cDNA was expressed in a yeast, Saccharomyces cerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid.

New discoveries in agrochemicals

Previously, the development of new agrochemicals has begun on the chemists bench, screened via bioassays, and brought to the marketplace following field trials. Since the evolution of molecular, combinatorial and green chemistry techniques, our approach to new discoveries in agrochemicals has fundamentally changed. The availability of biopesticides, transgenic crops, natural products, pest control agents with innovative modes of action and structures that allow rapid conversion to nontoxic metabolites allows us to use these products in a sustainable and effective manner called best management strategies. These tools have issued in a new generation of pest control based on human safety, environmental stewardship, and resistance management. New Discoveries in Agrochemicals will overview each of these technologies and present findings from leading international academic and industry researchers. This book covers the following topics: biopesticides and transgenic crops, combinatorial chemistry, mode of action, natural products, green chemistry, and human vector control.

Structures and Functions of Novel Cytochrome P450 Species Expressed During Flowering

ABSTRACT Cytochrome P450 monooxygenases consisting of a number of cytochrome P450 (P450 or CYP) species and a generic NADPH-cytochrome P450 oxidoreductase (P450 reductase) play an important role in the biosynthesis of a variety of secondary metabolites as well as in the metabolism of xenobiotics including herbicides in higher plants. It was reported that flavonoid 3′ 5′-hydroxylase (CYP75A3) involved in the biosynthesis of flower pigments was expressed in the flower. CYP78A2 was specifically expressed in the pollen tubes of phalaenopsis. Also, CYP78A1 was reported to be preferentially expressed in developing maize tassels. We found that CYP703A1 was specifically expressed in the early stage of flower development of Petunia. We attempted to express each of both maize CYP78A1 and petunia CYP703A1 cDNA clones in the yeast Saccharomyces cerevisiae AH22 cells to identify enzymatic functions of both P450 species. CYP78A1 was expressed in the yeast microsomes together with yeast P450 reductase. The microsomes metabolized lauric acid to give 12-hydroxylauric acid. Thus, CYP78A1 was found to be lauric acid 12-hydroxylase. CYP703A1 was also expressed in the yeast microsomes, which was found to metabolize lauric acid, although the metabolites produced were not identified yet. Based on these results, both maize CYP78A1 and petunia CYP703A1 specifically expressed in the early stage of flowering were found to be involved in the metabolism of fatty acids. So, we will discuss on relationship between fatty acid metabolism and flowering.