Hideyuki Inui

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.

Expression of Human Cytochromes P450 1A1 and P450 1A2 as Fused Enzymes with Yeast NADPH-cytochrome P450 Oxidoreductase in Transgenic Tobacco Plants

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

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

Abstract  A 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.

A Major Latex-Like Protein Is a Key Factor in Crop Contamination by Persistent Organic Pollutants

A latex-like protein binds and transports polychlorinated biphenyls into the aerial part of plants. This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.

Congener Specificity in the Accumulation of Dioxins and Dioxin-Like Compounds in Zucchini Plants Grown Hydroponically

Zucchini cultivars Cucurbita pepo subsp. ovifera cv. Patty Green and subsp. pepo cv. Gold Rush were cultivated hydroponically in a nutrient solution supplemented with a mixture of dioxins and dioxin-like compounds. Patty Green and Gold Rush showed low and high accumulation of these compounds in the aerial parts respectively. In both cultivars, the accumulation of each congener negatively depended on its hydrophobicity. This suggests that desorption and solubilization were partly responsible for congener specificity of accumulation, since this was not found in soil experiments. In contrast, no clear difference in accumulation in the roots was observed between the cultivars, whereas the translocation factors, which are indicators of efficient translocation from the roots to the aerial parts, differed among the congeners hydrophobicity-dependently. There were positive correlations between accumulation in the roots and the hydrophobicity of the polychlorinated biphenyl congeners in both cultivars. These results indicate that translocation was also partly responsible for the congener specificity and accumulation concentrations.

Phytomonitoring and Phytoremediation of Agrochemicals and Related Compounds Based on Recombinant Cytochrome P450s and Aryl Hydrocarbon Receptors (AhRs)

Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

A scFv Antibody-Based Immunoaffinity Chromatography Column for Clean-Up of Bisphenol A-Contaminated Water Samples

This is a report on the development of immunoaffinity chromatography using a column of silica gel with an immobilized single-chain variable fragment (scFv) antibody specific to bisphenol A (BPA) for cleanup of BPA-contaminated water samples. The BBA-2187 scFv antibody specific to BPA was purified from the periplasmic fractions of the recombinant Escherichia coli. After a sample of BPA-contaminated river water was applied to the immunoaffinity column, the background signal intensity observed in high-performance liquid chromatography (HPLC) analysis of the eluates was markedly lower than that observed in HPLC analysis of the eluates from an Oasis HLB cartridge treated with the same sample. The immunoaffinity column efficiently concentrated BPA from actual river water samples with different matrices. Our results demonstrate that the immunoaffinity column with immobilized BBA-2187 scFv antibody is efficient for the cleanup of BPA-contaminated water samples from different sources.

Overexpression of Arabidopsis thaliana LOV KELCH REPEAT PROTEIN 2 promotes tuberization in potato (Solanum tuberosum cv. May Queen)

Potato tuberization is induced under short‐day conditions and repressed under long‐day conditions. In this study, we produced transgenic potatoes overexpressing either Arabidopsis thaliana LOV KELCH PROTEIN 2 (35S:LKP2) or CONSTANS fused with a transcription repressor motif (35S:CO‐Rep). In an in vitro tuberization assay, the average number of tubers per plant was greater in 35S:LKP2 plants than in vector‐control plants, but lower in 35S:CO‐Rep plants. Under long‐day conditions in soil, all 35S:LKP2 plants tuberized, whereas most control plants and 35S:CO‐Rep plants did not. These results suggest genes involved in flowering time regulation can be used to control potato tuber production.

Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants

A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity.