rge amounts within the thylakoid membranes of chloroplasts and play a part in defending chlorophylls from active oxygen and peroxides. Hence, the decrease in carotenoids causes the loss of their protective impact against the generation250 S. Yamamoto et al.Journal of Pesticide Scienceof active oxygen by light inside the plant, resulting in bleaching and major to death.4) mGluR7 Source fenquinotrione is assumed to be an HPPD inhibitor due to the fact its chemical structure and herbicidal symptoms are extremely comparable to those of HPPD inhibitors. In this study, we examined the mode of action of fenquinotrione by examining its inhibitory effects on HPPD activity. The aspects accountable for the excellent rice selectivity of fenquinotrione are also discussed.had been purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Rice plants (Oryza sativa L. var. Kinmaze) and RGS4 list Arabidopsis plants (Arabidopsis thaliana, ecotype Columbia-0) have been applied in this study. 2. Bioresource for building in the HPPD enzyme assay Pseudomonas aeruginosa strain PAO1 for isolation in the homogentisate dioxygenase (HGD) gene was obtained in the Biological Resource Center, NITE (NBRC, Tokyo, Japan). 3. Cloning and expression of Arabidopsis HPPD (AtHPPD) The AtHPPD gene (At1g06570) was amplified from Arabidopsis cDNA using the Phusion Hot Begin II DNA Polymerase (Thermo Fisher Scientific, MA, USA). The primers utilized for amplification from the AtHPPD gene were 5-TCG AAG GTC GTC ATA TGG GC C ACC AAA ACG CCG CC-3 (forward primer) and 5-GTT AG C AGC CGG ATC CTC ATC CCA CTA ACT GTT TG-3 (reverse primer). The PCR solution was ligated in to the Escherichia coli expression pET-16b vector (Novagen, WI, USA) digested with Nde I and BamH I applying an In-Fusion HD Cloning Kit (TaKaRa Bio Inc., Shiga, Japan). The resultant vector was introduced in to the E. coli BL21 star (DE3) strain (Thermo Fisher Scientific) working with the heat shock system and then plated on Luria ertani (LB) agar medium supplemented with 100 /mL ampicillin for transformant selection. The transformed E. coli cells were picked out and grown to OD600=0.five.six in two T medium supplemented with 100 /mL ampicillin at 37 . The expression of N-terminal His-tagged AtHPPD was induced by 1 mM IPTG and cultured at 16 for 24 hr. Escherichia coli cells were har-Materials and methods1. Chemicals and plants Fenquinotrione and its derivatives and metabolites had been synthesized by the Kumiai Chemical Industry Co., Ltd. (Shizuoka, Japan). The structure of fenquinotrione, nuclear magnetic resonance (NMR) data, and mass spectrometry (MS) data for authentic standards are shown in Table 1. Three 14C-labeled compounds of fenquinotrione have been applied in the metabolic study: a 1-position label of a cyclohexenyl moiety (certain activity four.94 MBq/mg, radiochemical purity 98.3 , abbreviated as [Cy-14C] FQ) synthesized by the Institute of Isotopes Co., Ltd. (Budapest, Hungary); the uniform label of a chlorophenyl ring (certain activity 5.63 MBq/mg, radiochemical purity 99.two , abbreviated as [Qu-14C] FQ); and the uniform label of a phenyl ring (certain activity 5.29 MBq/mg, radiochemical purity 99.6 , abbreviated as [Bz-14C] FQ) synthesized by the Sekisui Medical Co., Ltd. (Ibaraki, Japan). The active type of benzobicyclon was synthesized by the Kumiai Chemical Industry Co., Ltd. Tefuryltrione, HPP, L(+)-ascorbic acid, iron(II) sulfate heptahydrate (FeSO4 H2O), and isopropylthio–galactoside (IPTG)Table 1. Compounds Fenquinotrione StructureH NMR information and MS data of authe
