Mechanism of action and selectivity of a novel herbicide, fenquinotrione

Abstract

Fenquinotrione is a novel herbicide that can control a wide range of broadleaf and sedge weeds with excellent rice selectivity. We revealed that fenquinotrione potently inhibited the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity in Arabidopsis thaliana with an IC₅₀ of 44.7 nM. The docking study suggested that the 1,3-diketone moiety of fenquinotrione formed a bidentate interaction with Fe(II) at the active site. Furthermore, π–π stacking interactions occurred between the oxoquinoxaline ring and the conserved Phe409 and Phe452 rings, indicating that fenquinotrione competes with the substrate, similar to existing HPPD inhibitors. A more than 16-fold difference in the herbicidal activity of fenquinotrione in rice and the sedge, Schoenoplectus juncoides, was observed. However, fenquinotrione showed high inhibitory activity against rice HPPD. Comparative metabolism study suggested that the potent demethylating metabolism followed by glucose conjugation in rice was responsible for the selectivity of fenquinotrione.

Introduction

In the present agricultural situation in Japan, cultivation systems have been diversified, including direct-seeding rice cultivation and transplanting rice. In addition, the planting of new demand rice varieties for feed and processing has been increasing. From the viewpoint of weed management, the following weeds, which show resistance to acetolactate synthase (ALS)–inhibiting herbicides, have become problematic in Japanese rice paddy fields: Schoenoplectus juncoides, Monochoria vaginalis, Monochoria korsakowii, Sagittaria trifolia, and Lindernia spp.¹⁾ Under these circumstances, there is a need for herbicides that have a long application period, can be applied to various cropping systems and cultivars, and can control the growth of a wide range of weeds, including ALS-resistant weeds, at low concentrations.

Fenquinotrione is a novel triketone-type herbicide with an oxoquinoxaline ring that was developed by the Kumiai Chemical Co., Ltd. When applied to paddy fields at 300 g a.i./ha, it showed excellent selectivity for rice and outstanding herbicide efficacy with bleaching symptoms against a wide range of broadleaf and sedge weeds, including ALS-resistant weeds. This herbicide also has sufficient residual activity and a high herbicidal effect against weeds at the high leaf stage.²⁾

Commercially available 4-hydroxyphenylpyruvate dioxygenase (HPPD)–inhibiting herbicides are classified into three groups based on their structural features: triketones, pyrazoles, and isoxazole.³⁾ HPPD (EC1.13.11.27) catalyzes the conversion of 4-hydroxyphenylpyruvic acid (HPP) into homogentisic acid in the catabolic pathway of the amino acid tyrosine. Homogentisic acid synthesized by HPPD is utilized as a precursor of tocopherol and plastoquinone. Plastoquinone is an essential cofactor that functions as an electron acceptor for the phytoene desaturase. Thus, the function of the phytoene desaturase in the carotenoid biosynthesis pathway is prevented indirectly when the biosynthesis of plastoquinone is arrested by the inhibition of HPPD. Carotenoids are present in large amounts in the thylakoid membranes of chloroplasts and play a role in protecting chlorophylls from active oxygen and peroxides. Thus, the decrease in carotenoids causes the loss of their protective effect against the generation of active oxygen by light in the plant, resulting in bleaching and leading to death.⁴⁾ Fenquinotrione is assumed to be an HPPD inhibitor because its chemical structure and herbicidal symptoms are very similar 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 factors responsible for the excellent rice selectivity of fenquinotrione are also discussed.

Materials and methods

1. Chemicals and plants

Fenquinotrione and its derivatives and metabolites were 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 were used in the metabolic study: a 1-position label of a cyclohexenyl moiety (specific activity 4.94 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 (specific activity 5.63 MBq/mg, radiochemical purity 99.2%, abbreviated as [Qu-¹⁴C] FQ); and the uniform label of a phenyl ring (specific 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 form of benzobicyclon was synthesized by the Kumiai Chemical Industry Co., Ltd. Tefuryltrione, HPP, L(+)-ascorbic acid, iron(II) sulfate heptahydrate (FeSO₄·7H₂O), and isopropylthio-β-galactoside (IPTG) were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Rice plants (Oryza sativa L. var. Kinmaze) and Arabidopsis plants (Arabidopsis thaliana, ecotype Columbia-0) were used in this study.

Table 1. ¹H NMR data and MS data of authentic compounds.

Compounds	Structure	1H NMR (δH, ppm)a)	MS (m/z)b)
Fenquinotrione		7.38 (1H, dd, J=7.8 Hz), 7.28 (2H, d, J=8.5 Hz), 7.26 (1H, m, J=6.1 Hz), 7.09 (2H, dd, J=7.7 Hz), 6.71 (1H, dd, J=8.5 Hz), 3.87 (3H, s), 2.75 (2H, t, J=6.4 Hz), 2.43 (2H, s), 2.02–2.08 (2H, m, J=6.4 Hz)	424 (M+)
M-1		9.73 (1H, s), 7.43 (1H, d, J=4.5 Hz), 7.35 (1H, dd, J=4.5 and 8.1 Hz), 7.05 (4H, dd, J=2.4 and 42.9 Hz), 6.66 (1H, d, J=8.1 Hz), 2.55–2.65 (2H, m), 2.60–2.48 (2H, m), 1.92–2.02 (2H, m)	411 (M+H+)
M-2		7.54 (2H, m), 7.27 (4H, dd, J=9.0 and 56.7 Hz), 6.61 (1H, d, J=8.4 Hz), 3.86 (3H, s)	331 (M+H+)

Authentic compounds were synthesized by Kumiai Chemical Industry Co., Ltd. (Shizuoka, Japan). ^a) 1H NMR spectrum of fenquinotrione (in CDCl₃) was measured on a JEOL JNM-LA-400 (400 MHz) spectrometer. ¹H NMR spectra of M-1 and M-2 (in DMSO d₆) were measured on JEOL JNM-LA-300 (300 MHz) spectrometer. ^b) EI-MS spectrum of fenquinotrione was measured on a JEOL JMS-SX-102. ESI-MS spectra fo M-1 and M-2 were measured on Thermo Fisher Scientific Q Exactive Focus Mass spectrometry.

2. Bioresource for construction of the HPPD enzyme assay

Pseudomonas aeruginosa strain PAO1 for isolation of the homogentisate dioxygenase (HGD) gene was obtained from 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 Start II DNA Polymerase (Thermo Fisher Scientific, MA, USA). The primers used for amplification of the AtHPPD gene were 5′-TCG AAG GTC GTC ATA TGG GCC ACC AAA ACG CCG CC-3′ (forward primer) and 5′-GTT AGC AGC CGG ATC CTC ATC CCA CTA ACT GTT TG-3′ (reverse primer). The PCR product was ligated into the Escherichia coli expression pET-16b vector (Novagen, WI, USA) digested with Nde I and BamH I using an In-Fusion HD Cloning Kit (TaKaRa Bio Inc., Shiga, Japan). The resultant vector was introduced into the E. coli BL21 star (DE3) strain (Thermo Fisher Scientific) using the heat shock method and then plated on Luria–Bertani (LB) agar medium supplemented with 100 µg/mL ampicillin for transformant selection. The transformed E. coli cells were picked out and grown to OD₆₀₀=0.5–0.6 in 2×YT medium supplemented with 100 µg/mL ampicillin at 37°C. The expression of N-terminal His-tagged AtHPPD was induced by 1 mM IPTG and cultured at 16°C for 24 hr. Escherichia coli cells were harvested by centrifugation (6,000 g at 4°C for 10 min) and stored at −80°C.

Escherichia coli cell pellets were suspended in a B-PER Bacterial Protein Extraction Reagent (Thermo Fisher Scientific) containing 0.2 mg/mL lysozyme, DTT (1 mM), a protease inhibitor cocktail (Sigma-Aldrich, MO, USA), and Cryonase™ Cold-active Nuclease (TaKaRa Bio Inc.). This suspension was centrifuged at 6,000 g at 4°C for 10 min. A recombinant His-tagged AtHPPD protein was purified by affinity chromatography using a HisTrap FF column (GE Healthcare Bioscience, NJ, USA).

4. Cloning and expression of rice HPPD (OsHPPD)

The OsHPPD gene (Os02g0168100) was amplified from rice cDNA using a Phusion Hot Start II DNA Polymerase. The primers used for amplification of the OsHPPD gene were 5′-GGG GCC CCT GGG ATC CAT GCC TCC CAC TCC CAC CC-3′ (forward primer) and 5′-GTC GAC CCG GGA ATT CCT AGG ATC CTT GAA CTG TA-3′ (reverse primer). The PCR product was ligated into the E. coli expression pGEX-6P-1 vector (GE Healthcare Bioscience) digested with BamH I and EcoR I using an In-Fusion HD Cloning Kit (TaKaRa Bio Inc.). The resultant vector was introduced into the E. coli BL21 star (DE3) strain using the heat shock method and then plated on an LB agar medium supplemented with 100 µg/mL ampicillin for transformant selection. The expression of OsHPPD in E. coli was performed following the procedure described for method 3. A recombinant GST–tagged OsHPPD protein was purified by affinity chromatography using a GSTrap FF column (GE Healthcare Bioscience), and GST tags were removed using a Precision Protease (GE Healthcare Bioscience).

5. Enzyme assay

HPPD activity was detected through the conversion of its product, homogentisate, to maleylacetoacetate, then catalyzed by HGD from Pseudomonas aeruginosa (PaHGD). The preparation of recombinant PaHGD protein was performed as previously mentioned.^5,6)

In this study, the assay for HPPD activity was carried out at a final volume of 1 mL in a semi-micro cuvette. The reaction mixture contained 980 µL of reaction solution (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.0), 2 mM L(+)-ascorbic acid, 10 µM FeSO₄, 50 nM HGD, 240 nM HPPD), and 20 µL of the substrate HPP. Reactions were initiated by adding the reaction solution to HPP in a semi-micro cuvette. The reactions were monitored at 320 nm using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan) at 25°C for 5 min. To evaluate the inhibitory activity of the compound on HPPD, 10 µL of the compound was added to the reaction mixture before adding the mixture to HPP. For a dose-response study, inhibitors were added at final concentrations of 1, 10, 30, 70, and 1,000 nM in the assay with the AtHPPD enzyme, and those were added at final concentrations of 1, 10, 25, 50, 70, 100, and 1,000 nM in the assay with the OsHPPD enzyme. The reaction mixture without HPPD was used as a negative control. A reaction mixture without the compound was used as a positive control. Inhibition of HPPD activity was determined by comparison with the positive control.

6. Molecular docking study

The AtHPPD crystal structure (PDB ID: 1TFZ) in complex with an existing inhibitor, DAS869⁷⁾ (2-tert-butyl-4-[3-(4-methoxyphenyl)-2-methyl-4-methylsulfonylbenzoyl]-1H-pyrazol-3-one), which was obtained from the Protein Data Bank, was used as the receptor protein. Docking simulation was performed using the CDOCKER module of Discovery Studio ver. 4.5 (Dassault Systems, Vélizy-Villacoublay, France). The receptor protein was prepared by eliminating the water molecules, adding hydrogen, and correcting the lacking amino acid residues using the “Clean Protein” tool in the “Prepare Protein” module. Later, the protein was assigned using a CHARMM force field. After removing DAS869 from the protein, its cavities were predicted using the “From Receptor Cavities” tool in the “Define and Edit Binding Site” module. Of all the predicted cavities, Site 1 was selected as the active site with reference to the position of DAS869 in 1TFZ. The obtained receptor was used as the “Input Receptor” molecule parameter. DAS869 and fenquinotrione were used as the “Input Ligand” parameters. All other parameters were the default settings.

7. Phylogenetic analysis of amino acid sequences

Phylogenetic analysis of the HPPD amino acid sequences of rice, Arabidopsis, and other plants such as corn, sorghum, wheat, barley, soybean, tomato, carrot, lettuce, rapeseed, millet, alfalfa, and velvetleaf was performed using the ClustalW algorithm.

8. Comparison of the physicochemical properties and biological effects of fenquinotrione derivatives on plants

The paddy soil was placed in a 50 cm² plastic pot. An appropriate amount of water was added to the soil. Monochoria vaginalis and Schoenoplectus juncoides were sown at a depth of 0.5 cm. Rice seedlings at the two-leaf stage were transplanted at a depth of 2 cm. The water was filled to a depth of 3 cm. The next day, water-dispersible powder containing 10 parts by weight of each of the compounds plus 0.5 parts by weight of polyoxyethylene octyl phenyl ether, 0.5 parts by weight of sodium salt from the β-naphthalenesulfonic acid-formalin condensate, 20 parts by weight of diatomaceous earth, and 69 parts by weight of clay was diluted with water and dropped onto the water surface so that the application amount of active ingredient (each compound) was 0.4, 1.6, 6.3, 25, and 100 g/10 a, respectively. The development and growth of weeds and rice plants were conducted in a greenhouse. The log P values used in this study were obtained experimentally using the shake-flask method.^8,9) The herbicidal-activity and rice-injury ratings were visually evaluated 28 days after the addition of the test dilution on a percentage scale, comparing the herbicidal symptoms of each observed pot with two reference pots that indicated 0% activity (no crop injury or herbicidal efficacy) and 100% activity (weed completely killed). The ED₉₀ or ED₂₀ values were calculated as the amount of active ingredient per hectare (g a.i./ha) required for 90% activity or 20% of the maximum injury to weeds or rice, respectively.

9. Estimating the metabolic pathway of fenquinotrione in rice seedlings

To investigate the dynamics of fenquinotrione in rice plants, metabolites in the rice plants were analyzed using three types of ¹⁴C-labeled compounds. A solution prepared by adding a predetermined amount of each acetone solution of [Cy-¹⁴C] FQ, [Qu-¹⁴C] FQ, and [Bz-¹⁴C] FQ to 50 mL of 0.1% HYPONeX® solution (HYPONeX JAPAN, Osaka, Japan) was used as a treatment solution of 0.32–0.35 ppm concentration. Fifteen rice seedlings at approximately the 2.5 leaf stage were placed in the prepared treatment solution and cultivated for 3 days. The collected plants were combined, 150 mL of extracting solvent (acetonitrile/distilled water/formic acid, 80/20/1) was added, and a Physcotron homogenizer was used to homogenize the plants. After filtering the residue, a portion of the extract was subjected to thin-layer chromatography (TLC) with authentic standards for the identification of metabolites. Two-dimensional development TLC using 60F254 silica gel plates (20×20 cm, 0.25 mm thick, Merck, Darmstatd, Germany) was performed with the developing solvent of ethyl acetate/chloroform/methanol/formic acid (6/6/1/1) and ethyl acetate/hexane/methanol/formic acid (6/6/1/1). The radioactivity on the TLC was detected using a bioimaging analyzer, Typhoon™ FLA7000 (GE Healthcare Bioscience). For the structural estimation of metabolites using the extracted sample treated with [¹⁴C-Bz] FQ, metabolites were subjected to LC/MS (TSQ Quantum (Thermo Fisher Scientific) with Radiomatic FSA 610TR (Perkin Elmer, MA, USA)) after isolation by TLC. Furthermore, to clarify the structure of the conjugated metabolite, the extracts of [Cy-¹⁴C] FQ– and [Qu-¹⁴C] FQ–treated samples were deconjugated with glucosidase, and the aglycone produced was qualitatively and quantitatively analyzed using LC/MS. In HPLC, an L-column (ODS, ϕ4.6×250 mm, CERI, Tokyo, Japan) was used, and gradient analysis was performed using acidic acetonitrile as the mobile phase. MS was measured in the ionization mode of ESI.

Results

1. Inhibition of plant HPPD activity by fenquinotrione and other HPPD inhibitors

To evaluate the inhibitory effect of HPPD-inhibiting herbicides including fenquinotrione on HPPD activity, we conducted an inhibition assay using recombinant HPPDs and calculated the concentration required for 50% inhibition (IC₅₀). Fenquinotrione inhibited recombinant AtHPPD activity (IC₅₀=44.7 nM) as strongly as the existing herbicides, benzobicyclon and tefuryltrione. In addition, fenquinotrione potently inhibited recombinant OsHPPD activity (IC₅₀=27.2 nM) (Table 2, Fig. 1).

Table 2. Inhibition of Arabidopsis HPPD and rice HPPD.

Compond	IC50 (nM)a)
	AtHPPD	OsHPPD
Fenquinotrione	44.7±12.7	27.2±1.5
Benzobicyclon (active form)	46.9±3.6	NT
Tefryltrione	35.8±2.7	NT

^a) The nano molar concentrations required for 50% inhibition of AtHPPD were calculated by the probit method. Data are expressed as the mean±S.D. of three independent experiments. NT, not tested

Fig. 1. Inhibitory effects of fenquinotrione on the HPPD activity of recombinant Arabidopsis and rice HPPD. Each data set was expressed as the mean±S.D. of three independent experiments.

2. Molecular docking study of fenquinotrione

We performed a docking study to investigate the characteristics of fenquinotrione as an HPPD inhibitor on a molecular basis. To validate the docking program, we first redocked DAS869 bound to the 1TFZ crystal structure. As a result, the top docking pose showed a binding pattern similar to that of DAS869 in 1TFZ, and the root-mean-square deviation (RMSD) was calculated to be 1.13 Å. In general, when the RMSD is less than or equal to 2 Å, docking is considered to be successful.¹⁰⁾

Thus, we performed a docking study to predict the binding mode of fenquinotrione using this docking condition. As a result, fenquinotrione fitted into the binding pocket of AtHPPD (Fig. 2A), and the distances from the two oxygen atoms of the 1,3-diketone moiety of fenquinotrione to Fe(II) were 2.05 and 2.26 Å, respectively, which were almost the same as for DAS869 (Fig. 2B-1). This result led to the conclusion that the 1,3-diketone moiety of fenquinotrione can form a bidentate interaction with Fe(II) at the active site. In addition, the result of the superposition showed that the binding style between DAS869 and fenquinotrione was similar, and π–π stacking interactions, which have been identified in the docking study of well-known HPPD inhibitors, were observed between the oxoquinoxaline ring and the conserved Phe409 and Phe452 rings (Fig. 2B-1, C, and D). Furthermore, hydrogen bonding between the oxygen atom of the oxoquinoxaline ring and Gln335 and π–π stacking between the methoxyphenyl group and Phe420 were observed as interactions unique to fenquinotrione (Fig. 2B-2 and C).

Fig. 2. Binding model of fenquinotrione to AtHPPD. (A) Predicted binding pose of fenquinotrione in the active site of AtHPPD. The yellow surface shows the binding pocket. Fenquinotrione is shown in molecular-stick format. (B) Close-up view of the active site and the binding mode of fenquinotrione. B-1 and -2 show a common binding mode for HPPD inhibitors and a specific binding mode for fenquinotrione, respectively. (C) 2D view of the interaction type of fenquinotrione with amino acids of the active site in AtHPPD. (D) The superposition of DAS869 (gray stick) and fenquinotrione (yellow stick). Key residues in the active site are shown in wireframe format, and the iron ion is shown in a blue sphere.

3. Comparison of amino acid sequences of HPPDs

Phylogenetic analysis of the amino acid sequence of plant HPPDs showed that monocotyledonous and dicotyledonous plants were divided into two clades (Fig. 3). More than 80% identity with rice HPPD among monocots and more than 70% identity with Arabidopsis HPPD among dicots was observed. In particular, there is a high level of homology of the amino acid residues at the active site. Among them, five amino acid residues, Phe409 and Phe452, which form a π–π stacking interaction with HPPD inhibitors, and His254, His336, and Glu422, which are essential for enzyme activity because they form a bidentate interaction with Fe(II), were completely conserved in the plants. Furthermore, two amino acid residues involved in interactions unique to fenquinotrione, Phe420 and Gln335, were also conserved (Supplemental Fig. S1).

Fig. 3. Phylogenetic tree for plant HPPDs based on amino acid sequences. Phylogenetic trees were constructed using a ClustalW algorithm. This percentage indicates amino acid identity with rice or Arabidopsis. HPPD proteins with GenBank (https://www.ncbi.nlm.nih.gov/genbank/) accession numbers are as follows: Oryza sativa (XP_015626163), Zea mays (NP_001105782), Sorghum bicolor (XP_002453359), Triticum aestivum (AAZ67144), Hordeum vulgare (CAA04245), Setaria italica (XP_004951787), Arabidopsis thaliana (NP_001154311), Brassica napus (AFB74218), Glycine max (ABQ96868), Daucus carota (AAC49815), Solanum lycopersicum (XP_004243609), Abutilon theophrasti (XP_004243609), Lactuca sativa (XP_023753058), and Medicago sativa (AQN69278). The identity and similarity of monocotyledons and dicotyledons were calculated on the basis of the rice and Arabidopsis HPPD, respectively.

Considering the high homology in the amino acid sequence of HPPD and the conservation of important amino acid residues in the active site, it was assumed that there was little difference in the affinity of the target enzyme, HPPD, to fenquinotrione among plants, as shown by the inhibition of recombinant HPPD activity (Fig. 1).

4. Comparison of physical properties and biological effects on plants among fenquinotrione analogs

To estimate the safety factors of fenquinotrione against rice from the viewpoint of molecular structure and physicochemical properties, the correlation between the structure of the fused ring and benzene ring moiety of fenquinotrione analogs and biological activity was confirmed (Table 3). There were no significant differences in logP values and HPPD inhibitory activities (IC₅₀ values) among fenquinotrione analogs. However, a more than 16-fold difference in biological activity between rice and S. juncoides was observed only in the structure of Cl or F for R1 and OMe for R2.

Table 3. Comparison of physical properties and biological effects on plants among the fenquinotrione analogs.

R1	R2	Log P	IC50a) (nM)	ED20b) Rice	ED90c)		ED20/ED90d)
					M. vaginalis	S. juncoides	Rice/S. juncoides
Cl	H	3.0	54	1.6	1.6	1.6	1
H	OMe	2.6	52	6.3	6.3	6.3	1
Me	OMe	2.8	19	100	6.3	25	4
Cl	OMe	2.9	45	>100	6.3	6.3	>16
F	OMe	2.3	93	>100	6.3	6.3	>16

^a) The 50% concentration of inhibition for Arabidopsis HPPD enzyme. ^b) Herbicidal activity of the 20% effective concentration (g a.i./10 a) for rice. ^c) Herbicidal activity of the 90% effective concentration (g a.i./10 a) for M. vaginalis and S. juncoides. ^d) The ratio of ED₂₀ to rice and ED₉₀ to S. juncoides was used as an index of selectivity between rice and S. juncoides. The structure having Cl for R1 and OMe for R2 represents fenquinotrione.

5. Estimating the metabolic pathway of fenquinotrione in rice seedlings

Three days after treatment with the labeled compounds, approximately 70% of the parent compound and about eight metabolites in the plants were detected by TLC (Supplemental Fig. S2), and the detected amount of these metabolites was less than 1% of the total radioactivity in the plant. Among these metabolites, M-1 and M-2 were identified by collation with an authentic standard (Table 1). Although the separation of these two compounds by TLC was insufficient, it was confirmed that both were detected by the [Qu-¹⁴C] FQ and [Bz-¹⁴C] FQ treatments (Supplemental Fig. S2A, B, D, and E). In contrast, only M-1 was detected; M-2, lacking the labeled site, was not detected via [Cy-¹⁴C] FQ treatment (Supplemental Fig. S2C and F). Other highly polar metabolites were detected at the origin of the TLC. In the LC/MS analysis of the extracts treated with [Bz-¹⁴C] FQ, an m/z 573 ion in positive mode (Fig. 4B and C) and an m/z 571 ion in negative mode (Fig. 4E and F) were detected at a retention time of 31.3 min, corresponding to the retention time of the ¹⁴C-metabolite peak (Fig. 4A and D). Therefore, a metabolite with a molecular weight of 572 was proposed as the glucose conjugate of M-1. To identify highly polar metabolites, glucosidase treatments were performed on the plant extracts treated with [Cy-¹⁴C] FQ and [Qu-¹⁴C] FQ, the results via LC/MS (Fig. 5A–E). As a result, an m/z 411 ion in positive mode at a retention time of 39.7 min (Fig. 5B and C) and an m/z 331 ion in positive mode at a retention time of 42.2 min (Fig. 5D and E) were detected, which were comparable to those of the authentic standards of M-1 and M-2, respectively. The amount of M-1 detected 3 days after the treatment was 4.7% in the plants after [Qu-¹⁴C] FQ treatment and 2.6% in the plants after [Cy-¹⁴C] FQ treatment. The amount of M-2 detected was 5.5% in the [Qu-¹⁴C] FQ treatment. M-1 and M-2 were found to exist freely and as glucose conjugates and were the major metabolites of fenquinotrione in rice plants.

Fig. 4. LC/MS analysis of the highly polar metabolite in rice seedlings treated with ¹⁴C-labeled fenquinotrione. (A–C) Analysis in the positive mode. (D–F) Analysis in the negative mode. (A, D) HPLC radiochromatograms. (B, E) LC/MS chromatograms of extracted ion m/z 573 (positive mode) and m/z 571 (negative mode). (C, F) Mass spectra of RT=31.3.

Fig. 5. LC/MS analysis of the aglycones derived from glucosidase-treatment extraction of rice in the positive mode. (A) HPLC radiochromatogram of the glucosidase-treated rice extract. (B) LC/MS chromatogram of extracted ion m/z 411. (C) Mass spectrum of M-1. (D) LC/MS chromatogram of extracted ion m/z 331. (E) Mass spectrum of M-2.

Discussion

We demonstrated here that fenquinotrione is a potent AtHPPD inhibitor similar to the existing HPPD-inhibiting herbicides. In addition, the docking study suggested that the 1,3-diketone moiety of fenquinotrione forms a bidentate interaction with Fe (II) in the active site, and π–π stacking interactions occur between the oxoquinoxaline ring and the conserved Phe409 and Phe452 rings. This indicates that fenquinotrione competes with the substrate, HPP, in the same manner as the existing HPPD-inhibiting herbicides. The docking study suggested that in addition to the interactions common to HPPD inhibitors, due to its unique oxoquinoxaline ring substituted at the 4-position with phenoxymethyl, fenquinotrione forms two strong interactions with AtHPPD: a π–π interaction with Phe420 and hydrogen bonding with Gln335.

Fenquinotrione also showed high inhibitory activity against OsHPPD. In addition, the high similarity in the amino acid sequence of HPPD among plants (Fig. 3) and the high conservation of fenquinotrione-binding sites of the HPPD protein (Supplemental Fig. S1) suggested that the selectivity between rice and weeds was not due to differences in affinity for the HPPD protein. Therefore, we compared the physical properties and biological effects of fenquinotrione derivatives on plants (Table 3). Both fenquinotrione and another compound, which have halogen at R1 and a methoxy group at R2, showed high selectivity with an ED₂₀/ED₉₀ ratio greater than 16, although there were no significant differences in physical properties or enzyme inhibitory activity among the compounds. Based on the results, we assumed that the high selectivity of fenquinotrione for rice was not due to its HPPD-inhibitory activity, nor its adsorption and translocation, but to its metabolism in rice. It is unclear why only the compounds with a halogen at R1 and a methoxy group at R2 showed high rice safety. Considering that the ED₂₀ for rice was lower when R1 was a hydrogen (even when R2 was a methoxy group), its substitution with halogen at R1 may have altered the electron density of the methoxy group at R2 and increased the compounds’ safety by becoming recognizable by a metabolic rice enzyme.

To estimate the metabolic mechanism of fenquinotrione, we examined the metabolites of fenquinotrione in rice. The major metabolites of fenquinotrione detected were M-1, M-2, and their glucose conjugates. M-2 is a hydrolysis product of the triketone moiety, and such metabolites are commonly found in existing HPPD inhibitors.^11–14) In contrast, M-1 is a demethylated form of methoxybenzene on the oxoquinoxaline ring unique to fenquinotrione. M-1 has a substructure that is essential for HPPD enzyme binding, suggesting that M-1 still has HPPD-inhibitory activity. Indeed, M-1 inhibited AtHPPD activity with an IC₅₀ of 171 nM that could control weeds, although its efficacy was lower than that of fenquinotrione (Supplemental Table 1). No clear bleaching symptoms were observed in rice, even when M-1 was applied at a four-fold higher concentration than the recommended label dose of fenquinotrione in pot trials (Supplemental Fig. S3). Furthermore, the safety level of M-1 for rice was higher than that of fenquinotrione in susceptibility tests on a solid culture medium in which the chemicals are absorbed directly from the roots (Supplemental Fig. S4). These results suggest that M-1 was detoxified in rice, similar to fenquinotrione. Considering the metabolism pathway of fenquinotrione, it was assumed that M-1 was detoxified by rapid conversion into glucose conjugates in rice.

Some forage rice cultivars have been reported to be susceptible to triketone-type herbicides; however, fenquinotrione has been found to be applicable to a wide variety of rice plants, including forage rice.²⁾ Therefore, we speculated that the safety of fenquinotrione against a wide range of rice cultivars, including forage rice, was related to its metabolism to M-1 and its glucose conjugate, which are specific to this herbicide. The detoxification of herbicides is generally divided into three phases.¹⁵⁾ Phase I involves the addition of functional groups to the herbicide by oxidation, reduction, or hydrolysis. Cytochrome P450 monooxygenase (P450) primarily mediates oxidation, including hydroxylation and demethylation. Phase II involves the conjugation of the metabolites produced in Phase I with endogenous compounds such as glutathione and glucose, resulting in water-soluble products that are easily excreted. Phase III involves the sequestration of soluble conjugates into organelles, such as the vacuole and/or cell wall. Considering the above metabolic system, the metabolism of fenquinotrione to M-1 by P450 in Phase I, followed by glucose conjugation in Phase II, was considered to be responsible for the safety of fenquinotrione in rice. Many factors are known to determine the rate and selectivity of substrate oxidation by P450, but the electron density distribution of the substrate is considered to be one of the more important factors.^16,17) Therefore, the reason only the analogs introduced with F and Cl showed high safety against rice may be that the methoxy group was recognized as a substrate in rice P450 due to the change in electron density. We will report further investigations of the detoxification mechanism of fenquinotrione in rice in a subsequent paper (manuscript in preparation).

In conclusion, the results of the present study demonstrate that fenquinotrione controls a wide range of broadleaf and sedge weeds by inhibiting HPPD. In addition, it is assumed that the excellent rice selectivity of fenquinotrione is due to its metabolism in rice plants. In recent years, the cultivation of paddy rice, such as forage rice and fermented rice roughage, has been promoted in Japan to expand the demand for rice and effectively utilize abandoned farmland, and the cultivation and dissemination of high-yield rice varieties suitable for these purposes have been progressing. We believe that fenquinotrione will meet the current demand for herbicides that can be applied to a variety of cultivation systems and rice varieties.

Electronic supplementary materials

The online version of this article contains supplementary materials (Supplemental Methods, Supplemental Figs. S1–S4, and Supplemental Table 1), which are available at https://www.jstage.jst.go.jp/browse/jpestics/.