Synthesis and Biological Evaluation of Novel Triazole Derivatives as

May 14, 2019 - Strigolactones (SLs) are one of the plant hormones that control several ... East, and Asia that maintain seed dormancy in the absence o...
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Article Cite This: J. Agric. Food Chem. 2019, 67, 6143−6149

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Synthesis and Biological Evaluation of Novel Triazole Derivatives as Strigolactone Biosynthesis Inhibitors Kojiro Kawada,† Ikuo Takahashi,‡ Minori Arai,† Yasuyuki Sasaki,† Tadao Asami,‡,§,∥ Shunsuke Yajima,† and Shinsaku Ito*,† †

Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan § Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ∥ Department of Biochemistry, King Abdulaziz University, Jeddah, Saudi Arabia

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S Supporting Information *

ABSTRACT: Strigolactones (SLs) are one of the plant hormones that control several important agronomic traits, such as shoot branching, leaf senescence, and stress tolerance. Manipulation of the SL biosynthesis can increase the crop yield. We previously reported that a triazole derivative, TIS108, inhibits SL biosynthesis. In this study, we synthesized a number of novel TIS108 derivatives. Structure−activity relationship studies revealed that 4-(2-phenoxyethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1yl)butan-1-one (KK5) inhibits the level of 4-deoxyorobanchol in roots more strongly than TIS108. We further found that KK5treated Arabidopsis showed increased branching phenotype with the upregulated gene expression of AtMAX3 and AtMAX4. These results indicate that KK5 is a specific SL biosynthesis inhibitor in rice and Arabidopsis. KEYWORDS: strigolactone, triazole derivative, structure−activity relationship, Arabidopsis, KK5

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INTRODUCTION Strigolactones (SLs) are terpenoid-derived signaling molecules that have been recognized as one group of plant hormones involved in various developmental phenomena, such as branching initiation and root development.1−3 In addition, SLs are also rhizosphere-signaling molecules that act as germination stimulants and hyphae-branching factors for root parasitic weeds and arbuscular mycorrhizal fungi, respectively.4,5 Root parasitic weeds, such as Orobanche spp. and Striga spp., are harmful plants in sub-Saharan Africa, the Middle East, and Asia that maintain seed dormancy in the absence of host plant.6 It has been reported that approximately 300 million people are affected economically by Striga spp. in Africa, with estimated losses of $US 7 billion.7 Because SL biosynthesis mutants protect against the infection of root parasitic weeds, SL biosynthesis inhibitors have potential to control damage from root parasitic weeds.2 In addition, chemicals that perturb SL biosynthesis are promising as chemical tools for analyzing the mechanisms of SL action. Genetic and biochemical studies have revealed that SLs are biosynthesized by several enzymes in rice and Arabidopsis.8−15 A carotenoid isomerase, D27, which catalyzes the first step of SL biosynthesis, converts all-trans-β-carotene to 9-cis-β-carotene. An important intermediate of SL biosynthesis, carlactone (CL), is synthesized by carotenoid cleavage dioxygenase 7 (CCD7) (AtMAX3 in Arabidopsis/D17 in rice) and carotenoid cleavage dioxygenase 8 (CCD8) (AtMAX4 in Arabidopsis/D10 in rice) from 9-cis-β-carotene. Although enzymatic activities are different between rice and Arabidopsis, the conversion of CL is catalyzed by CYP711A family enzymes. CL is oxidized by AtMAX1 (Arabidopsis) and Os900 (rice) to carlactonoic acid © 2019 American Chemical Society

(CLA) and 4-deoxyorobanchol (4DO), respectively. Os1400 encoding the orobanchol synthase catalyzes orobanchol formation from 4DO. CLA is methylated by unknown methyltransferase to methyl carlactonoate (MeCLA) in Arabidopsis. Until now, around 25 SLs, including 4DO, orobanchol, and MeCLA, have been identified from various plant species.16 The perception of SLs depends upon the SL receptor (AtD14 in Arabidopsis/D14 in rice) and F-box protein (AtMAX2 in Arabidopsis/D3 in rice).17−20 SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKEs (SMXLs), which encodes a substrate of the SCFMAX2 complex, was reported to be a repressor of SL signaling.21 Chemicals with nitrogen-containing heterocycle, such as triazole and imidazole, are known to act as inhibitors of various P450 enzymatic activities. For example, uniconazole-P, which is known as a plant growth regulator, inhibits the activities of P450 enzymes, including gibberellin, brassinosteroid, and cytokinin biosynthesis and abscisic acid metabolism.22−24 Specific P450 inhibitors, such as brassinazole and abscinazole, have been developed by a structure−activity relationship study using uniconazole-P as the lead chemical. In our early work, we screened for the chemicals that induce the elongation of a second tiller, which is the phenotype of SLdeficient mutants, and found TIS13 to be the lead chemical in SL biosynthesis inhibition.25 A structure−activity relationship study revealed TIS108 as a potent inhibitor of SL biosynthesis.26 Received: Revised: Accepted: Published: 6143

February 24, 2019 May 9, 2019 May 14, 2019 May 14, 2019 DOI: 10.1021/acs.jafc.9b01276 J. Agric. Food Chem. 2019, 67, 6143−6149

Article

Journal of Agricultural and Food Chemistry

7.0 Hz), 6.93 (1H, t, J = 7.5 Hz), 6.86 (2H, dd, J = 1.0 and 9.0 Hz), 6.06 (1H, dd, J = 5.0 and 9.5 Hz), 3.92 (2H, t, J = 6.3 Hz), 2.09−2.26 (2H, m), 1.71−1.76 (2H, m), 1.26−1.47 (6H, m). (E)-6-Phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)hex-4-en-1one (KK3). 1H NMR (CDCl3): δ 8.30 (s, 1H), 7.95 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 7.25 (t, J = 7.7 Hz, 2H), 6.93 (t, J = 7.3 Hz, 1H), 6.81 (d, J = 8.3 Hz, 2H), 6.09 (dd, J = 8.7 and 5.1 Hz, 1H), 5.80−5.63 (m, 2H), 4.39 (d, J = 4.4 Hz, 2H), 3.16−2.95 (m, 1H), 2.95−2.85 (m, 1H). 4-(2-Phenoxyethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK5). To a suspension of sodium hydride (0.85 g) in dimethylformamide (5 mL) was added 1-phenyl-2-(1H-1,2,4-triazol-1yl)ethanone (1.08 g) in dimethylformamide (5 mL) at 0 °C under nitrogen. After the solution stirred at 0 °C for 10 min, (2-(2bromoethoxy)ethoxy)benzene (2.06 g) in dimethylformamide (5 mL) was added at 0 °C. The mixture was warmed to 70 °C and stirred for 5 h. The reaction was quenched by adding distilled water on ice. The aqueous phase was extracted with ethyl acetate 3 times. The combined organic phases were dried over anhydrous Na2SO4 and concentrated in vacuo. Purification by silica gel column chromatography (hexane/ethyl acetate as the eluent) gave KK5 as a white solid (5.5% yield). 1 H NMR (CDCl3): δ 8.31 (s, 1H), 7.94 (d, J = 7.5 Hz, 2H), 7.91 (s, 1H), 7.56 (t, J = 7.3 Hz, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.9 Hz, 2H), 7.00−6.89 (m, 3H), 6.31 (dd, J = 9.9 and 5.2 Hz, 1H), 4.11 (t, J = 4.6 Hz, 2H), 3.81−3.68 (m, 2H), 3.68−3.59 (m, 1H), 3.33−3.24 (m, 1H), 2.62−2.50 (m, 1H), 2.41−2.30 (m, 1H). 13C NMR (400 MHz, CDCl3): δ 194.1, 159.2, 151.8, 143.7, 134.2 134.3, 134.2, 129.6, 129.1, 128.8, 121.2, 114.7, 69.9, 67.2, 66.4, 60.4, 32.7. HRMS (m/z): [M + H]+ calculated for C20H22N3O3+, 352.1656; found, 352.1662. 3-Methyl-6-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)hexan-1-one (KK6). 1H NMR (CDCl3): δ 8.42 (s, 1H), 8.00 (d, J = 7.6 Hz, 2H), 7.91 (s, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.8 Hz, 2H), 7.25 (t, J = 7.9 Hz, 2H), 6.91 (t, J = 7.1 Hz, 1H), 6.80 (d, J = 8.0 Hz, 2H), 5.91 (d, J = 8.7 Hz, 1H), 3.89−3.78 (m, 2H), 2.64−2.51 (m, 1H), 1.94−1.81 (m, 1H), 1.45−1.35 (m, 1H), 1.29−1.16 (m, 2H), 1.01 (d, J = 6.8 Hz, 3H). 4-(2-(2,6-Dichlorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4triazol-1-yl)butan-1-one (KK12). 1H NMR (CDCl3): δ 8.42 (s, 1H), 7.99 (d, J = 7.1 Hz, 2H), 7.95 (s, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.99 (t, J = 8.0 Hz, 1H), 6.41 (dd, J = 9.9 and 4.4 Hz, 1H), 4.19 (t, J = 4.4 Hz, 2H), 3.84−3.75 (m, 2H), 3.70−3.65 (m, 1H), 3.34−3.28 (m, 1H), 2.63−2.55 (m, 1H), 2.42−2.34 (m, 1H). 4-(2-(3-Chlorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK13). 1H NMR (CDCl3): δ 8.32 (s, 1H), 7.92 (d, J = 7.9 Hz, 2H), 7.89 (s, 1H), 7.53 (t, J = 7.1 Hz, 1H), 7.39 (t, J = 7.7 Hz, 2H), 7.16 (t, J = 8.1 Hz, 1H), 6.92−6.77 (m, 3H), 6.29 (dd, J = 9.5 and 5.2 Hz, 1H), 4.00 (t, J = 4.4 Hz, 2H), 3.76−3.64 (m, 2H), 3.61− 3.56 (m, 1H), 3.30−3.25 (m, 1H), 2.59−2.51 (m, 1H), 2.37−2.29 (m, 1H). 4-(2-(4-Bromophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK14). 1H NMR (CDCl3): δ 8.31 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.89 (s, 1H), 7.54 (t, J = 7.2 Hz, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 9.2 Hz, 2H), 6.29 (dd, J = 9.2 and 5.2 Hz, 1H), 4.02 (t, J = 4.4 Hz, 2H), 3.75−3.56 (m, 3H), 3.31−3.24 (m, 1H), 2.58−2.48 (m, 1H), 2.36−2.49 (m, 1H). 4-(2-(4-Methoxyphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK15). 1H NMR (CDCl3): δ 8.34 (s, 1H), 7.95 (d, J = 7.1 Hz, 2H), 7.95 (s, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.99 (t, J = 8.0 Hz, 1H), 6.41 (dd, J = 9.9 and 4.4 Hz, 1H), 4.19 (t, J = 4.4 Hz, 2H), 3.84−3.75 (m, 2H), 3.70−3.65 (m, 1H), 3.34−3.28 (m, 1H), 2.63−2.55 (m, 1H), 2.42−2.34 (m, 1H). 4-(2-(2,6-Dimethylphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4triazol-1-yl)butan-1-one (KK16). 1H NMR (CDCl3): δ 8.38 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.92 (s, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.91 (t, J = 7.6 Hz, 1H), 6.39 (dd, J = 9.9 and 4.7 Hz, 1H), 3.91 (t, J = 4.4 Hz, 2H), 3.74−3.62 (m, 3H), 3.34−3.28 (m, 1H), 2.64−2.55 (m, 1H), 2.42−2.34 (m, 1H), 2.29 (s, 6H).

As shown in the analysis of SL function in a non-model plant,27,28 it is worthwhile developing more specific and potent SL biosynthesis inhibitors. In this paper, we synthesized TIS108 derivatives and estimated the effects of synthetic chemicals to look for specific inhibitors of SL biosynthesis.

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MATERIALS AND METHODS

Plant Materials and Growth Conditions. We used rice variety ( Oryza sativa ‘Shiokari’) as the wild type (WT). Rice seedlings were grown hydroponically as described in a previous study.2 Surfacesterilized rice seeds were incubated in sterile water at 25 °C in the dark for 2 days. The germinated seeds were transferred to hydroponic culture medium solidified with 0.7% agar and cultured at 25 °C under fluorescent white light with a 14 h light and 10 h dark photoperiod for 6 days. To determine the 4DO level in rice roots and root exudates, each seedling was transferred to a brown glass vial containing 12 mL of hydroponic culture media and grown under the same conditions for 6 days. The 15-day-old seedlings were then transferred to a new brown glass vial containing 12 mL of hydroponic culture media with or without tested chemicals. On the following day, roots and hydroponic culture media were collected to measure 4DO levels and the Striga germination rate. To measure the length of second leaf sheath, the 8-day-old seedlings were transferred to a brown glass vial containing 12 mL of hydroponic culture media with or without tested chemicals and grown under the same conditions for 7 days. We used Arabidopsis ecotype Col-0 as the WT. Seeds were sterilized in 70% ethanol for 30 min and then placed on half-strength Murashige and Skoog (MS) medium containing 0.8% sucrose and 0.8% agar (pH 5.7). For the branching assay, after stratification at 4 °C for 2 days, plants were grown at 22 °C under constant light for 7 days. The 7-dayold seedlings were transferred to a plastic pot containing Arabidopsis hydroponic culture solution with or without chemicals and grown under the same conditions for 4 weeks. The solution was added and renewed every 3 and 7 days, respectively. We measured the number of rosette branches over 2 mm. To measure the hypocotyl length, after stratification at 4 °C for 2 days, plants were grown at 22 °C under dark conditions for 7 days. Then, we measured the hypocotyl length of germinated plants within 2 days using ImageJ. For the gene expression assay, stratified seeds were cultured at 22 °C under constant light for 4 weeks. Plants were incubated in sterile water with or without chemicals for 1 day under the same conditions. Total RNA was extracted from roots. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis. Total RNA was extracted from roots using Plant RNA Isolation Reagent (Invitrogen, Waltham, MA, U.S.A.), according to the protocol of the manufacturer. cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA eraser (Takara Bio, Shiga, Japan). Quantitative polymerase chain reaction (qPCR) was performed with a Thermal Cycler Dice Real Time System II (Takara Bio) and SYBR Premix Ex Taq (Takara Bio). The transcript levels were normalized against those of UBC, using primers specific for MAX3 (5′GTGTATTTAAGATGCCACCGA-3′ and 5′-CTTGAATTCCGAATCATACTCAC-3′), MAX4 (5′-GTTTTACCCGATGCTAGGATC-3′ and 5′-TGATGCTGCACATATCCATCG-3′), MAX2 (5′CCGGAGAACGATATGAGCACAG-3′ and 5′TTGGTCCTCGAATCGGCTACAC-3′), and UBC (5′-TAGCATTGATGGCTCATCCT-3′ and 5′-GGCGAGGCGTGTATACATTT3′). Chemicals. TIS108 and triazole derivatives were synthesized as described previously.26 7-Phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)heptan-1-one (KK1). 1H NMR (CDCl3): δ 8.39 (1H, s), 7.99 (2H, d, J = 7.8 Hz), 7.94 (1H, s), 7.63 (1H, t, J = 7.5 Hz), 7.51 (2H, t, J = 7.8 Hz), 7.26 (2H, dd, J = 7.5 and 9.0 Hz), 6.93 (1H, t, J = 7.5 Hz), 6.85 (2H, d, J = 7.5 Hz), 6.09 (1H, dd, J = 5.0 and 10.0 Hz), 3.91 (2H, t, J = 6.3 Hz), 2.10−2.29 (2H, m), 1.69−1.77 (2H, m), 1.24−1.60 (4H, m). 8-Phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)octan-1-one (KK2). 1H NMR (CDCl3): δ 8.37 (1H, s), 7.98 (2H, d, J = 7.5 Hz), 7.94 (1H, s), 7.64 (1H, t, J = 6.5 Hz), 7.51 (2H, t, J = 6.8 Hz), 7.27 (2H, t, J = 6144

DOI: 10.1021/acs.jafc.9b01276 J. Agric. Food Chem. 2019, 67, 6143−6149

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Journal of Agricultural and Food Chemistry 4-(2-(3-Trifluorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK17). 1H NMR (CDCl3): δ 8.33 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.43− 7.36 (m, 3H), 7.21 (d, J = 7.6 Hz, 1H), 7.12 (br s, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.31 (dd, J = 9.1 and 4.8 Hz, 1H), 4.11 (t, J = 4.9 Hz, 2H), 3.79−3.70 (m, 2H), 3.65−3.61 (m, 1H), 3.36−3.31 (m, 1H), 2.62− 2.53 (m, 1H), 2.39−2.32 (m, 1H). 4-(2-(2-Fluorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK18). 1H NMR (CDCl3): δ 8.39 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.12−6.91 (m, 4H), 6.35 (dd, J = 9.6 and 4.8 Hz, 1H), 4.17 (t, J = 4.5 Hz, 2H), 3.82−3.63 (m, 3H), 3.28−3.22 (m, 1H), 2.60− 2.52 (m, 1H), 2.42−2.33 (m, 1H). 4-(2-(4-Phenoxyphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK19). 1H NMR (CDCl3): δ 8.34 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.35−7.24 (m, 3H), 7.15−6.88 (m, 6H), 6.33 (dd, J = 9.6 and 5.2 Hz, 1H), 4.07 (t, J = 4.5 Hz, 2H), 3.78−3.60 (m, 3H), 3.33− 3.28 (m, 1H), 2.67−2.31 (m, 2H). 4-(2-([1,1′-Biphenyl]-4-yloxy)ethoxy)-1-phenyl-2-(1H-1,2,4triazol-1-yl)butan-1-one (KK20). 1H NMR (CDCl3): δ 8.33 (s, 1H), 7.94 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 7.56−7.51 (m, 5H), 7.40 (m, 4H), 7.29 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 8.0 Hz, 2H), 6.33 (dd, J = 9.6 and 4.8 Hz, 1H), 4.15 (t, J = 4.8 Hz, 2H), 3.81−3.71 (m, 2H), 3.67− 3.62 (m, 1H), 3.34−3.28 (m, 1H), 2.62−2.53 (m, 1H), 2.40−2.32 (m, 1H). Quantification of the Endogenous 4DO Level. We used deuterium-labeled 5-deoxystrigol (5DS-d6) as the internal standard.29 For 4DO analysis in root exudates, the hydroponic culture medium was extracted twice with ethyl acetate after the addition of 5DS-d6 (300 pg). The ethyl acetate layer was dried under reduced pressure. For 4DO analysis in roots, the roots were homogenized in acetone containing 5DS-d6. The filtrates were concentrated in vacuo and dissolved in 10% acetone. The extracts were subjected to Oasis HLB 3 mL cartridges (Waters), washed with 6 mL of water, eluted with 6 mL of acetone, concentrated in vacuo, and dissolved in 1 mL of 15% (v/v) ethyl acetate in hexane. The extracts were subjected to a Sep-Pak silica 1 mL cartridge (Waters), washed with 2 mL of the same solvent, and eluted with 3 mL of 35% (v/v) ethyl acetate in hexane. SL-containing fractions were dried in vacuo.30 For liquid chromatography−tandem mass spectrometry (LC−MS/ MS) analysis, dried SL-containing fractions were dissolved in acetonitrile and subjected to LC−MS/MS analysis. LC−MS/MS analysis was conducted as reported previously.30 Striga Germination Assay. A germination assay using Striga hermonthica was performed as described previously.25 For the bioassay, deionized water was used as a negative control.

Figure 1. Synthesis of TIS108 derivatives: (A) 1,2,4-triazole, K2CO3, and acetone, (B) K2CO3 and acetone, and (C) 60% NaH, dimethylformamide (DMF), and reflux.

manner within the concentration range of 10−100 nM (Figure 2B). The extension of the carbon chain length from 4 to 6 (TIS108, KK1, and KK2) exhibited decreased activity of the inhibition of 4DO levels (Figure 2A). Introduction of branched chain (KK6) also reduced the 4DO inhibitory activity. Surprisingly, the introduction of an oxygen atom to the carbon chain (KK5) increased the 4DO inhibitory activity at 10 nM (Figure 2B). Second, we estimated the effect of the modification of the benzene ring on the 4DO inhibitory activity in the treatment of 10 nM chemicals. Although no chemicals inhibited the 4DO production at a statistically significant level, KK13 showed the strongest inhibitory activity in the tested chemicals. However, the modification of the benzene ring hardly affected the inhibitory activity of 4DO production in comparison to KK5. 1H-1,2,4-triazole derivatives, such as uniconazole-P and paclobutrazol, inhibit a variety of cytochrome P450s, because the nitrogen atom in the triazole group binds to heme iron in cytochrome P450. In plants, various triazole derivatives inhibit gibberellin biosynthesis, because there are two types of P450s (CYP701A and CYP88A) in the gibberellin biosynthesis pathway.32 One of the SL biosynthesis inhibitors, TIS13, shows a dwarf phenotype as a side effect, and this is rescued by co-application of gibberellin with TIS13.25 On the basis of this result, we estimated the effect of the synthesized compounds on gibberellin biosynthesis. We tested five compounds (KK5, KK12, KK13, KK16, and KK18). All of the compounds did not inhibit the length of the second leaf sheath in rice at 50 μM (Figure 3A). Furthermore, we measured Arabidopsis hypocotyl length grown under dark conditions, because brassinosteroid, which regulates dark-induced photomorphogenesis, is synthesized by some P450 enzymes and some triazole derivatives inhibit brassinosteroid biosynthesis. Although KK5, KK12, and KK18 did not change the hypocotyl length at the concentrations

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RESULTS Synthesis of TIS108 Derivatives. To investigate the structure−activity relationship of TIS108 derivatives, we synthesized 14 TIS108 derivatives (Figure 1). Especially, we focused on the carbon chain at the R1 position and the substitution pattern of the benzene ring (R2). Selection of Novel SL Biosynthesis Inhibitors. To determine the ability of the synthesized chemicals to inhibit SL biosynthesis, we measured the level of 4DO, a major endogenous SL in rice, in root exudates using LC−MS/MS, because the level of 4DO in roots was correlated with that in root exudates. Because SL levels in roots and root exudates are upregulated when inorganic phosphate is reduced in the culture media,2,31 we examined the effects of TIS108 derivatives on 4DO levels under phosphate deficiency. First, we estimated the effect of the substitution at the R1 position on 4DO inhibitory activity, as the extension of the carbon chain length from 3 to 4 at the R1 position increased the 4DO inhibitory activity26 (panels A and B of Figure 2). As described in our previous report, TIS108 showed 4DO inhibitory activity in a dose-dependent 6145

DOI: 10.1021/acs.jafc.9b01276 J. Agric. Food Chem. 2019, 67, 6143−6149

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of synthesized chemicals on 4DO production in rice root exudates. 4DO levels in rice root exudates of (A) 100 nM and (B and C) 10 nM chemical-treated seedlings determined by LC−MS/MS. The data are means ± standard deviation (SD) (n = 3). (∗) Statistically different from that of the control plants (Dunnett’s test; p < 0.05).

Figure 3. Effects of synthesized chemicals on rice second leaf sheath length and Arabidopsis hypocotyl length. (A) Second leaf sheath length of 50 μM chemical-treated 1-week-old rice. The data are means ± SD (n = 17−20). (B) Hypocotyl length of 1 μM (white bar) or 3 μM (gray bar) chemicaltreated Arabidopsis seedlings. Stratified seeds were grown at 22 °C under dark conditions for 7 days. The data are means ± SD (n = 50−60). (∗) Statistically different from that of the control plants (Dunnett’s test; p < 0.05).

Figure 4. Comparison of the inhibitory activity of SL biosynthesis between TIS108 and KK5 4DO levels in rice (A) root exudates and (B) roots. White bars indicate TIS108 treatment. Gray bars indicate KK5 treatment. The data are means ± SD (n = 3). (∗) Statistically different from that of the control plants (Dunnett’s test; p < 0.05).

of 1 and 3 μM, KK13 and KK16 inhibited the elongation of the hypocotyl in Arabidopsis at 3 μM (Figure 3B). Effect of KK5 on SL Biosynthesis. Because KK5 showed strong inhibitory activity of 4DO production in rice root exudates and weak side effects on gibberellin and brassinosteroid

biosynthesis, we selected KK5 as a candidate for a novel SL biosynthesis inhibitor and used it in the following tests. To estimate whether KK5 actually inhibits SL biosynthesis, we analyzed the endogenous 4DO levels in roots. KK5-treated rice showed the reduction of endogenous 4DO in both roots and 6146

DOI: 10.1021/acs.jafc.9b01276 J. Agric. Food Chem. 2019, 67, 6143−6149

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Journal of Agricultural and Food Chemistry root exudates in a dose-dependent manner (3−30 nM) (Figure 4). In addition, the inhibitory activity of 4DO biosynthesis of KK5 was 10-fold stronger than that of TIS108. This result suggests that KK5 inhibits SL biosynthesis in rice. In Arabidopsis, SL biosynthesis mutants exhibit a more branching phenotype. KK5-treated WT plants dose-dependently showed an increased branching phenotype at the concentration range of 0.1−3 μM (Figure 5). Next, we

Figure 7. Striga germination assay. Germination stimulant levels in root exudates from 1 μM KK5-treated rice. Mock, culture media of mocktreated rice; KK5, culture media of 1 μM KK5-treated rice; and KK5 + GR24, mixture of 1 μM GR24 and culture media of 1 μM KK5-treated rice. The data are means ± SD of four samples. Different letters signify differences at p < 0.05, using Tukey’s test.

application of GR24 with the culture media of KK5-treated rice recovered the germination activity, suggesting that the reduced germination activity of the culture media of KK5-treated rice is not caused by the direct inhibition of Striga germination but the reduction of SL levels in culture media.

Figure 5. Effect of KK5 on the number of branches in 5-week-old Arabidopsis. The data are means ± standard error (SE) (n = 18−39). (∗∗) Statistically different from that of the 0 μM KK5 treatment (t test; p < 0.01).

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DISCUSSION In this study, to find novel SL biosynthesis inhibitors, we synthesized TIS108 derivatives and estimated synthesized chemicals. KK5 inhibits the endogenous level of 4DO in roots and root exudates of rice. In addition, KK5-treated Arabidopsis showed SL-deficient mutant-like morphology. Until now, some SL biosynthesis inhibitors have been reported (Figure S1 of the Supporting Information). Abamine, which is an ABA biosynthesis inhibitor, inhibits SL biosynthesis in rice and sorghum at 100 μM.35 Some of the hydroxamic acid compounds show the inhibition of OsD27, AtCCD7, and AtCCD8 at the concentration range of 10−100 μM. 36 TIS13, TIS108, and tebuconazole derivatives, which have triazole moiety, also inhibit SL biosynthesis in rice at 10, 0.1, and 10 μM, respectively.25,26,37 On the other hand, KK5 showed inhibitory activity of SL biosynthesis in rice at 10−100 nM. Thus, KK5 appears to be the most potent inhibitor of all reported SL biosynthesis inhibitors; however, these inhibitors need to be tested under the same assay conditions to compare their effectiveness. Especially, while KK5 inhibited SL biosynthesis in rice at the nanomolar order, micromolar treatment is needed to show the more branching phenotype in Arabidopsis. This contradiction may be caused by the difference in affinity between the target proteins in each plant. Because KK5 is a

estimated the effect of KK5 on Arabidopsis gene expression. Previous studies have revealed that the transcription levels of several genes related to SL biosynthesis, such as MAX3 (At2g44990) and MAX4 (At4g32810), were upregulated in several SL biosynthesis and SL-insensitive mutants and TIS108treated plants.2,33,34 On the basis of these findings, we performed RT-qPCR analysis to estimate the expression level of two SL biosynthesis genes (MAX3 and MAX4) and one SL signaling gene (MAX2) in Arabidopsis roots treated with or without 5 μM KK5. MAX3 and MAX4 genes were significantly upregulated in KK5-treated plants. On the other hand, the expression level of the MAX2 gene, which is not affected by the endogenous SL level, did not change (Figure 6). Because previous studies have revealed that MAX3 and MAX4 were regulated by SL signaldependent feedback regulation,33 KK5 could also inhibit SL biosynthesis in Arabidopsis. These results suggest the possibility that KK5 inhibits SL biosynthesis in various plants. Striga Germination Assay. SLs are seed germination stimulants for the root parasitic weeds Striga and Orobanche. We checked the S. hermonthica germination rate of the root exudates from KK5-treated rice. In accordance with the results of the 4DO analysis in roots and root exudates, the culture media of KK5-treated rice showed less germination stimulating activity than those of mock-treated rice (Figure 7). In addition, co-

Figure 6. Effect of KK5 on SL biosynthesis gene expression. The data are means ± SE (n = 3). (∗∗) Statistically different from that of the 0 μM KK5 treatment (t test; p < 0.01). 6147

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(5) Akiyama, K.; Matsuzaki, K.; Hayashi, H. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 2005, 435 (7043), 824−827. (6) Spallek, T.; Mutuku, M.; Shirasu, K. The genus Striga: A witch profile. Mol. Plant Pathol. 2013, 14 (9), 861−869. (7) Parker, C. Observations on the current status of Orobanche and Striga problems worldwide. Pest Manage. Sci. 2009, 65 (5), 453−459. (8) Alder, A.; Jamil, M.; Marzorati, M.; Bruno, M.; Vermathen, M.; Bigler, P.; Ghisla, S.; Bouwmeester, H.; Beyer, P.; Al-Babili, S. The path from β-carotene to carlactone, a strigolactone-like plant hormone. Science 2012, 335 (6074), 1348−1351. (9) Arite, T.; Iwata, H.; Ohshima, K.; Maekawa, M.; Nakajima, M.; Kojima, M.; Sakakibara, H.; Kyozuka, J. DWARF10, an RMS1/MAX4/ DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J. 2007, 51 (6), 1019−1029. (10) Ishikawa, S.; Maekawa, M.; Arite, T.; Onishi, K.; Takamure, I.; Kyozuka, J. Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol. 2005, 46 (1), 79−86. (11) Zhang, Y.; van Dijk, A. D.; Scaffidi, A.; Flematti, G. R.; Hofmann, M.; Charnikhova, T.; Verstappen, F.; Hepworth, J.; van der Krol, S.; Leyser, O.; Smith, S. M.; Zwanenburg, B.; Al-Babili, S.; Ruyter-Spira, C.; Bouwmeester, H. J. Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis. Nat. Chem. Biol. 2014, 10 (12), 1028−1033. (12) Abe, S.; Sado, A.; Tanaka, K.; Kisugi, T.; Asami, K.; Ota, S.; Kim, H. I.; Yoneyama, K.; Xie, X.; Ohnishi, T.; Seto, Y.; Yamaguchi, S.; Akiyama, K.; Yoneyama, K.; Nomura, T. Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (50), 18084−18089. (13) Seto, Y.; Sado, A.; Asami, K.; Hanada, A.; Umehara, M.; Akiyama, K.; Yamaguchi, S. Carlactone is an endogenous biosynthetic precursor for strigolactones. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (4), 1640− 1645. (14) Yoneyama, K.; Mori, N.; Sato, T.; Yoda, A.; Xie, X.; Okamoto, M.; Iwanaga, M.; Ohnishi, T.; Nishiwaki, H.; Asami, T.; Yokota, T.; Akiyama, K.; Yoneyama, K.; Nomura, T. Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis. New Phytol. 2018, 218 (4), 1522−1533. (15) Lin, H.; Wang, R.; Qian, Q.; Yan, M.; Meng, X.; Fu, Z.; Yan, C.; Jiang, B.; Su, Z.; Li, J.; Wang, Y. DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 2009, 21 (5), 1512−1525. (16) Xie, X. Structural diversity of strigolactones and their distributeon in the plant kingdom. J. Pestic. Sci. 2016, 41 (4), 175−180. (17) Jiang, L.; Liu, X.; Xiong, G.; Liu, H.; Chen, F.; Wang, L.; Meng, X.; Liu, G.; Yu, H.; Yuan, Y.; Yi, W.; Zhao, L.; Ma, H.; He, Y.; Wu, Z.; Melcher, K.; Qian, Q.; Xu, H. E.; Wang, Y.; Li, J. DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 2013, 504 (7480), 401−405. (18) Zhou, F.; Lin, Q.; Zhu, L.; Ren, Y.; Zhou, K.; Shabek, N.; Wu, F.; Mao, H.; Dong, W.; Gan, L.; Ma, W.; Gao, H.; Chen, J.; Yang, C.; Wang, D.; Tan, J.; Zhang, X.; Guo, X.; Wang, J.; Jiang, L.; Liu, X.; Chen, W.; Chu, J.; Yan, C.; Ueno, K.; Ito, S.; Asami, T.; Cheng, Z.; Wang, J.; Lei, C.; Zhai, H.; Wu, C.; Wang, H.; Zheng, N.; Wan, J. D14-SCF(D3)dependent degradation of D53 regulates strigolactone signalling. Nature 2013, 504 (7480), 406−410. (19) Yao, R.; Ming, Z.; Yan, L.; Li, S.; Wang, F.; Ma, S.; Yu, C.; Yang, M.; Chen, L.; Chen, L.; Li, Y.; Yan, C.; Miao, D.; Sun, Z.; Yan, J.; Sun, Y.; Wang, L.; Chu, J.; Fan, S.; He, W.; Deng, H.; Nan, F.; Li, J.; Rao, Z.; Lou, Z.; Xie, D. DWARF14 is a non-canonical hormone receptor for strigolactone. Nature 2016, 536 (7617), 469−473. (20) de Saint Germain, A.; Clavé, G.; Badet-Denisot, M.-A.; Pillot, J.P.; Cornu, D.; Le Caer, J.-P.; Burger, M.; Pelissier, F.; Retailleau, P.; Turnbull, C.; Bonhomme, S.; Chory, J.; Rameau, C.; Boyer, F.-D. An histidine covalent receptor and butenolide complex mediates strigolactone perception. Nat. Chem. Biol. 2016, 12 (10), 787−794. (21) Wang, L.; Wang, B.; Jiang, L.; Liu, X.; Li, X.; Lu, Z.; Meng, X.; Wang, Y.; Smith, S. M.; Li, J. Strigolactone Signaling in Arabidopsis

triazole-type inhibitor, the CYP711 family can be one of the potential target proteins (Figure S2 of the Supporting Information). In the near future, we will estimate inhibitory activity against the CYP711 family. Triazole-containing chemicals inhibit various P450-catalyzed enzymatic reactions. Uniconazole-P is known as an inhibitor of gibberellin and brassinosteroid biosynthesis. TIS13 inhibits not only SL biosynthesis but also gibberellin biosynthesis. On the other hand, KK5, KK12, and KK18 did not inhibit gibberellin and brassinosteroid biosynthesis in physiological assays in Arabidopsis and rice, respectively. Thus, these chemicals can be specific SL biosynthesis inhibitors. Because biosynthetic inhibitors of plant hormones can control their endogenous levels in various plants, occasionally in a specific developmental stage and tissue, SL biosynthesis inhibitors will play an important role in investigations into the function of SLs in tissue, organs, and biochemical processes. The use of TIS108 revealed the role of SL in AM fungi-inoculated Sesbania cannabina; SL production levels affect the alleviation of salt stress.27,28 In addition, because SLs are also germination stimulants for root parasitic weeds, KK5 can be a useful tool for analyzing SL function and controlling the damage of root parasitic weeds.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01276.

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Structures of known SL biosynthesis inhibitors (Figure S1) and SL biosynthesis pathway and predicted target site of KK5 (Figure S2) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-3-5477-2365. E-mail: [email protected]. ORCID

Shinsaku Ito: 0000-0002-2747-1799 Funding

This work was supported, in part, by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (S; Grant 18H5266). Notes

The authors declare no competing financial interest.

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