Synthesis and Biological Evaluation of Novel Triazole Derivatives as

Agricultural and Environmental Chemistry

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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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01276 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Journal of Agricultural and Food Chemistry

1

Title

2

Synthesis and biological evaluation of novel triazole derivatives as

3

strigolactone biosynthesis inhibitors

4 5

Running title

6

Novel strigolactone biosynthesis inhibitors

7 8

Corresponding author

9

Shinsaku Ito

10

Department

11

Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

12

Phone, +81-3-5477-2365

13

E-mail address: [email protected]

of

Bioscience,

Tokyo

University

of

Agriculture,

1-1-1

14 15

Authors

16

Kojiro Kawada1, Ikuo Takahashi2, Minori Arai1, Yasuyuki Sasaki1, Tadao

17

Asami2,3,4, Shunsuke Yajima1, Shinsaku Ito1*

18 19

Author addresses

20

1

21

Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

22

2

23

1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

24

3

25

4

26

Arabia

Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Department of Applied Biological Chemistry, The University of Tokyo, 1-1JST, CREST, Saitama, Japan Department of Biochemistry, King Abdulaiz University, Jeddah, Saudi

27 28 29 30 31 32 33 34 35 1 ACS Paragon Plus Environment


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ABSTRACT

37

Strigolactones (SLs) are one of the plant hormones that control several

38

important agronomic traits, such as shoot branching, leaf senescence and

39

stress tolerance. Manipulation of the SL biosynthesis can increase the crop

40

yield. We previously reported that a triazole derivative, TIS108, inhibits SL

41

biosynthesis. In this study, we synthesized a number of novel TIS108

42

derivatives. Structure-activity relationship studies revealed that 4-(2-

43

phenoxyethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK5) inhibits

44

the level of 4-deoxyorobanchol in roots more strongly than TIS108. We further

45

found that KK5-treated Arabidopsis showed increased branching phenotype

46

with the up-regulated gene expression of AtMAX3 and AtMAX4. These

47

results indicate that KK5 is a specific SL biosynthesis inhibitor in rice and

48

Arabidopsis.

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 2 ACS Paragon Plus Environment

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71


Introduction

72 73

Strigolactones (SLs) are terpenoid-derived signaling molecules that have

74

been recognized as one group of the plant hormones involved in various

75

developmental phenomena such as branching initiation and root

76

development 1-3. In addition, SLs are also rhizosphere-signaling molecules

77

that act as germination stimulants and hyphae-branching factors for root

78

parasitic weeds and arbuscular mycorrhizal fungi, respectively4,5. Root

79

parasitic weeds, such as Orobanche spp. and Striga spp., are harmful plants

80

in sub-Saharan Africa, the Middle East and Asia that maintain seed

81

dormancy in the absence of host plant6. It has been reported that

82

approximately 300 million people are affected economically by Striga spp. in

83

Africa, with estimated losses of $US 7 billion7. Because SL biosynthesis

84

mutants protect against the infection of root parasitic weeds, SL

85

biosynthesis inhibitors have a potential to control damage from root

86

parasitic weeds2. In addition, chemicals that perturb SL biosynthesis are

87

promising as chemical tools for analyzing the mechanisms of SL action.

88

Genetic and biochemical studies have revealed that SLs are

89

biosynthesized by several enzymes in rice and Arabidopsis8-15. A carotenoid

90

isomerase, D27, which catalyzes the first step of SL biosynthesis, converts all-

91

trans-beta-carotene to 9-cis-beta-carotene. An important intermediate of SL

92

biosynthesis, carlactone (CL), is synthesized by carotenoid cleavage

93

dioxygenase 7 (CCD7) (AtMAX3 in Arabidopsis/D17 in rice) and CCD8

94

(AtMAX4 in Arabidopsis/D10 in rice) from 9-cis-beta-carotene. Although

95

enzymatic activities are different between rice and Arabidopsis, the

96

conversion of CL is catalyzed by CYP711A family enzymes. CL is oxidized by

97

AtMAX1 (Arabidopsis) and Os900 (rice) to carlactonoic acid (CLA) and 4-

98

deoxyorobanchol (4DO), respectively. Os1400 encoding the orobanchol

99

synthase catalyzes orobanchol formation from 4DO. CLA is methylated by

100

unknown methyltransferase to methyl carlactonoate (MeCLA) in Arabidopsis.

101

Until now, around 25 SLs including 4DO, orobanchol, and MeCLA have been

102

identified from various plant species16. The perception of SLs depends on the

103

SL receptor (AtD14 in Arabidopsis/D14 in rice) and F-box protein (AtMAX2

104

in Arabidopsis/D3 in rice)

105

GROWTH2-LIKEs (SMXLs), which encodes a substrate of the SCFMAX2

17-20.

SUPPRESSOR OF MORE AXILLARY

3 ACS Paragon Plus Environment


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complex, was reported to be a repressor of SL signaling21.

107

Chemicals with nitrogen-containing heterocycle, such as triazole and

108

imidazole, are known to act as inhibitors of various P450 enzymatic activities.

109

For example, uniconazole-P, which is known as plant growth regulator,

110

inhibits the activities of P450 enzymes including gibberellin, brassinosteroid

111

and cytokinin biosynthesis, and abscisic acid metabolism

112

inhibitors such as brassinazole and abscinazole have been developed by a

113

structure activity relationship study using uniconazole-P as the lead chemical.

114

In our early work, we screened for the chemicals that induce the

115

elongation of a 2nd tiller, which is the phenotype of SL deficient mutants, and

116

found TIS13 to be the lead chemical in SL biosynthesis inhibition25. A

117

structural activity relationship study revealed TIS108 as a potent inhibitor

118

of SL biosynthesis26. As shown in the analysis of SL function in a non-model

119

plant27,28, it is worthwhile developing more specific and potent SL

120

biosynthesis inhibitors. In this paper, we synthesized TIS108 derivatives and

121

estimated the effects of synthetic chemicals to look for specific inhibitor of SL

122

biosynthesis.

22-24.

Specific P450

123 124

Materials and Methods

125

Plant materials and growth condition

126

We used rice variety (Oryza sativa ‘Shiokari’) as wild-type (WT). Rice

127

seedlings were grown hydroponically as described in a previous study2.

128

Surface-sterilized rice seeds were incubated in sterile water at 25°C in the

129

dark for 2 days. The germinated seeds were transferred to hydroponic culture

130

medium solidified with 0.7% agar and cultured at 25°C under fluorescent

131

white light with a 14-hour light and 10-hour dark photoperiod for 6days. To

132

determine 4DO level in rice roots and root exudates, each seedling was

133

transferred to a brown glass vial containing 12 mL of hydroponic culture

134

media and grown under the same conditions for 6 days. 15-day-old seedlings

135

were then transferred to a new brown glass vial containing 12 mL of

136

hydroponic culture media with or without tested chemicals. On the following

137

day, roots and hydroponic culture media were collected to measure 4DO levels

138

and Striga germination rate. To measure the length of second leaf sheath, 8-

139

day-old seedlings were transferred to a brown glass vial containing 12 mL of

140

hydroponic culture media with or without tested chemicals and grown under 4 ACS Paragon Plus Environment

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141


the same conditions for 7 days.

142

We used Arabidopsis ecotype Col-0 as the WT. Seeds were sterilized

143

in 70% ethanol for 30 min and then placed on half-strength Murashige and

144

Skoog (MS) medium containing 0.8% sucrose and 0.8% agar (pH 5.7). For

145

branching assay, after stratification at 4°C for 2 days, plants were grown at

146

22°C under constant light for 7 days. 7-day-old seedlings were transferred to

147

a plastic pot containing Arabidopsis hydroponic culture solution with or

148

without chemicals and grown under the same conditions for 4 weeks. The

149

solution was added and renewed every 3 days and 7 days, respectively. We

150

measured the number of rosette branches over 2 mm. To measure the

151

hypocotyl length, after stratification at 4 ºC for 2 days, plants were grown at

152

22 ºC under dark condition for 7 days. Then we measured the hypocotyl length

153

of germinated plants within 2 days by using the ImageJ. For the gene

154

expression assay, stratified seeds were cultured at 22°C under constant light

155

for 4 weeks. Plants were incubated in sterile water with or without chemicals

156

for 1 day under the same conditions. Total RNA was extracted from roots.

157 158

RT-PCR analysis

159

Total RNA was extracted from roots using Plant RNA Isolation reagent

160

(Invitrogen, Waltham, MA, USA), according to the manufacture’s protocol.

161

cDNA was synthesized by using PrimeScript RT Reagent Kit with gDNA

162

eraser (Takara Bio, Shiga, Japan). Quantitative PCR was performed with

163

Thermal Cycler Dice Real Time System II (Takara Bio) and SYBR Premix Ex

164

Taq (Takara Bio). The transcript levels were normalized against those of UBC,

165

using primers specific for MAX3 (5′-GTGTATTTAAGATGCCACCGA-3′ and

166

5′-

167

GTTTTACCCGATGCTAGGATC-3′ and 5′- TGATGCTGCACATATCCATCG-

168

3′),

169

TTGGTCCTCGAATCGGCTACAC-3′)

170

TAGCATTGATGGCTCATCCT-3′ and 5′- GGCGAGGCGTGTATACATTT-3′).

CTTGAATTCCGAATCATACTCAC-3′), MAX2

(5′-

MAX4

CCGGAGAACGATATGAGCACAG-3′ and

UBC

(5′and

5′(5’-

171 172

Chemicals

173

TIS108 and triazole derivatives were synthesized as described previously 26.

174 175

7-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)heptan-1-one (KK1) 5 ACS Paragon Plus Environment


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NMR (CDCl3) 8.39 (1H, s), 7.99(2H, d J=7.8Hz), 7.94 (1H, s), 7.63 (1H, t

176

1H

177 179

J=7.5Hz), 7.51 (2H, t J=7.8Hz), 7.26 (2H, dd J=7.5, 9.0Hz), 6.93 (1H, t J=7.5Hz), 6.85 (2H, d J=7.5Hz), 6.09 (1H, dd J=5.0, 10.0Hz), 3.91 (2H, t J=6.3Hz), 2.10-2.29 (2H, m), 1.69-1.77 (2H, m), 1.24-1.60 (4H, m).

180

8-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)octan-1-one (KK2)

181

1H

182 183

J=6.5Hz), 7.51 (2H, t J=6.8Hz), 7.27 (2H, t J=7.0Hz), 6.93 (1H, t J=7.5Hz), 6.86 (2H, dd J=1.0, 9.0Hz), 6.06 (1H, dd J=5.0, 9.5Hz), 3.92 (2H, t J=6.3Hz),

184

2.09-2.26 (2H, m), 1.71-1.76 (2H, m), 1.26-1.47 (6H, m).

185

(E)-6-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)hex-4-en-1-one (KK3)

186

1H

187

= 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

188

Hz, 1H), 6.81 (d, J =

189

(m, 2H), 4.39 (d, J = 4.4 Hz, 2H), 3.16-2.95 (m, 1H), 2.95-2.85 (m, 1H).

190

4-(2-phenoxyethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one (KK5)

191

To a suspension of sodium hydride (0.85 g) in dimethylformamide (5 mL) was

192

added

193

dimethylformamide (5 mL) at 0 ºC under nitrogen. After the solution stirred

194

at 0 ºC for 10 min, (2-(2-bromoethoxy)ethoxy)benzene (2.06 g) in

195

dimethylformamide (5 mL) was added at 0 ºC. The mixture was warmed to

196

70 ºC and stirred for 5 h. The reaction was quenched by adding distilled water

197

on ice. The aqueous phase was extracted with ethyl acetate three times. The

198

combined organic phases were dried over anhydrous Na2SO4, and

199

concentrated in vacuo. Purification by silica gel column chromatogramphy

200

(hexane/ethyl acetate as eluent) gave the KK5 as a white solid (5.5% yield).

201

1H

202

= 7.3, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.9 Hz, 2H), 7.00-6.89 (m, 3H),

203

6.31 (dd, J = 9.9, 5.2 Hz, 1H), 4.11 (t, J = 4.6 Hz, 2H), 3.81-3.68 (m, 2H), 3.68-

204

3.59 (m, 1H), 3.33-3.24 (m, 1H), 2.62-2.50 (m, 1H), 2.41-2.30 (m, 1H). 13C

205

NMR (400 MHz CDCl3):

206

129.6, 129.1, 128.8, 121.2, 114.7, 69.9, 67.2, 66.4, 60.4, 32.7. HRMS (m/z):

207

[M+H]+ calcd. for C20H22N3O3+, 352.1656; found 352.1662.

208

3-methyl-6-phenoxy-1-phenyl-2-(1H-1,2,4-triazol-1-yl)hexan-1-one (KK6)

209

1H

210

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 =

178

NMR (CDCl3) 8.37 (1H, s), 7.98 (2H, d J=7.5Hz), 7.94 (1H, s), 7.64 (1H, t

NMR (CDCl3): 8.30 (s, 1H), 7.95 (d, J =7.6 Hz, 2H), 7.92 (s, 1H), 7.61 (t, J 8.3 Hz, 2H), 6.09 (dd, J = 8.7, 5.1 Hz, 1H), 5.80-5.63

1-Phenyl-2-(1H-1,2,4-triazol-1-yl)ethanone

(1.08

g)

in

NMR (CDCl3): 8.31 (s, 1H), 7.94 (d, J = 7.5 Hz, 2H), 7.91 (s, 1H), 7.56 (t, J

d 194.1, 159.2, 151.8, 143.7, 134.2 134.3, 134.2,

NMR (CDCl3): 8.42 (s, 1H), 8.00 (d, J = 7.6 Hz, 2H), 7.91 (s, 1H), 7.61 (t,


Page 7 of 29


211

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,

212

2H), 2.64-2.51 (m, 1H), 1.94-1.81 (m, 1H), 1.45-1.35 (m, 1H), 1.29-1.16 (m,

213

2H), 1.01 (d, J = 6.8 Hz, 3H).

214

4-(2-(2,6-dichlorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-

215

one (KK12)

216

1H

217

= 7.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.99 (t, J =

218

8.0 Hz, 1H), 6.41 (dd, J = 9.9, 4.4 Hz, 1H), 4.19 (t, J = 4.4 Hz, 2H), 3.84-3.75

219

(m, 2H), 3.70-3.65 (m, 1H), 3.34-3.28 (m, 1H), 2.63-2.55 (m, 1H), 2.42-2.34

220

(m, 1H).

221

4-(2-(3-chlorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one

222

(KK13)

223

1H

224

= 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,

225

3H), 6.29 (dd, J = 9.5, 5.2 Hz, 1H), 4.00 (t, J = 4.4 Hz, 2H), 3.76-3.64 (m, 2H),

226

3.61-3.56 (m, 1H), 3.30-3.25 (m, 1H), 2.59-2.51 (m, 1H), 2.37-2.29 (m, 1H).

227

4-(2-(4-bromophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one

228

(KK14)

229

1H

230

= 7.2 Hz, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 6.77 (d, J =

231

9.2 Hz, 2H), 6.29 (dd, J = 9.2, 5.2 Hz, 1H), 4.02 (t, J = 4.4 Hz, 2H), 3.75-3.56

232

(m, 3H), 3.31-3.24 (m, 1H), 2.58-2.48 (m, 1H), 2.36-2.49 (m, 1H).

233

4-(2-(4-methoxyphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-

234

one (KK15)

235

1H

236


237

8.0 Hz, 1H),6.41 (dd, J = 9.9, 4.4 Hz, 1H), 4.19 (t, J = 4.4 Hz, 2H), 3.84-3.75

238

(m, 2H), 3.70-3.65 (m, 1H), 3.34-3.28 (m, 1H), 2.63-2.55 (m, 1H), 2.42-2.34

239

(m, 1H).

240

4-(2-(2,6-dimethylphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-

241

1-one (KK16)

242

1H

243


244

7.6 Hz, 1H), 6.39 (dd, J = 9.9, 4.7 Hz, 1H), 3.91 (t, J = 4.4 Hz, 2H), 3.74-3.62

245

(m, 3H), 3.34-3.28 (m, 1H), 2.64-2.55 (m, 1H), 2.42-2.34 (m, 1H), 2.29 (s, 6H).








246

4-(2-(3-trifluorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-

247

one (KK17)

248

1H

249

= 7.6 Hz, 1H), 7.43-7.36 (m, 3H), 7.21 (d, J = 7.6 Hz, 1H), 7.12 (br s, 1H),

250

7.08 (d, J = 8.3 Hz, 1H), 6.31 (dd, J = 9.1, 4.8 Hz, 1H), 4.11 (t, J = 4.9 Hz,

251

2H), 3.79-3.70 (m, 2H), 3.65-3.61 (m, 1H), 3.36-3.31 (m, 1H), 2.62-2.53 (m,

252

1H), 2.39-2.32 (m, 1H).

253

4-(2-(2-fluorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-one

254

(KK18)

255

1H

256

= 7.6 Hz, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.12-6.91 (m, 4H), 6.35 (dd, J = 9.6,

257

4.8 Hz, 1H), 4.17 (t, J = 4.5 Hz, 2H), 3.82-3.63 (m, 3H), 3.28-3.22 (m, 1H),

258

2.60-2.52 (m, 1H), 2.42-2.33 (m, 1H).

259

4-(2-(4-phenoxyphenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-

260

one (KK19)

261

1H

262

= 8.0 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 7.35-7.24 (m, 3H), 7.15-6.88 (m, 6H),

263

6.33 (dd, J = 9.6, 5.2 Hz, 1H), 4.07 (t, J = 4.5 Hz, 2H), 3.78-3.60 (m, 3H),

264

3.33-3.28 (m, 1H), 2.67-2.31 (m, 2H).

265

4-(2-([1,1'-biphenyl]-4-yloxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-

266

1-one (KK20)

267

1H

268

7.51 (m, 5H), 7.40 (m, 4H), 7.29 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 8.0 Hz, 2H),

269

6.33 (dd, J = 9.6, 4.8 Hz, 1H), 4.15 (t, J = 4.8 Hz, 2H), 3.81-3.71 (m, 2H),

270

3.67-3.62 (m, 1H), 3.34-3.28 (m, 1H), 2.62-2.53 (m, 1H), 2.40-2.32 (m, 1H).




NMR (CDCl3): 8.33 (s, 1H), 7.94 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 7.56-

271 272

Quantification of endogenous 4DO level

273

We used deuterium-labeled 5-deoxystrigol (d6-5DS) as internal standard29.

274

For 4DO analysis in root exudates, the hydroponic culture medium was

275

extracted twice with ethyl acetate after the addition of d6-5DS (300 pg). The

276

ethyl acetate layer was dried under reduced pressure. For 4DO analysis in

277

roots, the roots were homogenized in acetone containing d6-5DS. The filtrates

278

were concentrated in vacuo, and dissolved in 10 % acetone. The extracts were

279

subjected to Oasis HLB 3-mL cartridges (Waters), washed with 6 mL water,

280

eluted with 6 mL acetone, concentrated in vacuo, and dissolved in 1 mL 15% 8 ACS Paragon Plus Environment

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281

(vol/vol) ethyl acetate in hexane. The extracts were subjected to Sep-Pak silica

282

1-mL cartridge (Waters), washed with 2 mL of the same solvent, eluted with

283

3 mL 35% (vol/vol) ethyl acetate in hexane. SL-containing fractions were dried

284

in vacuo. 30

285

For LC-MS/MS analysis, dried SL-containing fractions were dissolved in

286

acetonitrile, and subjected to LC-MS/MS analysis. LC-MS/MS analysis was

287

conducted as reported previously.30

288 289

Striga germination assay

290

Germination assay using Striga hermonthica was performed as described

291

previously25. For bioassay, de-ionized water was used as negative control.

292 293

Results

294

Synthesis of TIS108 derivatives

295

To investigate the structure-activity relationship of TIS108 derivatives, we

296

synthesized 14 TIS108 derivatives (Fig. 1). Especially, we focused on the

297

carbon chain at the R1 position and the substitution pattern of the benzene

298

ring (R2).

299 300

Selection of novel SL biosynthesis inhibitors

301

To determine the ability of the synthesized chemicals to inhibit SL

302

biosynthesis, we measured the level of 4DO, a major endogenous SL in rice,

303

in root exudates using the LC-MS/MS, as the level of 4DO in root was

304

correlated with that in root exudates. Since SL levels in roots and root

305

exudates are upregulated when inorganic phosphate is reduced in the

306

culture media2,31, we examined the effects of TIS108 derivatives on 4DO

307

levels under phosphate deficiency. First, we estimated the effect of the

308

substitution at the R1 position on 4DO inhibitory activity, as the extension

309

of carbon chain length from three to four at R1 position increased the 4DO

310

inhibitory activity26 (Fig. 2A and B). As described in our previous report,

311

TIS108 showed 4DO inhibitory activity in a dose-dependent manner within

312

the concentration range of 10–100 nM (Fig. 2B). The extension of carbon

313

chain length from 4 to 6 (TIS108, KK1 and KK2) exhibited decreased

314

activity of the inhibition of 4DO levels (Fig. 2A). Introduction of branched

315

chain (KK6) also reduced the 4DO inhibitory activity. Surprisingly, the 9 ACS Paragon Plus Environment


316

introduction of an oxygen atom to the carbon chain (KK5) increased the

317

4DO inhibitory activity at 10 nM (Fig. 2B). Second, we estimated the effect

318

of the modification of benzene ring on 4DO inhibitory activity in the

319

treatment of 10 nM chemicals. Although no chemicals inhibited the 4DO

320

production at statistically significant level, KK13 showed the strongest

321

inhibitory activity in the tested chemicals. However, the modification of the

322

benzene ring hardly affected the inhibitory activity of 4DO production in

323

comparison with KK5.

324

1H-1,2,4-triazole derivatives such as uniconazole-P and

325

paclobutrazol inhibit a variety of cytochrome P450s, because the nitrogen

326

atom in the triazole group binds to heme iron in cytochrome P450. In plants,

327

various triazole derivatives inhibit gibberellin biosynthesis, because there

328

are two types of P450s (CYP701A and CYP88A) in the gibberellin

329

biosynthesis pathway32. One of the SL biosynthesis inhibitors, TIS13, shows

330

dwarf phenotype as a side effect and this is rescued by co-application of

331

gibberellin with TIS1325. Based on this result, we estimated the effect of the

332

synthesized compounds on gibberellin biosynthesis. We tested five

333

compounds (KK5, 12, 13, 16, and 18). All the compounds did not inhibit the

334

length of second leaf sheath in rice at 50 µM (Fig. 3A). Furthermore, we

335

measured Arabidopsis hypocotyl length grown under dark conditions,

336

because brassinosteroid, which regulates dark-induced photomorphogenesis,

337

is synthesized by some P450 enzymes, and some triazole derivatives inhibit

338

brassinosteroid biosynthesis. Although KK5, 12, and 18 did not change the

339

hypocotyl length at the concentration of 1 µM and 3 µM, KK13 and KK16

340

inhibited the elongation of the hypocotyl in Arabidopsis at 3 µM (Fig. 3B).

341 342

Effect of KK5 on strigolactone biosynthesis

343

Because KK5 showed strong inhibitory activity of 4DO production in rice

344

root exudates and weak side effects on gibberellin and brassinosteroid

345

biosynthesis, we selected KK5 as a candidate for a novel SL biosynthesis

346

inhibitor, and used it in following tests.

347

To estimate whether KK5 actually inhibits SL biosynthesis, we

348

analyzed the endogenous 4DO levels in roots. KK5-treated rice showed the

349

reduction of the endogenous 4DO in both roots and root exudates in dose-

350

dependent manner (3-30 nM) (Fig. 4). In addition, the inhibitory activity of 10 ACS Paragon Plus Environment

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351

4DO biosynthesis of KK5 was 10-fold stronger than that of TIS108. This

352

result suggests that KK5 inhibits SL biosynthesis in rice.

353

In Arabidopsis, SL biosynthesis mutants exhibit a more branching

354

phenotype. KK5-treated wildtype plants dose-dependently showed an

355

increased branching phenotype at the concentration range of 0.1-3 µM (Fig.

356

5A). Next, we estimated the effect of KK5 on Arabidopsis gene expression.

357

Previous studies have revealed that the transcription levels of several genes

358

related to SL biosynthesis, such as MAX3 (At2g44990) and MAX4

359

(At4g32810), were upregulated in several SL biosynthesis and SL

360

insensitive mutants, and TIS108-treated plants2,33,34. Based on these

361

findings, we performed RT-qPCR analysis to estimate the expression level of

362

two SL biosynthesis genes (MAX3 and MAX4) and one SL signaling gene

363

(MAX2) in Arabidopsis roots treated with or without 5 µM KK5. MAX3 and

364 365

MAX4 genes were significantly upregulated in KK5-treated plants. On the other hand, the expression level of MAX2 gene, which is not affected by the

366

endogenous SL level, did not change (Fig. 5). As previous studies have

367

revealed that MAX3 and MAX4 were regulated by SL signal-dependent

368

feedback regulation33, KK5 could also inhibits SL biosynthesis in

369

Arabidopsis. These results suggest the possibility that KK5 inhibits SL

370

biosynthesis in various plants.

371 372

Striga germination assay

373

SLs are seed germination stimulants for the root parasitic weeds Striga and

374

Orobanche. We checked the Striga hermonthica germination rate of the root

375

exudates from KK5-treated rice. In accordance with the results of the 4DO

376

analysis in root and root exudates, the culture media of KK5-treated rice

377

showed less germination stimulating activity than those of mock-treated rice

378

(Fig. 7). In addition, co-application of GR24 with the culture media of KK5-

379

treated rice recovered the germination activity, suggesting that the reduced

380

germination activity of the culture media of KK5-treated rice is not caused by

381

the direct inhibition of Striga germination but the reduction of SL levels in

382

culture media.

383 384

Discussion



385

In this study to find novel SL biosynthesis inhibitors, we synthesized

386

TIS108-derivatives and estimated synthesized chemicals. KK5 inhibits

387

endogenous level of 4DO in roots and root exudates of rice. In addition,

388

KK5-treated Arabidopsis showed SL-deficient mutant-like morphology.

389

Until now, some SL biosynthesis inhibitors have been reported (Fig. S1).

390

Abamine, which is an ABA biosynthesis inhibitor, inhibits SL biosynthesis

391

in rice and sorghum at 100 µM35. Some of the hydroxamic acid compounds

392

show the inhibition of OsD27, AtCCD7, and AtCCD8 at the concentration

393

range of 10-100 µM36. TIS13, TIS108 and tebuconazole derivatives, which

394

have triazole moiety, also inhibit SL biosynthesis in rice at 10, 0.1, and 10

395

µM, respectively25,26,37. On the other hand, KK5 showed inhibitory activity of

396

SL biosynthesis in rice at 10-100 nM. Thus, KK5 appears to be the most

397

potent inhibitor of all reported SL biosynthesis inhibitors; however, these

398

inhibitors need to be tested under the same assay conditions to compare

399

their effectiveness. Especially, while KK5 inhibited SL biosynthesis in rice

400

at nanomolar order, micromolar treatment is needed to show the more

401

branching phenotype in Arabiodpsis. This contradiction may be caused by

402

the difference in affinity between the target proteins in each plant. As KK5

403

is a triazole-type inhibitor, the CYP711 family can be one of the potential

404

target proteins (Fig. S2). In the near future, we will estimate inhibitory

405

activity against the CYP711 family.

406

Triazole-containing chemicals inhibit various P450-catalyzed

407

enzymatic reactions. Uniconazole-P is known as an inhibitor of gibberellin

408

and brassinosteroid biosynthesis. TIS13 inhibits not only SL biosynthesis,

409

but also gibberellin biosynthesis. On the other hand, KK5, KK12, and KK18

410

did not inhibit gibberellin and brassinosteroid biosynthesis in physiological

411

assays in Arabidopsis and rice, respectively. Thus, these chemicals can be

412

specific SL biosynthesis inhibitors.

413

Because biosynthetic inhibitors of plant hormones can control their

414

endogenous levels in various plants, occasionally in a specific developmental

415

stage and tissue, SL biosynthesis inhibitors will play an important role in

416

investigations into the function of SLs in tissue, organs, and biochemical

417

processes. The use of TIS108 revealed the role of SL in AM fungi-inoculated

418

Sesbania cannabina; SL production levels affect the alleviation of salt

419

stress27,28. In addition, as SLs are also germination stimulants for root 12 ACS Paragon Plus Environment

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420

parasitic weeds, KK5 can be a useful tool for analyzing SL function and

421

controlling the damage of root parasitic weeds.

422 423

Funding information

424

This work was supported, in part, by a JSPS Grant-in-Aid for Scientific

425

Research (S; grant number 18H5266).

426 427

References

428

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429

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de Saint Germain, A.; Clavé, G.; Badet-Denisot, M. A.; Pillot, J. P.; Cornu, D.; Le

Wang, L.; Wang, B.; Jiang, L.; Liu, X.; Li, X.; Lu, Z.; Meng, X.; Wang, Y.; Smith, S.

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Sasaki, E.; Ogura, T.; Takei, K.; Kojima, M.; Kitahata, N.; Sakakibara, H.; Asami,

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Regulation of Strigolactone Biosynthesis by Gibberellin Signaling. Plant Physiol 2017, 174 (2),

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root parasites. Planta 2007, 227 (1), 125-32.

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Yoneyama, K.; Suzuki, Y.; Asami, T., Feedback-regulation of strigolactone biosynthetic genes

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and strigolactone-regulated genes in Arabidopsis. Biosci Biotechnol Biochem 2009, 73 (11),

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biosynthesis inhibitor TIS108 on Arabidopsis. Plant Signal Behav 2013, 8 (5), e24193.

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Asami, T. Abamine as a basis for new designs of regulators of strigolactone production. J

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Pestic Sci 2011, 36(1), 53-7.

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Bugg, T. D., Biochemical characterization and selective inhibition of β-carotene cis-trans

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isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic

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pathway. FEBS J 2015, 282 (20), 3986-4000.

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deriveatives are potent inhibitors of strigolactone biosynthesis. J Pestic Sci 2013, 38(3), 147-

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51.

Ueno, K.; Hanada, A.; Yamaguchi, S.; Asami, T., Preparation of multideuterated 5-

Ito, S.; Yamagami, D.; Umehara, M.; Hanada, A.; Yoshida, S.; Sasaki, Y.; Yajima,

Yoneyama, K.; Xie, X.; Kusumoto, D.; Sekimoto, H.; Sugimoto, Y.; Takeuchi, Y.,

Hedden, P.; Thomas, S. G., Gibberellin biosynthesis and its regulation. Biochem J Mashiguchi, K.; Sasaki, E.; Shimada, Y.; Nagae, M.; Ueno, K.; Nakano, T.;

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Harrison, P. J.; Newgas, S. A.; Descombes, F.; Shepherd, S. A.; Thompson, A. J.;

Ito, S.; Umehara, M.; Hanada, A.; Yamaguchi, S.; Asami, T. Tebuconazole

549 550

Figure Legends

551 552

Fig. 1 Synthesis of TIS108 derivatives

553

(A) 1,2,4-triazole. K2CO3, acetone (B) K2CO3, acetone (C) 60% NaH, dimethylformamide

554

(DMF), reflux 16 ACS Paragon Plus Environment

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555 556

Fig. 2 Effect of synthesized chemicals on 4DO production in rice root exudates.

557

4DO levels in rice root exudates of 100 nM (A), 10 nM (B and C) chemical-treated seedlings

558

determined by LC-MS/MS. The data are means ± SD (n = 3). * means statistically different

559

from that of the control plants (Dunnett’s test, P < 0.05).

560 561

Fig. 3 Effects of synthesized chemicals on rice second leaf sheath length and Arabidopsis

562

hypocotyl length.

563

(A) Second leaf sheath length of 50 µM chemical- treated one-week-old rice. The data are

564

means ± SD (n = 17-20). (B) Hypocotyl length of 1 µM (white bar) or 3 µM (gray bar)

565

chemical-treated Arabidopsis seedlings. Stratified seeds were grown at 22°C under dark

566

condition for 7 days. The data are means ± SD (n = 50-60). * means statistically different

567


568 569

Fig. 4 A comparison of the inhibitory activity of SL biosynthesis between TIS108 and KK5

570

4DO levels in rice root exudates (A) and roots (B). White bars indicate TIS108 treatment. Gray

571

bars indicate KK5 treatments. The data are means ± SD (n = 3). * means statistically different

572


573 574

Fig. 5 Effect of KK5 on the number of branches in 5-week-old Arabidopsis.

575

The data are means ± SE (n = 18-39). ** means statistically different from that of the 0

576

µM KK5-treatment (t-test, P < 0.01).

577 578

Fig. 6 Effect of KK5 on SL biosynthesis gene expression. The data are means ±

579

SE (n = 3). ** means statistically different from that of the 0 µM KK5-treatment (t-test,

580

P < 0.01)).

581 582

Fig. 7 Striga germination assay.

583

Germination stimulant levels in root exudates from 1 µM KK5-treated rice.

584

Mock, culture media of mock-treated rice; KK5, culture media of 1 µM KK5-

585

treated rice; KK5 + GR24, mixture of 1 µM GR24 and culture media of 1 µM

586

KK5-treated rice. The data are means ± SD of four samples. Different letters

587

mean signify differences at P < 0.05, Tukey’s test.

588 589 17 ACS Paragon Plus Environment


590

Figures

591 592

Fig. 1 Synthesis of TIS108 derivatives

593

(A) 1,2,4-triazole. K2CO3, acetone (B)K2CO3, acetone (C) 60% NaH, dimethylformamide

594

(DMF), reflux

595


Page 18 of 29

Page 19 of 29


596 597

Fig. 2 Effect of synthesized chemicals on 4DO production in rice root exudates.

598

4DO levels in rice root exudates of 100 nM (A), 10 nM (B and C) chemical-treated seedlings

599

determined by LC-MS/MS. The data are means ± SD (n = 3). * means statistically different

600


601 602

603 604

Fig. 3 Effects of synthesized chemicals on rice second leaf sheath length and Arabidopsis

605

hypocotyl length.

606

(A) Second leaf sheath length of 50 µM chemical- treated one-week-old rice. The data are

607

means ± SD (n = 17-20). (B) Hypocotyl length of 1 µM (white bar) or 3 µM (gray bar)

608

chemical-treated Arabidopsis seedlings. Stratified seeds were grown at 22°C under dark 19 ACS Paragon Plus Environment


609

condition for 7 days. The data are means ± SD (n = 50-60) * means statistically different

610


Page 20 of 29

611

612 613

Fig. 4 A comparison of the inhibitory activity of SL biosynthesis between TIS108 and KK5

614

4DO levels in rice root exudates (A) and roots (B). White bars indicate TIS108 treatment. Gray

615

bars indicate KK5 treatments. The data are means ± SD (n = 3). * means statistically different

616


617 618

619 620

Fig. 5 Effect of KK5 on the number of branches in 5-week-old Arabidopsis.

621

The data are means ± SE (n = 18-39). ** means statistically different from that of the 0

622

µM KK5-treatment (t-test, P < 0.01)).

623 20 ACS Paragon Plus Environment

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624 625

Fig. 6 Effect of KK5 on SL biosynthesis gene expression. The data are means ±

626

SE (n = 3). ** means statistically different from that of the 0 µM KK5-treatment (t-test,

627

P < 0.01)).

628

629 630

Fig. 7 Striga germination assay.

631

Germination stimulant levels in root exudates from 1 µM KK5-treated rice.

632

Mock, culture media of mock-treated rice; KK5, culture media of 1 µM KK5-

633

treated rice; KK5 + GR24, mixture of 1 µM GR24 and culture media of 1 µM

634

KK5-treated rice. The data are means ± SD of four samples. Different letters

635

mean signify differences at P < 0.05, Tukey’s test.

636 637 638 639 640 21 ACS Paragon Plus Environment


641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657

TOC Graphic

658

659 660


Page 22 of 29

Page 23 of 29


N

N

(A)

N

N

N

Br

(C)

O

O

R2

(B)

HO

Br

　 TIS108 KK1 KK2 KK3 KK5 KK6 KK12 KK13 KK14 KK15 KK16 KK17 KK18 KK19 KK20

R1

N R1

O

O

R2 O

R1 -CH2CH2CH2CH2- -CH2CH2CH2CH2CH2- -CH2CH2CH2CH2CH2CH2- -CH2CH=CHCH2- -CH2CH2OCH2CH2- -CH(CH3)CH2CH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2- -CH2CH2OCH2CH2-

ACS Paragon Plus Environment

R2 H H H H H H 2,6-dichloro 3-chloro 4-bromo 4-methoxy 2,6-dimethyl 3-trifluoro 2-fluoro 4-phenoxy 4-phenyl

R2


15 10

*

5

KK6

KK5

KK3

TIS108

0

*

*

20 18 16 14 12 10 8 6 4 2 0


control KK5 KK12 KK13 KK14 KK15 KK16 KK17 KK18 KK19 KK20

20

control

Chemicals (100 nM)

4DO (pg/mL)

25

*

(C)

10 nM 30 nM 100 nM

30

4DO (pg/mL)

3.5 3 2.5 2 1.5 1 0.5 0

(B)

control TIS108 KK1 KK2

4DO (pg/mL)

(A)

Page 24 of 29

Chemicals (10 nM)

Page 25 of 29


(B)


1 µM 3 µM

KK18

KK16

KK13

KK12

KK5

* *

TIS108

4 3.5 3 2.5 2 1.5 1 0.5 0

control

Hypocotyl length (cm) KK18

KK16

KK13

KK12

KK5

TIS108

** PAC

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

Control

Length of second leaf sheath (cm)

(A)


(A)

(B) 250

12 TIS108

10

KK5

8

4DO (pg/gFW)

4DO (pg/mL)

Page 26 of 29

6 4

**

2

**

0 0

**

** **

TIS108

200

KK5

150 100

** **

50

**

0

3 10 30 Concentration (nM)


0

** **

3 10 30 Concentration (nM)

Page 27 of 29


Number of branches

4 3.5

**

3 2.5 2 1.5 1 0.5 0 0 0.1 1 3 KK5 conc. (µM)


Relative transcript levels


AtMAX3 8 6

AtMAX4 **

4 2 0

3

AtMAX2 **

1.5

2

1

1

0.5

0 0 5 KK5 conc. (µM)

Page 28 of 29

0 0 5 KK5 conc. (µM)


0 5 KK5 conc. (µM)


Striga germination (%)

Page 29 of 29

80 70 60 50 40 30 20 10 0

b b

c

a Water Mock KK5

KK5 +GR24

Root exudate


Synthesis and Biological Evaluation of Novel Triazole Derivatives as

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