Synthesis and Biological Evaluation of Novel Triazole Derivatives as Strigolactone Biosynthesis Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b01276

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 tha

Full text of DOI:10.1021/acs.jafc.9b01276

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Agricultural and Environmental Chemistry
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
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Title
Synthesis and biological evaluation of novel triazole derivatives as
strigolactone biosynthesis inhibitors
Running title
Novel strigolactone biosynthesis inhibitors
Corresponding author
Shinsaku Ito
10 Department of Bioscience, Tokyo University of Agriculture, 1-1-1
11 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
12 Phone, +81-3-5477-2365
13 E-mail address: s4ito@nodai.ac.jp
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
1
20
Department of Bioscience, Tokyo University of Agriculture, 1-1-1
21 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
22
2 Department of Applied Biological Chemistry, The University of Tokyo, 1-1-
23 1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
3
24
25
JST, CREST, Saitama, Japan
4
Department of Biochemistry, King Abdulaiz University, Jeddah, Saudi
26 Arabia
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36 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
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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) 17-20. SUPPRESSOR OF MORE AXILLARY
105 GROWTH2-LIKEs (SMXLs), which encodes a substrate of the SCFMAX2
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106 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 22-24. Specific P450
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.
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
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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′-
CTTGAATTCCGAATCATACTCAC-3′),
MAX4
(5′-
167 GTTTTACCCGATGCTAGGATC-3′ and 5′- TGATGCTGCACATATCCATCG-
168 3′),
MAX2
(5′-
CCGGAGAACGATATGAGCACAG-3′
and UBC
and
5′-
169 TTGGTCCTCGAATCGGCTACAC-3′)
(5’-
170 TAGCATTGATGGCTCATCCT-3′ and 5′- GGCGAGGCGTGTATACATTT-3′).
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)
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176
177
178
179
1H NMR (CDCl3) 8.39 (1H, s), 7.99(2H, d J=7.8Hz), 7.94 (1H, s), 7.63 (1H, t
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
182
1H NMR (CDCl3) 8.37 (1H, s), 7.98 (2H, d J=7.5Hz), 7.94 (1H, s), 7.64 (1H, t
J=6.5Hz), 7.51 (2H, t J=6.8Hz), 7.27 (2H, t J=7.0Hz), 6.93 (1H, t J=7.5Hz),
183 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 NMR (CDCl3): 8.30 (s, 1H), 7.95 (d, J =7.6 Hz, 2H), 7.92 (s, 1H), 7.61 (t, J
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 = 8.3 Hz, 2H), 6.09 (dd, J = 8.7, 5.1 Hz, 1H), 5.80-5.63
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
1-Phenyl-2-(1H-1,2,4-triazol-1-yl)ethanone
(1.08
g)
in
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 NMR (CDCl3): 8.31 (s, 1H), 7.94 (d, J = 7.5 Hz, 2H), 7.91 (s, 1H), 7.56 (t, J
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): d 194.1, 159.2, 151.8, 143.7, 134.2 134.3, 134.2,
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
210
1H NMR (CDCl3): 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 =
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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 NMR (CDCl3): 8.42 (s, 1H), 7.99 (d, J = 7.1 Hz, 2H), 7.95 (s, 1H), 7.57 (t, J
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 NMR (CDCl3): 8.32 (s, 1H), 7.92 (d, J = 7.9 Hz, 2H), 7.89 (s, 1H), 7.53 (t, J
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 NMR (CDCl3): 8.31 (s, 1H), 7.92 (d, J = 7.2 Hz, 2H), 7.89 (s, 1H), 7.54 (t, J
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 NMR (CDCl3): 8.34 (s, 1H), 7.95 (d, J = 7.1 Hz, 2H), 7.95 (s, 1H), 7.57 (t, J
236 = 7.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.99 (t, J =
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 NMR (CDCl3): 8.38 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.92 (s, 1H), 7.56 (t, J
243 = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 6.91 (t, J =
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).
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246 4-(2-(3-trifluorophenoxy)ethoxy)-1-phenyl-2-(1H-1,2,4-triazol-1-yl)butan-1-
247 one (KK17)
248
1H NMR (CDCl3): 8.33 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J
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 NMR (CDCl3): 8.39 (s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J
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 NMR (CDCl3): 8.34 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.55 (t, J
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 NMR (CDCl3): 8.33 (s, 1H), 7.94 (d, J = 7.6 Hz, 2H), 7.92 (s, 1H), 7.56-
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).
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%
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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
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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
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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
MAX4 genes were significantly upregulated in KK5-treated plants. On the
365 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
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
Striga germination assay
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
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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
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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
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539 35. Kitahata, N.; Ito, S.; Kato, A.; Ueno, K.; Nakano, T.; Yoneyama, K.; Yoneyama, K.;
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548 51.
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
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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 from that of the control plants (Dunnett’s test, P < 0.05).
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 from that of the control plants (Dunnett’s test, P < 0.05).
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
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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
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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 from that of the control plants (Dunnett’s test, P < 0.05).
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
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609 condition for 7 days. The data are means ± SD (n = 50-60) * means statistically different
610 from that of the control plants (Dunnett’s test, P < 0.05).
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 from that of the control plants (Dunnett’s test, P < 0.05).
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
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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
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641
642
643
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650
651
652
653
654
655
656
657 TOC Graphic
658
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660
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N
N
N
N
O
(A)
N
N
Br
O
R1
(C)
R2
O
O
(B)
R2
R2
R1
HO
Br
O
R1
R2
H
H
H
H
H
H
TIS108
KK1
-CH2CH2CH2CH2-
-CH2CH2CH2CH2CH2-
KK2
-CH2CH2CH2CH2CH2CH2-
-CH2CH=CHCH2-
KK3
KK5
-CH2CH2OCH2CH2-
-CH(CH3)CH2CH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
-CH2CH2OCH2CH2-
KK6
KK12
KK13
KK14
KK15
KK16
KK17
KK18
KK19
KK20
2,6-dichloro
3-chloro
4-bromo
4-methoxy
2,6-dimethyl
3-trifluoro
2-fluoro
4-phenoxy
4-phenyl
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(A)
3.5
(B)
(C)
20
10 nM
30 nM
100 nM
30
25
20
15
10
5
18
16
14
12
10
8
3
2.5
2
1.5
1
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6
0.5
0
4
*
*
2
*
0
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Chemicals
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(B)
(A)
1 µM
3 µM
4
3.5
3
2.5
2
1.5
1
0.5
0
4.5
4
3.5
3
2.5
2
1.5
1
*
*
**
0.5
0
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 29
(A)
(B)
250
12
10
8
TIS108
KK5
TIS108
200
KK5
150
6
100
**
**
4
**
**
**
30
50
**
**
2
**
**
**
0
0
0
3
10
0
3
10
30
Concentration (nM)
Concentration (nM)
ACS Paragon Plus Environment

Page 27 of 29
Journal of Agricultural and Food Chemistry
4
3.5
3
**
2.5
2
1.5
1
0.5
0
0 0.1 1
3
KK5 conc. (µM)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 29
AtMAX3
AtMAX4
AtMAX2
8
6
4
2
0
3
1.5
1
**
5
**
5
2
1
0
0.5
0
0
0
0
5
KK5 conc. (µM)
KK5 conc. (µM)
KK5 conc. (µM)
ACS Paragon Plus Environment

Page 29 of 29
Journal of Agricultural and Food Chemistry
b
80
70
60
50
40
30
20
10
0
b
c
a
Water Mock KK5 KK5
+GR24
Root exudate
ACS Paragon Plus Environment