The Plant Journal (2016) 87, 245–257 doi: 10.1111/tpj.

13197

RESOURCE

Aminooxy-naphthylpropionic acid and its derivatives are

inhibitors of auxin biosynthesis targeting L-tryptophan
aminotransferase: structure–activity relationships
Megumi Narukawa-Nara1,†,‡, Ayako Nakamura1,†, Ko Kikuzato1, Yusuke Kakei1, Akiko Sato1, Yuka Mitani1, Yumiko
Yamasaki-Kokudo2, Takahiro Ishii2,§, Ken-ichiro Hayashi4, Tadao Asami5, Takehiko Ogura3, Shigeo Yoshida1,3,
Shozo Fujioka3,6, Takashi Kamakura7, Tsutomu Kawatsu8,9, Masanori Tachikawa8, Kazuo Soeno2 and Yukihisa Shimada1,3,*
1
Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa 244-0813, Japan,
2
Western Region Agricultral Reserch Center (WARC), National Agricultural Food Research Organization (NARO), Zentsuji,
Kagawa 765-8508, Japan,
3
RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan,
4
Department of Biochemistry, Okayama University of Science, Okayama, Okayama 700-0005, Japan,
5
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan,
6
RIKEN, Advanced Science Institute, Wako, Saitama 351-0198, Japan,
7
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba
278-8510, Japan,
8
Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa 236-0027,
Japan, and
9
Institute for Molecular Science, National Institutes of Natural Science, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan

Received 11 December 2015; revised 12 April 2016; accepted 15 April 2016; published online 18 July 2016.
*For correspondence (e-mail yshimada@yokohama-cu.ac.jp).
†
These authors contributed equally to this work.
‡
Present address: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
§
Present address: Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.

SUMMARY
We previously reported L-a-aminooxy-phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis,
but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN
AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from
AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy
groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed
that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These
active compounds reduced the endogenous indole-3-acetic acid (IAA) content upon application to Arabidop-
sis seedlings. Among the compounds, we selected 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid
(KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169
showed typical auxin-deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo
experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine
ammonia-lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we
added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased
the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum
reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly
developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.

Keywords: auxin, auxin biosynthesis inhibitor, L-a-aminooxy-phenylpropionic acid, structure–activity

relationships, Arabidopsis thaliana.

© 2016 The Authors 245

The Plant Journal © 2016 John Wiley & Sons Ltd
246 Megumi Narukawa-Nara et al.
INTRODUCTION
The phytohormone auxin has a broad range of physiologi-
cal effects on almost every aspect of plant growth and
development (Davies, 2004). Indole-3-acetic acid (IAA) is
the most abundant auxin in higher plants. It is synthesized
through pathways that depend on L-tryptophan (Trp) and
others that do not (Wright et al., 1991; Normanly et al.,
1993; Zhao, 2014). However, less is known about the Trp-
independent pathways. Trp-dependent IAA biosynthetic
pathways have been investigated intensively, and several
have been proposed, for example, the indole-3-pyruvic
acid (IPyA) pathway, the indole-3-acetoamide (IAM) path-
way, the tryptamine (TAM) pathway and the indole-3-acet-
aldoxime (IAOx) pathway (Fig. S1 in the Supporting
Information; Zhao, 2014). In the IPyA pathway, TRYPTO-
PHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1)
and TRYPTOPHAN AMINOTRANSFERASE RELATEDs
(TARs) catalyze the production of IPyA from Trp (Stepa-
nova et al., 2008; Tao et al., 2008) and YUCCAs (YUCs) cat-
alyze the conversion of IPyA into IAA (Mashiguchi et al.,
2011). Mutation of multiple TAA1/TARs or YUC genes
results in a serious IAA-deficient phenotype that exhibits,
for example, gravity defects, shorter primary roots and
hypocotyls, defective vascular development and abnormal
flower development (Cheng et al., 2006, 2007; Stepanova
et al., 2008; Tao et al., 2008). These findings led to the pro-
posal that the IPyA pathway constitutes one of the main
pathways for IAA biosynthesis in Arabidopsis (Mashiguchi
et al., 2011; Won et al., 2011). However, these proposals
have not been sufficiently supported by alternative
approaches, and the importance of the IPyA pathway
remains unclear.
Because auxin biosynthesis is mediated by enzymes
encoded by multiple auxin biosynthetic genes, no single
mutation has a marked effect on auxin biosynthesis and
plant development in Arabidopsis. Although auxin is
essential for plant growth and development, multiple
mutations of genes involved in IAA biosynthesis may lead
to lethality, poor growth and male sterility (reviewed in
Zhao, 2014). Thus, it is difficult to evaluate auxin functions
and IAA biosynthesis pathways using genetic approaches.
A chemical genetic approach using small molecular tools
represents a useful alternative method (Dejoughe and
Russinova, 2014; Rigal et al., 2014). Chemical treatment
can be performed with any tissue and at any time in the
lifecycle using appropriate concentrations of chemical
agents. Several auxin inhibitors have been reported to
date, some of which are competitive inhibitors of IAA, for
example p-chloro-phenoxy-isobutyric acid (PCIB), and
some of which are transport inhibitors, for example 2,3,5-
triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic
acid (NPA) (De Rybel et al., 2009; Rigal et al., 2014). How-
ever, when applied to wild-type (WT) Arabidopsis, these
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
classic auxin inhibitors tend to exhibit insufficient activity
or specificity; that is, they do not repress auxin-inducible
gene expression (Soeno et al., 2010).
Because of the complexity of the proposed auxin biosyn-
thesis pathways, the application of any single compound is
unlikely to block auxin biosynthesis in plants. Therefore,
few studies have been conducted on inhibitors of IAA
biosynthesis. The first auxin biosynthesis inhibitor was dis-
covered through analyses of large-scale transcriptome data
(Soeno et al., 2010). L-a-(2-aminoethoxyvinyl)glycine (AVG)
and L-a-aminooxyphenylpropionic acid (AOPP) may inhibit
IAA biosynthesis by targeting Trp aminotransferase. AOPP
decreases the levels of IAA in seedlings of several plant
species (Soeno et al., 2010), and seedlings treated with
AOPP or its derivatives show an auxin-deficient phenotype
in Arabidopsis (Soeno et al., 2010) and tomato (Higashide
et al., 2014). Another small molecule, L-kynurenine (Kyn), is
an alternative substrate and competitive inhibitor of TAA1
(He et al., 2011). Other compounds are also reported to tar-
get YUCs (Nishimura et al., 2014; Kakei et al., 2015) or an
unknown factor (Ili
c et al., 1999; Ludwig-Mu
€ ller et al., 2010;
Ishida et al., 2014). Among them, the boronic acid inhibitors
4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic
acid (PPBo) are effective inhibitors of auxin biosynthesis in
both eudicot and monocot plant species (Kakei et al., 2015).
We previously showed that AOPP and AVG inhibited
biosynthesis of IAA, but the precise molecular target
remained elusive (Soeno et al., 2010). Because these com-
pounds blocked Trp aminotransferase activity in Arabidop-
sis and wheat extracts, we expected that TAA1 would be
the primary target protein of AOPP. AOPP is not an ideal
inhibitor because it is unstable in plant growth medium
[e.g. Murashige–Skoog (MS) medium] (Soeno et al., 2010),
and is an inhibitor of phenylalanine ammonia-lyase (PAL)
(Amrhein et al., 1976; Amrhein and Godeke, 1977; Amrhein
and Hollander, 1979).
In this study, we designed and synthesized novel AOPP
derivatives and investigated their structure–activity rela-
tionships in terms of inhibition of auxin biosynthesis
in vitro and in vivo. We also investigated one representa-
tive of these compounds with regard to whether its activity
was specific to auxin and other pathways, both in vitro and
in vivo. We also tested AOPP derivatives to which protec-
tion groups had been introduced to obtain compounds
with greater inhibitory activity.
RESULTS
Structural requirements for TAA1 inhibitors
To investigate the molecular target of AOPP, we performed
an in vitro enzymatic activity assay using recombinant
TAA1 produced in Escherichia coli. AOPP inhibited TAA1
© 2016 The Authors
 Auxin biosynthesis inhibitors targeting TAA1 247

in vitro (Fig. 1b). Then we synthesized AOPP derivatives (a)

(Fig. 1a) and examined their inhibitory activity in order to
identify which functional groups of AOPP are critical for
this inhibition. To evaluate the contribution of the ami-
nooxy group to this inhibitory activity we examined the
inhibitory activity of L-phenylalanine (Phe) and 2-naphthy-
lalanine (2-Nal). Phe and 2-Nal did not show inhibitory
activity against TAA1 (Fig. 1b). These results suggest that
the aminooxy group is necessary for the inhibition of
TAA1 in vitro. We also tested KOK1169 [2-(aminooxy)-3-
(naphthalen-2-yl) propanoic acid, or AONP] in which the
phenyl group of AOPP was substituted for a naphthyl
group, to investigate the importance of the phenyl group
of AOPP. KOK1169 showed stronger inhibition of TAA1
than AOPP. Thus, substitution of the phenyl group of
AOPP does not decrease its activity. Because KOK1169
exhibited marked inhibition of TAA1, we synthesized
KOK3048 and KOK3094, in which the carboxyl group of
KOK1169 was substituted for a hydrogen atom or a hydro-
xyl group, respectively, to investigate the importance of
the carboxy groups of AOPP derivatives in TAA1 inhibition. (b) 0.4
Both exhibited little or no inhibition of the recombinant
TAA1 (Fig. 1b). These results suggest that the carboxy
0.3
group of AOPP is essential for inhibition of TAA1 in vitro.
We also synthesized KOK1154 by adding a methyl group at
A330

the b position of AOPP and investigated its inhibitory activ- 0.2 **

ity. It did not exhibit inhibition (Fig. 1b). This suggests that
a methyl group at the b position of AOPP may disrupt its
binding to TAA1.
Structure–activity relationships of AOPP derivatives for
inhibition of Trp aminotransferase in vitro, and auxin
biosynthesis in vivo
Based on the above results, we synthesized AOPP deriva-
tives in which the phenyl group was replaced with various
substituents to identify more effective TAA1 inhibitors.
Because KOK1169 inhibits TAA1 activity, we replaced the
R1 moiety of AOPP with a larger substituent than the origi-
nal phenyl group. This resulted in synthesis of 14 novel
compounds (designated Type I compounds; Fig. S2).
First, we performed an in vitro enzymatic inhibition
assay to identify compounds with strong inhibitory activity
against recombinant TAA1. Because the result of this assay
varied depending on the TAA1 preparation used as well as
the AOPP lot number (AOPP being a relatively unstable
compound), the two series of results are presented sepa-
rately in two graphs (Fig. 2a, b). Among the compounds,
KOK1167, KOK1169, KOK1176, KOK1180, KOK1187,
KOK2027, KOK2118, KOK2122, KOK2155 and KOK2173
showed stronger inhibitory activity against TAA1 than did
AOPP (Fig. 2a). KOK2031 and KOK2036, in which the phe-
nyl groups were substituted by phenoxyphenyl and quino-
lyl groups, respectively, and KOK2090, para-brominated
AOPP, showed weaker inhibitory activity against TAA1
0.1 **
0
Figure 1. Inhibitory activities of L-a-aminooxy-phenylpropionic acid (AOPP)
and its derivatives against TRYPTOPHAN AMINOTRANSFERASE of ARABI-
DOPSIS 1 (TAA1).
(a) Chemical structures of the tested compounds. The substituted groups
are indicated by circles. 2-Nal, 2-naphthylalanine; Phe, L-phenylalanine.
(b) The inhibitory activity of the compounds against recombinant TAA1.
Indole-3-pyruvic acid (IPyA)–borate complex production was assessed by
monitoring absorption at 330 nm. Data represent the mean  SE (n = 3) of
three independent experiments. Statistically significant differences relative
to the control sample are indicated by asterisks (Student’s t-test; *P < 0.05;
**P < 0.01). 2-Nal, 2-naphthylalanine.
than did AOPP (Fig. 2a). KOK2111, in which the phenyl
group of AOPP was substituted for an N-phenylpiperazine
group, showed negligible activity against TAA1 (Fig. 2a).
Second, because AOPP also inhibits PAL, we performed
AtPAL2 inhibition assays to identify undesirable inhibitory
activities. We found that all compounds with a two-ring
structure (i.e. KOK1167, KOK1169, KOK2027 and KOK2173)
did not inhibit, or only very weakly inhibited, AtPAL2 activ-
ity (Fig. 2b). In contrast, compounds with a one-ring struc-
ture (i.e. KOK1176, KOK1180, KOK1187, KOK2090,

© 2016 The Authors

The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
248 Megumi Narukawa-Nara et al.

(a) A330 (b) A290 Figure 2. Effects of the compounds on TRYPTO-

PHAN AMINOTRANSFERASE of ARABIDOPSIS 1
0 0.1 0.2 0.3 0 0.1 0.2
(TAA1) and Arabidopsis phenylalanine ammonia-
Mock Mock lyase 2 (AtPAL2) activity in vitro and endogenous
AOPP ** AOPP ** IAA level in vivo.
(a) Inhibition of recombinant TAA1 activity in vitro.
KOK2111 The test compound (1 lM) was added to the enzyme
KOK2111
KOK2031 reaction mixture, and the production of the indole-
KOK2036 ** KOK2027 3-pyruvic acid (IPyA)–borate complex was assessed
KOK2031 ** KOK2036 by monitoring absorption at 330 nm.
KOK2090 ** KOK1169 (b) Inhibition of recombinant AtPAL2 activity
KOK1180 ** KOK1154
in vitro. The test compound (15 nM) was added to
KOK1176 ** KOK1167
the enzyme reaction mixture, and the amount of t-
KOK1187 ** * cinnamic acid was determined by monitoring
KOK2090 * absorption at 290 nm.
KOK1169 ** KOK2118 ** (c) The endogenous IAA level of 7-day-old Ara-
KOK1167 ** KOK1176 ** bidopsis seedlings treated with 30 lM of the test
KOK2027 ** KOK1180 ** compounds for 3 h. Values are relative to those of
KOK2118 ** KOK1187 ** mock-treated plants (5.9 pg/mg fresh weight) and
0 0.1 0.2 are the mean  SE of at least three independent
biological replicates. Statistically significant differ-
Mock ences relative to the control sample are indicated
** AOPP ** by asterisks (Student’s t-test; *P < 0.05; **P < 0.01).

** KOK2173
** KOK2155 **
** KOK2122 **

(c) Relative IAA level

0 0.2 0.4 0.6 0.8 1
Mock
AOPP **
KOK2031 *
KOK2111 **
KOK2027 **
KOK2036 **
KOK1176 *
KOK1169 **
KOK2173 **
KOK2122 **
KOK2090 **
KOK2118 **
KOK1180 **
KOK1167 **
KOK1187 **
KOK2155 **

KOK2118, KOK2122 and KOK2155) exhibited stronger inhi-
bitory activity against AtPAL2 (Fig. 2b). Compounds with a
modified phenyl group at the R1 position did not exhibit
significantly reduced AtPAL2 inhibitory activity, even if a
halogen atom or methyl group was introduced to the phe-
nyl ring (Figs 2b and S2).
Third, to evaluate the auxin biosynthesis inhibitory activ-
ity of the compounds in vivo, we measured the endoge-
nous IAA content in 7-day-old Arabidopsis seedlings
treated with each compound at 30 lM for 3 h. As shown in
Fig. 2(c), all of the newly synthesized compounds
decreased the endogenous IAA level compared with the
mock treatment. Compounds with a phenyl group at the R1
moiety, such as KOK2155 and KOK1187, showed strong
inhibitory activities. However, because these compounds
were also strong inhibitors of PAL (Figs 2b) they were
excluded from subsequent analysis.
Based on these observations, we concluded that
KOK1167, KOK1169, KOK2027 and KOK2173 have potential
as auxin biosynthesis inhibitors targeted at TAA1, because
they efficiently inhibited TAA1 activity, had low inhibitory
activity against AtPAL2 and reduced the endogenous auxin
level in Arabidopsis. Among these, the activity of KOK1167
was unexpected. Arabidopsis seedlings grown in its pres-
ence showed a typical auxin-overproducing phenotype with
more (an increase in number but not in size) lateral roots
and root hairs (Fig. S3). Thus we excluded it from the candi-
date auxin biosynthesis inhibitors. KOK1167 is an analog of
NAA, which is a typical synthetic auxin. It is unclear whether
KOK1167 itself has auxin activity or is metabolized to 1-NAA.

© 2016 The Authors

The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257

Molecular-docking studies of AOPP derivatives as TAA1
inhibitors
Next, we estimated the binding energy between TAA1 and
the newly synthesized compounds, and then analyzed the
correlation between the binding energy and inhibitor activ-
ity. The docking software AUTODOCK 4.2 (Morris et al., 2009)
was used to generate docking models of the synthesized
compounds with TAA1 (Table S1). Two hundred docking
models were generated and then we used PEARLS software
(Han et al., 2006) to predict the compound–target interac-
tion energies, the total compound–target interaction
energy, van der Waals interactions, electrostatic interac-
tions, hydrogen bonding, the solvation free energy and the
conformational entropy (kcal mol 1). Among the 200 mod-
els, the lowest values of each parameter corresponding to
the strongest docking results are shown in Table S1. In
vitro enzyme inhibition activities for TAA1 are also shown
in Table S1. When TAA1 inhibitory activity and binding
energy were compared, a correlation with total ligand–re-
ceptor interaction energy was found [Pearson’s correlation
coefficient (PCC) = 0.61]. Next, we investigated the multiple
factors that constitute the total interaction energy. Among
them, ligand–receptor conformational entropy and ligand–
receptor hydrogen bond energy were highly correlated
(PCC = 0.83 and 0.79, respectively). Although the confor-
mational entropy showed the highest PCC value with inhi-
bitory activity, effective inhibitors cannot be distinguished
from ineffective inhibitors using this value. In contrast, a
hydrogen bond energy of less than 2.3 is indicative of
strong inhibition of TAA1 activity (>50% inhibition at
30 lM).
In vitro enzyme kinetic studies on TAA1, AtPAL2 and
AtACS8, and in vivo inhibition of anthocyanin and
ethylene biosynthesis
Based on the above results, we selected KOK1169 as a pos-
sible specific inhibitor of IAA biosynthesis, and further
investigated its activities in detail. To examine the mecha-
nism by which KOK1169 inhibits TAA1, we first conducted
an enzyme kinetic assay. Recombinant enzyme activity was
analyzed in the presence of various concentrations of the
inhibitor and Trp. The results of a Dixon plot analysis
(Dixon, 1953) suggested that KOK1169 inhibits TAA1 in a
competitive manner (Fig. 3).
Because aminooxy compounds have inhibitory activity
against ammonia lyases such as PAL as well as against
pyridoxal phosphate (PLP)-dependent enzymes such as
1-aminocyclopropane-1-carboxylic acid synthase (ACS)
(Captini et al., 2003), we determined the inhibition constant
(Ki) values of KOK1169, AOPP and AVG against recombi-
nant TAA1, AtPAL2 and AtACS8 (Figs 3 and S4). The
results are summarized in Table 1. Of the three com-
pounds, KOK1169 had the greatest affinity for TAA1. The Ki
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
Auxin biosynthesis inhibitors targeting TAA1 249
300
250
1/(A330/min)
200
150
100
50 µM Trp
50 100 µM Trp
150 µM Trp
0
[KOK1169], (nM)
Figure 3. Dixon plot of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid
(KOK1169) based on analysis of TRYPTOPHAN AMINOTRANSFERASE of
ARABIDOPSIS 1 (TAA1) kinetics.
Inhibition of recombinant TAA1 activity was analyzed in vitro. The enzyme
reaction mixture contained the inhibitors at the indicated concentrations.
L-tryptophan (Trp) was used at concentrations of 50, 100 and 150 lM. The Ki
values were determined from Dixon plots. Values are the mean  SD of
three independent experiments.
value for inhibition of TAA1 by KOK1169 was 76.8 nM and
was considerably lower than the Ki value (11.52 lM) for
inhibition by Kyn (He et al., 2011). Moreover, KOK1169 had
a markedly lower affinity for AtPAL2 than AOPP (Table 1).
In addition, KOK1169 showed considerably lower affinity
for AtACS8 than AVG, and the highest inhibitory activity
(smallest Ki value) against TAA1 of the three enzymes. On
the other hand, AOPP had stronger affinity for AtPAL2, and
AVG had a stronger affinity for AtACS8. These results sug-
gest that KOK1169 is an effective and specific inhibitor of
TAA1 in vitro.
Next, we investigated the in vivo activities of KOK1169
in Arabidopsis seedlings. Because AOPP disturbs antho-
cyanin biosynthesis by blocking PAL activity, we measured
the anthocyanin content in Arabidopsis seedlings in the
presence of the inhibitors to evaluate the inhibition of PAL
activity in vivo (Noe et al., 1980; MacDonald and D’Cunha,
2007). a-(Phenylethyl-2-one)-indole 3-acetic acid (PEO-IAA)
is a competitive inhibitor of IAA (Hayashi et al., 2012). We
used PEO-IAA as a comparison for inhibition of auxin sig-
naling. Sucrose treatment induces PAL activity, and thus
accumulation of anthocyanin, in Arabidopsis (Solfanelli
et al., 2006). Therefore, Arabidopsis seedlings were cul-
tured in medium containing 10% sucrose in the presence
250 Megumi Narukawa-Nara et al.
Table 1 Ki values determined by Dixon plot analysis
AOPP (nM) KOK1169 (nM) AVG (nM)
TAA1 350.4  21.5 76.8  1.5 8600  100
AtPAL2 3.9  0.3 245.8  5.7 N.D.
AtACS8 848.0  3.0 244.5  1.3 37.6  0.6
AOPP, L-a-aminooxy-phenylpropionic acid; KOK1169, 2-(aminooxy)-
3-(naphthalen-2-yl)propanoic acid; AVG, L-a-(2-aminoethoxyvinyl)
glycine; TAA1, TRYPTOPHAN AMINOTRANSFERASE of ARABIDOP-
SIS 1; AtPAL2, Arabidopsis phenylalanine ammonia-lyase 2;
AtACS8, Arabidopsis 1-aminocyclopropane-1-carboxylic acid syn-
thase 8; N.D., inhibition was not detected.
Values are the mean  SD of three independent experiments.
or absence of inhibitors. AOPP inhibited the accumulation
of anthocyanin in a dose-dependent manner, whereas nei-
ther KOK1169 nor PEO-IAA inhibited the accumulation of
anthocyanin in the Arabidopsis seedlings (Fig. S5a). This
indicates that KOK1169 does not inhibit anthocyanin syn-
thesis in vivo.
To evaluate the inhibition of ACS by KOK1169 in vivo,
we assayed ethylene generation in Arabidopsis seedlings.
ACS catalyzes the conversion of S-adenosylmethionine
(AdoMet) into 1-aminocyclopropane-1-carboxylic acid
(ACC); this conversion is generally the rate-limiting step in
ethylene biosynthesis in plants (Yang and Hoffman, 1984;
Kende, 1993; Tsuchisaka et al., 2009). We used AVG as a
typical ACS inhibitor for comparison. Because auxin treat-
ment induces ethylene biosynthesis, Arabidopsis seedlings
were grown in the presence of excess IAA (100 lM) in the
presence or absence of AVG, PEO-IAA, AOPP and
KOK1169. This experimental system also has the advan-
tage of canceling the effect of endogenous auxin for ethy-
lene biosynthesis, because inhibitors of auxin biosynthesis
decrease the endogenous IAA content and the endogenous
auxin level affects ethylene biosynthesis (Woeste et al.,
1999; Swarup et al., 2007). KOK1169, AOPP and PEO-IAA
showed markedly weaker inhibition of ethylene biosynthe-
sis than AVG (Fig. S5b). These data suggest that KOK1169
has little inhibitory activity against ethylene synthesis
in vivo. Based on in vivo assays and the Ki values of the
inhibitors in vitro, we concluded that KOK1169 has no
undesirable inhibitory activity against anthocyanin or ethy-
lene biosynthesis.
Phenotypic analysis of seedlings grown in the presence of
KOK1169
To clarify the mechanism underlying the effect of KOK1169
on plant growth and development, Arabidopsis seedlings
were grown vertically on half-strength MS medium supple-
mented with 1–100 lM KOK1169 or AOPP for 8 days. The
IAA content in seedlings decreased as the concentration of
inhibitors increased (Fig. S6). KOK1169 resulted in a
greater reduction in the level of endogenous IAA than
AOPP. Next, we compared the phenotypes of seedlings
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
treated with KOK1169 with those treated with PEO-IAA and
AOPP. Seedlings treated with PEO-IAA had multiple abnor-
mal phenotypes similar to auxin-defective mutants (Haya-
shi et al., 2012), namely, impaired root gravitropism, a
shorter main root, reduced lateral roots, reduced root hairs
and growth inhibition in the aerial part. The phenotype of
seedlings treated with 100 lM KOK1169 was similar to that
of seedlings treated with 30 lM PEO-IAA, and the pheno-
type with KOK1169 was partially reversed in the presence
of 10 nM IAA (Fig. 4a) while the phenotype with PEO-IAA
was not. In contrast, change in phenotype was not evident
when seedlings were grown in the presence of 100 lM
AOPP (Fig. 4a). The difference in phenotype severity was
clear between the seedlings treated with KOK1169 and
those treated with AOPP. KOK1169, AOPP and PEO-IAA
shortened the primary root length of seedlings in a dose-
dependent manner (Fig. 4b). AOPP was the least-effective
inhibitor of primary root elongation. To confirm that the
defective primary root elongation was caused by the
reduced content of endogenous IAA, we grew the seed-
lings in the presence of the inhibitors in combination with
IAA. We found that IAA at 1 or 10 nM significantly restored
the primary root growth inhibited by 100 lM KOK1169 and
AOPP, whereas even 100 nM IAA was not sufficient to
reverse the inhibition of root growth by 30 lM PEO-IAA
(Fig. 4e). We also investigated the effects of KOK1169 on
lateral root formation and shoot development. Both were
inhibited by KOK1169 in a dose-dependent manner
(Fig. 4c, d), and this inhibition was overcome by the addi-
tion of IAA (Fig. 4f, g).
Next, we investigated the sensivity of wei8-1 (a TAA1-
knockout mutant) (Stepanova et al., 2008) to KOK1169.
Five-day-old seedlings of the WT or wei8-1 mutant were
grown in the presence of 1–100 lM KOK1169 for 4 days,
and the length of primary roots was analyzed. The wei8-1
mutants were more sensitive to KOK1169 than the WT
plants (Fig. S7a, b). This finding suggests that KOK1169
has inhibitory activity against TAA1 and TARs. Further-
more, 8-day-old WT seedlings treated with 300 lM
KOK1169 mimicked the morphology of wei8-1/tar2-1 dou-
ble mutants (a knockout mutant of TAA1 and its homolo-
gous gene TAR2; Stepanova et al., 2008), which exhibit
upwardly curved cotyledons and defective shoot and root
development (Fig. S7c). These results suggest that TAA1/
TARs are the primary targets of KOK1169.
Effect of KOK1169 on the expression of Aux/IAA genes
We previously demonstrated that the expression of auxin-
responsive genes was downregulated as a result of auxin
deficiency caused by treatment with an inhibitor of auxin
biosynthesis (Soeno et al., 2010; Ishida et al., 2013). Thus,
to investigate the effects of inhibitors on auxin signaling,
we analyzed expression levels of the auxin-responsive
Aux/IAA1 and Aux/IAA5 genes in response to treatment
© 2016 The Authors
 Auxin biosynthesis inhibitors targeting TAA1 251

(a)

(b) (c) (d)

(e) (f) (g)

Figure 4. Effects of inhibitors on growth of Arabidopsis seedlings, and recovery by exogenous indole-3-acetic acid (IAA).
(a) Gross morphology of 8-day-old seedlings grown on half-strength MS medium containing 30 lM a-(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA), 100 lM
2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169) or 100 lM L-a-aminooxy-phenylpropionic acid (AOPP), with or without 10 nM IAA. Scale bar = 1 cm.
The length of the primary root (b), the number of lateral roots (c), and the weight of shoots (d) of 8-day-old seedlings grown on a medium containing the inhibi-
tors at various concentrations. The recovery of each plant feature is shown in panels (e–g), respectively. Plants were grown on a medium containing 100 lM
KOK1169, 100 lM AOPP or 30 lM PEO-IAA (e), 1 lM KOK1169 (f) and 30 lM KOK1169 (g) with the indicated concentrations of IAA for 8 days. Values are the
mean  SE (n = 10) of one of three independent experiments, which yielded similar results. Statistically significant differences relative to the control sample are
indicated by asterisks (Student’s t-test; *P < 0.05; **P < 0.01).

with KOK1169, AOPP and PEO-IAA. We grew WT Arabidop- 100 lM) for 3 h. Then the expression levels of Aux/IAA
sis seedlings on half-strength MS medium for 8 days and genes were determined by quantitative real-time PCR
then incubated them in the presence of inhibitors (1– (qRT-PCR). The expression levels of Aux/IAA1 and Aux/
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
252 Megumi Narukawa-Nara et al.

IAA5 were downregulated after treatment with inhibitors
for 3 h (Fig. 5a). Next, we investigated whether the inhibi-
tion of gene expression caused by inhibitor treatment
would be restored in the presence of 10–1000 nM IAA. The
inhibition of gene expression caused by treatment
with KOK1169 was canceled in the presence of 10 nM IAA,
whereas that by PEO-IAA was not. Inhibition by
PEO-IAA was only canceled in the presence of 1 lM IAA
(Fig. 5b).
Chemical knockdown of auxin biosynthesis and analysis
of auxin biosynthesis intermediates
To further investigate the mode of action of KOK1169, we
analyzed intermediates of IAA biosynthesis in Arabidopsis
seedlings given labeled [15N2]-Trp in the presence or
absence of KOK1169. Trp, IPyA and TAM were analyzed as
major intermediates of IAA biosynthesis pathways. We
incubated 8-day-old seedlings in deionized water contain-
ing 30 lM [15N2]-Trp with or without 30 lM KOK1169 for
3 h, and then analyzed the relative amounts of IAA and its
precursors by ultra-performance liquid chromatography-
tandem mass spectroscopy (UPLC-MS/MS). Seedlings
treated with [15N2]-Trp accumulated [15N2]-Trp, [15N2]-TAM,
[15N]-IPyA and [15N]-IAA (Fig. 6, Table S5). Seedlings trea-
ted with both [15N2]-Trp and KOK1169 accumulated lower
levels of [15N]-IPyA and [15N]-IAA than those treated with
[15N2]-Trp alone (Fig. 6). In contrast, seedlings treated with
both [15N2]-Trp and 30 lM KOK1169 accumulated similar
levels of [15N2]-Trp and [15N2]-TAM to those treated with
[15N2]-Trp alone (Fig. 6). These findings suggest that
KOK1169 acts on the IPyA pathway of auxin biosynthesis.
Derivatives of Type I compounds have increased inhibitory
activity and reduce IAA levels in vivo
Bioactive inhibitors should be stable and have moderate
hydrophobicity, which is important for membrane perme-
ability. To investigate the relationship between the
hydrophobicity of the inhibitors and their auxin inhibition
in vivo, we modified the 14 AOPP derivatives (Type I com-
pounds). To increase hydrophobicity, we introduced alkyl
groups to the Type I compounds at the R2 moiety (Fig. S8).
We designated these Type II compounds (Fig. S8). The cal-
culated partition coefficient (log P) values of all Type II
compounds were higher than those of the original Type I
compounds (Table S2). When 7-day-old Arabidopsis seed-
lings were treated with Type II compounds at 30 lM for
3 h, 11 of the 15 Type II compounds showed increased
inhibition of the endogenous auxin level compared with
the original Type I compounds (Fig. S10).
Higashide et al. (2014) recently showed that KOK1101,
which is an AOPP derivative and has both a methylated
carboxyl group and a phthaloyl group instead of the ami-
nooxy group, effectively inhibited the accumulation of IAA
in tomato and Arabidopsis seedlings. Based this finding,

(a) (b)
Aux/IAA5
Aux/IAA5

*
*
Relative expression

*
Relative expression

** ** * **
****
Aux/IAA1
Aux/IAA1

*
* * **
**
*
**
** **
**** * **

Figure 5. Effect of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169) on the expression of auxin-responsive genes.

(a) The expression of the auxin-inducible genes Aux/IAA1 and Aux/IAA5 was determined by quantitative real-time PCR (qRT-PCR). Eight-day-old seedlings were
treated with the indicated inhibitors for 3 h. The expression levels of the mock-treated seedlings were used to normalize the data. Glyceraldehyde-3-P dehydro-
genase C subunit 1 (GAPDH) was used as the internal control and data are presented as mean  SE of three independent experiments.
(b) Aux/IAA1 and Aux/IAA5 expression was determined by qRT-PCR. Eight-day-old seedlings were treated with the inhibitors for 2 h and then with 10–1000 nM
indole-3-acetic acid (IAA) for a further 1 h. The expression levels of the mock-treated seedlings were used to normalize the data. GAPDH was used as the internal
control and data are presented as mean  SE of three independent experiments. Statistically significant differences relative to the control sample are indicated
by asterisks (Student’s t-test; *P < 0.05; **P < 0.01).

© 2016 The Authors

The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257

Figure 6. Effects of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid
(KOK1169) on the levels of endogenous intermediates and uptake of 15N-
labels from [15N2]-L-tryptophan (Trp) in auxin biosynthesis.
Eight-day-old seedlings were treated with 30 lM [15N2]-Trp in the presence
or absence of 30 lM KOK1169 for 3 h. Then the relative levels of endoge-
nous indole-3-acetic acid (IAA) and intermediates [tryptamine (TAM) and
indole-3-pyruvic acid (IPyA)] were analyzed by ultra-performance liquid
chromatography-tandem mass spectroscopy. The actual amounts of Trp,
TAM and IAA are shown in Table S5. Data are mean  SE of three indepen-
dent experiments, each using 15 seedlings. Statistically significant differ-
ences relative to the control sample are indicated by asterisks (Student’s t-
test; *P < 0.05; **P < 0.01).
we further modified Type II compounds by adding a pro-
tection group of the aminooxy moiety; these were classi-
fied as Type III compounds (Fig. S9). The log P values of
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
Auxin biosynthesis inhibitors targeting TAA1 253
all Type III compounds, with the exception of KOK2015,
were higher than those of the original Type II compounds
(Table S2). Most of the Type III compounds exhibited
stronger inhibition of endogenous IAA accumulation than
the corresponding Type I compounds (Fig. S10). In the
most striking case, the endogenous IAA level was
decreased by about 90% 3 h after treatment (Fig. S10).
DISCUSSION
Structure–activity relationship between TAA1 and its
inhibitors
To identify the molecular target of AOPP, we performed an
in vitro TAA1 activity assay using recombinant protein pro-
duced in E. coli. We found that AOPP inhibited the activity
of recombinant TAA1 in vitro (Fig. 1b). To evaluate the
structure–activity relationship between TAA1 and its inhibi-
tors, we investigated the inhibitory activity of AOPP deriva-
tives against TAA1 in vitro (Fig. 1). The results supported
the hypothesis that the aminooxy and carboxy moieties
are essential for the inhibitory activity of the compounds.
Analysis of 14 derivatives of AOPP (Fig. S2) suggested that
the inhibitory activities against TAA1 and AtPAL2 differ
markedly among the Type I compounds (Fig. 2). KOK1169
and KOK2173, in which the phenyl moiety of AOPP was
substituted for a naphthyl group and its derivative, respec-
tively, showed strong inhibitory activity against TAA1
(Fig. 2). KOK2027, in which the phenyl moiety was substi-
tuted for a biphenyl group, was also a good inhibitor of
TAA1. This suggests that the naphthyl and biphenyl
groups at the R1 moiety increase binding to TAA1 (Fig. 2a,
Table S1), and thus their inhibitory activity. In contrast,
KOK2036 and KOK2111 were weaker inhibitors of TAA1
than AOPP (Fig. 2a). This indicates that compounds with a
nitrogen-containing six-member ring at the R1 moiety are
less effective inhibitors of TAA1 than AOPP. Although halo-
genated AOPP derivatives showed strong inhibitory activ-
ity against TAA1 (Fig. 2a), they also showed marked
inhibition of AtPAL2 (Fig. 2b). These results also suggest
that the structure of the R1 substituent is an important
determinant of the specificity of inhibitors of TAA1 and
AtPAL2. Endogenous IAA analysis suggested that all Type I
compounds act as inhibitors of auxin biosynthesis
(Fig. 2c).
Comparison of in silico, in vitro and in vivo activities
In the molecular docking simulation analysis, the total
ligand–receptor interaction energy was correlated with the
actual inhibition of TAA1 (Table S1). Among the factors
constituting the total energy, only the hydrogen bond
energy and conformational entropy showed high PCC val-
ues, suggesting that the other factors are less important
for binding. Therefore, we conclude that the total interac-
tion energy and the hydrogen bond energy can be used to
254 Megumi Narukawa-Nara et al.
design efficient inhibitors of TAA1, because if these energy
values are lower than appropriate thresholds ( 6.9 for the
total interaction energy and 2.3 for the hydrogen bond
energy), ligands are likely to have an inhibitory effect on
TAA1 (e.g. 50% inhibition at 30 lM). Thus screening using
in silico docking simulation is a cost-effective approach for
designing and testing novel structures. In contrast to the
good correlation between in silico binding affinity and
in vitro TAA1 inhibition activity, the inhibition of IAA
biosynthesis was not strongly correlated with the inhibi-
tory activity against TAA1 (compare panels a and c in
Fig. 2). The in vitro activity of small-molecule inhibitors is
frequently not congruent with their in vivo effects (Lipinski,
2003). Such disparity between in vivo and in vitro findings
can be caused by several factors, and chemical stability
and membrane permeability are major determinants. This
is discussed below.
KOK1169 is an improved inhibitor of auxin biosynthesis
We further characterized the activities of KOK1169 (AONP)
in vitro and in vivo. Consistent with the results of in vitro
analyses, KOK1169 resulted in a greater decrease in the
endogenous IAA level than AOPP did (Fig. S6). Depending
on the target enzyme, aminooxy compounds display prop-
erties of reversible competitive or irreversible inhibitors by
forming a stable oxime with the PLP cofactor (Eliot and
Kirsch, 2004). Dixon plots suggested that KOK1169 inhibits
TAA1 in a competitive manner (Fig. 3). In vitro analyses
using recombinant TAA1, AtPAL2 and AtACS8 suggested
that KOK1169 showed higher affinity for TAA1, and consid-
erably lower affinity for AtPAL2 and AtACS8, compared
with AOPP and AVG, respectively (Table 1). The off-target
effects of KOK1169 in vivo, including inhibition of antho-
cyanin and ethylene synthesis, were also modest (Fig. S5).
Therefore, in vitro and in vivo experiments indicated that
KOK1169 is more specific for TAA1.
We analyzed intermediates of IAA biosynthesis in Ara-
bidopsis seedlings given labeled [15N2]-Trp in the pres-
ence or absence of KOK1169. Because IAM and IAOx are
present at lower levels than IAA in Arabidopsis (Sugawara
et al., 2009; Novak et al., 2012), we analyzed Trp, TAM
and IPyA as potential intermediates (Figs 6 and S1). We
found that the IPyA level was decreased in the presence
of KOK1169. These results support our hypothesis that
KOK1169 is an inhibitor of auxin biosynthesis that targets
Trp aminotransferases. This finding is consistent with a
previous report that the IPyA pathway is the primary
auxin biosynthetic pathway in Arabidopsis (Mashiguchi
et al., 2011).
Kyn was identified as a competitive inhibitor of TAA1/
TARs (He et al., 2011). It is also an alternative substrate of
TAA1 (He et al., 2011). The inhibitory activity of Kyn
against enzymes involved in amino acid metabolism
remains unknown. YUCASIN was identified as an inhibitor
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
of YUC enzymes, but its inhibitory activity was not very
effective against WT Arabidopsis (Nishimura et al., 2014).
Recently, we identified PPBo and BBo as effective inhibi-
tors of YUC enzymes (Kakei et al., 2015). These inhibitors
are effective for WT Arabidopsis seedlings. We addressed
the activity of KOK1169 in this study, and showed that it
can be used as an alternative compound in future studies
of auxin biosynthesis.
Protection of aminooxy and carboxy groups increases
inhibitor activity in vivo
To investigate the relationship between the hydrophobicity
of the compounds and their activity as inhibitors of auxin
biosynthesis, we introduced protection groups at the R2
moiety, designated Type II compounds (Fig. S8). The calcu-
lated log P values of Type II compounds were higher than
those of the corresponding Type I compounds (Table S2).
Moreover, most Type II compounds exhibited greater inhi-
bition of IAA accumulation in vivo compared with the cor-
responding Type I compounds (Fig. S10, Table S2). The
partition coefficient (e.g. the value of log P) can be used to
predict uptake of a compound by plant roots and its
translocation to shoot tissue (Dettenmaier et al., 2009).
Thus an increased value of log P compared with the origi-
nal aminooxy Type I compounds was indicative of
increased inhibitory activity. To further increase the log P
values, the chemically reactive aminooxy group from the
Type II compounds was also substituted for protection
groups. These were classified as Type III compounds
(Fig. S9). Several Type III compounds had a stronger inhi-
bitory effect on IAA biosynthesis than the corresponding
Type I and Type II compounds in vivo (Fig. S10). Because
aminooxy residues are highly reactive, the Type III com-
pounds may be more chemically stable. Thus the Type III
compounds were expected to be more stable and more
hydrophobic than the Type I compounds, and also to exert
greater inhibition of auxin biosynthesis. In fact, several of
the Type III compounds resulted in a marked decrease in
the endogenous auxin level; indeed, the greatest decrease
in auxin level was about 90% (Fig. S10). These observa-
tions are striking, because the decrease in endogenous
auxin in the hypocotyls and roots of the wei8-1/tar2-1
double mutant was about 50–60% that of the WT (Stepa-
nova et al., 2008). It should be noted that Type II and III
compounds showed no inhibition of TAA1 in vitro (Fig
S11), probably due to their lack of an aminooxy group
and/or a carboxy group. Consistent with these results,
Type II and III compounds did not strongly bind to TAA1
in the in silico analysis (Table S3). Therefore, there was
no correlation between the in vitro and in vivo activity of
the Type II and III compounds. Thus whether the in vivo
activity of Type III compounds is due to conversion to
Type I compounds or to other unknown active structures
remains unclear.
© 2016 The Authors

CONCLUDING REMARKS
The newly developed compounds described here consti-
tute a class of auxin biosynthesis inhibitors that target
TAA1. These compounds, including Type II and III com-
pounds, are designated ‘pyruvamine’. We selected and
evaluated KOK1169 (AONP) as a representative of these
auxin biosynthesis inhibitors. Pyruvamine will be commer-
cially available for research use in the near future.
EXPERIMENTAL PROCEDURES
Details of the chemical synthetic procedures and mass spectro-
scopic and NMR data are provided in Methods S1. The experimen-
tal procedures are described in brief below. Full details of the
methods are available in Methods S2.
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Col-0) was used as the WT. Seeds
were surface sterilized and grown on solid half-strength MS med-
ium containing 1% sucrose and 0.6% gelite (pH 5.7) supplemented
or not with test compounds. Seedlings were grown vertically for
8 days under continuous white light at 22°C.
Plant growth conditions for endogenous auxin
measurement
Arabidopsis seedlings were grown on solid medium for 6 days
under continuous white light at 22°C, transferred to fresh liquid
medium, and then cultured for an additional 24 h. They were then
cultured in the presence of test compounds (final concentration
30 lM) for 3 h. They were weighed and immediately frozen in liq-
uid nitrogen and stored at 70°C until analysis of endogenous
IAA levels. UPLC-MS/MS was used to quantify IAA in the seed-
lings, as described by Soeno et al. (2010).
TAA1 inhibition assay
The in vitro TAA1 inhibition assay was performed as described
previously (Tao et al., 2008) with minor modifications. The
enzyme reaction mixture was prepared in 500 ll of 500 mM borate
buffer (pH 8.5) containing 300 lM Trp, 1 mM sodium pyruvate,
10 lM pyridoxal phosphate, 1 lg purified TAA1 recombinant pro-
tein and 1 lM of the test compound. The mixture was incubated at
35°C for 30 min and the reaction was stopped by adding 20 ll of
6 N HCl. Absorbance at 330 nm was measured to monitor the
amount of IPyA–borate complex. Reaction mixtures without TAA1
were used as controls and 500 mM borate buffer was used as the
blank for the spectrophotometric assay. In addition, we tested 50,
100 and 150 lM Trp to determine Ki values using a Dixon plot.
Quantitative real-time PCR analysis
Arabidopsis seedlings were grown vertically for 7 days under con-
tinuous white light at 22°C and then transferred to fresh liquid
half-strength MS medium and cultured for 24 h. Then they were
cultured in the presence or absence of compounds for another
2 h. IAA or DMSO (for mock treatment) was added to the medium
for 1 h. The seedlings were collected and frozen in liquid nitrogen.
Total RNA was isolated using an RNeasy Plant Mini Kit according
to the manufacturer’s instructions (Qiagen, https://www.qiagen.-
com/). Single-stranded cDNA was synthesized using a Superscript
II Reverse Transcriptase (Invitrogen, http://www.thermofisher.com/
) with random primers. PCR reactions were performed in a final
© 2016 The Authors
The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 87, 245–257
Auxin biosynthesis inhibitors targeting TAA1 255
volume of 25 ll according to the manufacturer’s instructions
(qPCR Mastermix plus for SYBR I Low ROX, Eurogentec, http://
www.eurogentec.com/).
Measurement of IAA intermediates and labeling
experiments
Endogenous IAA was measured according to the method
described by Soeno et al. (2010), with modifications. Arabidopsis
seedlings were grown on half-strength MS agar medium contain-
ing 1% sucrose for 7 days, cultured in deionized water for 1 day
and then given 30 lM of [15N2]-Trp with or without 30 lM KOK1169
treatment for 3 h. To analyze relative amounts of IAA and its inter-
mediates, samples were extracted using the modified QuEChERS
method (http://quechers.cvua-stuttgart.de/), purified in OASIS HLB
and MCX cartridge columns, and then analyzed by UPLC-MS/MS
(ACQUITY Ultra-performance Liquid Chromatography-TQ Detec-
tor, Waters, http://www.waters.com/) in multiple reaction monitor-
ing (MRM) mode (Table S4). For analysis of total IPyA, extracts
were subjected to methoximation with methoxiamine-HCl before
purification.
ACKNOWLEDGEMENTS
We thank Ms Sanae Tashiro-Shimada, Yasuyo Kagawa, Satoko
Kubo, Marie Mitsui, Yuki Shikata and Chihiro Takeuchi for techni-
cal assistance, Drs Shinji Kamisuki and Fumio Sugawara for tech-
nical assistance and skillful advice, and Drs Yusuke Jikumaru and
Yuji Kamiya for help with the ethylene analysis. The paper is con-
tribution no. 1012 from The Kihara Institute for Biological
Research, Yokohama City University. This work was supported by
a grant from the Scientific Technique Research Promotion Pro-
gram for Agriculture, Forestry, Fisheries and Food industry (to
YS). We also thank the Arabidopsis Biological Resource Center for
providing the wei8-1 and wei8-1/tar2-1 mutant lines.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online ver-
sion of this article.
Figure S1. Auxin biosynthetic pathway and the predicted target of
L-a-aminooxy-phenylpropionic acid (AOPP).
Figure S2. Chemical structures of 14 L-a-aminooxy-phenylpropio-
nic acid (AOPP) derivatives (Type I compounds).
Figure S3. Effect of KOK1167 on the phenotype of Arabidopsis
seedlings.
Figure S4. Dixon plot analysis of inhibitors based on enzyme
kinetic analysis of TRYPTOPHAN AMINOTRANSFERASE of ARABI-
DOPSIS 1 (TAA1), Arabidopsis phenylalanine ammonia-lyase 2
(AtPAL2) and Arabidopsis 1-aminocyclopropane-1-carboxylic acid
synthase 8 (AtACS8).
Figure S5. Effects of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic
acid (KOK1169) on anthocyanin and ethylene biosynthesis.
Figure S6. Endogenous indole-3-acetic acid (IAA) levels in seed-
lings treated with 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid
(KOK1169) or L-a-aminooxy-phenylpropionic acid (AOPP).
Figure S7. Effects of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic
acid (KOK1169) on auxin-deficient mutants.
Figure S8. Chemical structures of the Type II compounds.
Figure S9. Chemical structures of the Type III compounds.
Figure S10. Effects of the protection groups on inhibition of auxin
biosynthesis.
Figure S11. Effects of Type II and III compounds on TRYPTOPHAN
AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1) activity in vitro.
256 Megumi Narukawa-Nara et al.
Table S1. Relationship between TRYPTOPHAN AMINOTRANSFER-
ASE of ARABIDOPSIS 1 (TAA1) inhibition activity and the free
energy of binding with TAA1 (Type I compounds).
Table S2. Hydrophobicity of the compounds and their inhibition of
endogenous auxin level in Arabidopsis.
Table S3. Relationship between TRYPTOPHAN AMINOTRANSFER-
ASE of ARABIDOPSIS 1 (TAA1) inhibition activity and the free
energy of binding with TAA1 (Type II and III compounds).
Table S4. Diagnostic multiple reaction monitoring transitions and
optimized instrument settings (Corne voltage and collision energy,
CE) and retention times of the ultra-performance liquid chro-
matography-tandem mass spectroscopy method.
Table S5. Effects of 2-(aminooxy)-3-(naphthalen-2-yl)propanoic
acid (KOK1169) and [15N2]-L-tryptophan treatment on the levels of
endogenous indole-3-acetic acid (IAA) and its intermediates.
Methods S1. Chemical synthesis and characterization of the com-
pounds.
Methods S2. Additional experimental procedures.
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