molecule auxin inhibitors that target YUCCA are ... - Wiley Online Library

The Plant Journal (2015) 84, 827–837

doi: 10.1111/tpj.13032

TECHNICAL ADVANCE

Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function Yusuke Kakei1,†, Chiaki Yamazaki1,†,‡, Masashi Suzuki1, Ayako Nakamura1, Akiko Sato1, Yosuke Ishida1, Rie Kikuchi1, Shouichi Higashi2, Yumiko Kokudo3, Takahiro Ishii3,§, Kazuo Soeno3 and Yukihisa Shimada1,* 1 Kihara Institute for Biological Research, Yokohama City University, Maiokacho 641-12, Totsuka, Yokohama, Kanagawa, 244-0813, Japan, 2 Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan, and 3 National Agriculture and Food Research Organization (NARO), Western Region Agricultural Research Center (WARC), Senyu, Zentsuji, Kagawa 765-8508, Japan Received 10 July 2015; revised 7 September 2015; accepted 11 September 2015; published online 24 September 2015. *For correspondence (e-mail [email protected]). † These authors contributed equally to this work. ‡ Present address: Japan Space Forum, 3-2-1 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. § Present address: Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan.

SUMMARY Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin-deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole-3-pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole-3-acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin-containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild-type Arabidopsis seedlings. Co-treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (Ki) of BBo and PPBo were 67 and 56 nM, respectively. In addition, PPBo did not interfere with the auxin response of auxin-marker genes when it was co-treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function. Keywords: auxin biosynthesis inhibitor, chemical biology, YUCCA, Arabidopsis thaliana, borate, Brachypodium distachyon, technical advance.

INTRODUCTION The plant hormone auxin plays a central role in the regulation of plant growth and development, including cell division and elongation, differentiation, tropisms, apical dominance, senescence, abscission, and flowering (Woodward and Bartel, 2005; Teale et al., 2006). Multiple pathways have been proposed for the biosynthesis of indole-3-acetic acid (IAA), the most common auxin in nat€ gl and Kostermans, 1934; Went and Thimann, ure (Ko 1937). A major, and the best-characterized, pathway in © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd

Arabidopsis thaliana L. is the indole-3-pyruvate (IPyA) pathway (Figure 1a). In this pathway, the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) and its close homologues, TRYPTOPHAN AMINOTRANSFERASE RELATED 1 (TAR1) and TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2), convert L-tryptophan (L-Trp) to IPyA (Stepanova et al., 2008; Tao et al., 2008; Yamada et al., 2009; Zhou et al., 2011). The YUCCA (YUC) enzymes subsequently synthesize IAA from IPyA (Mashiguchi et al., 827

828 Yusuke Kakei et al.

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Figure 1. IAA biosynthetic pathway and structures of YUC inhibitors. (a) Auxin (IAA) biosynthesis pathways with proposed enzymes and intermediates. L-Trp, L-tryptophan; IPyA, indole-3-pyruvic acid; IAA, indole-3-acetic acid; IAM, indole-3-acetamide; IAOx, indole-3-acetaldoxime; IAN, indole-3-acetonitrile; TAM, tryptamine; IAAld, indole-3-acetaldehyde; TAA1, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1; TARs, TRYPTOPHAN AMINOTRANSFERASE RELATEDs; YUC, YUCCA, flavin-containing monooxygenase; AMI1, AMIDASE1; NIT, nitrilase; AOPP, L-amino-oxyphenylpropionic acid; Kyn, L-kynurenine. (b) Structures of YUC inhibitors. The presented compounds and other compounds in Figure S1 with assigned index (numbers) are collected and used for focused screening.

2011; Won et al., 2011). A biochemical study of YUC6 showed that YUC catalyzes oxidative decarboxylation of IPyA and phenyl pyruvate (Dai et al., 2013). The Arabidopsis YUC family has 11 members. The overexpression of YUC1 (yuc1-D) produces long hypocotyls, epinastic cotyledons, and short primary roots with long root hairs. YUC1, YUC2, YUC4 and YUC6 are required for the development of floral organs and vascular tissues (Cheng et al., 2006). YUC1, YUC4, YUC10 and YUC11 are essential for embryogenesis and leaf formation (Cheng et al., 2007). YUC3, YUC5, YUC7, YUC8, and YUC9 function in the root gravitropic response and in root development (Chen et al., 2014). Recently, VAS1 was reported to convert IPyA to L-Trp and act as a coordinator of this pathway and ethylene biosynthesis (Zheng et al., 2013). There are other auxin biosynthesis pathways that have been reported in a variety of plants and plant pathogens. Some pathways are dependent on Trp and others are Trp-independent (Woodward and Bartel, 2005; Chandler, 2009; Normanly, 2010). For example, known Trp-dependent pathways are the tryptamine (TAM) and indole-3-acetaldoxiame (IAOx) pathways (Woodward and Bartel, 2005; Pollmann et al., 2006; Chandler, 2009; Mano et al., 2010; Normanly, 2010; Zhao, 2010) and the indole-3-acetamide (IAM) pathway (Lehmann et al., 2010; Figure 1a). Each pathway comprises different proposed enzymes and genes that were identified using molecular or genetic approaches. Mutations of genes involved in auxin signaling disrupt development and are sometimes fatal; these mutations are MONOPTEROS (Berleth and Jurgens, 1993; Przemeck et al., 1996), BODENLOS (Hamann et al., 1999) and AUXINRESISTANT6 (Hobbie et al., 2000). Furthermore, mutations of genes in an auxin biosynthesis gene family may not affect IAA content or phenotype, due to the functional

redundancies of gene family members (reviewed by Sauer et al., 2013; Zhao, 2014). This makes it difficult to analyze and understand the function of auxin and IAA biosynthesis pathway using genetic approaches. Chemical genetics is a powerful alternative strategy that employs small molecules as probes to dissect biological processes (Dejonghe and Russinova, 2014; Rigal et al., 2014). These small chemicals can be applied to any tissue and anytime in the life cycle, with appropriate concentrations of chemicals, to knock down a targeted protein. Therefore, chemical genetics is advantageous in studying essential functions and tissue specificity of target molecules. These chemicals can be applied to analyze various species of vegetables, trees and crops. In this manner, chemical genetics is a remarkably useful approach to analyze growth and development of plants, especially when limited genetic mutant or manipulation tools are available. Numerous plant mutants have been identified as chemical-resistant mutants for auxin or auxin inhibitors and have been widely used in auxin research. They can serve as a tool that is complementary to genetic and genomic methods, facilitating the identification of an array of components modulating auxin metabolism, transport and signaling. Classical chemical tools were utilized to identify auxin-resistant (axr) mutants and transport inhibitor response (tir) mutants in auxin (De Rybel et al., 2009; Ma and Robert, 2013). Recent studies have introduced advanced chemical tools in auxin biology (De Rybel et al., 2009; Ma and Robert, 2013). a-(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA) and auxinole inhibit auxin signaling by interacting with TIR1/AFB–Aux/IAA co-receptors and are valuable tools to interrogate auxin transcriptional responses (Hayashi et al., 2008, 2012). The 1-naphthylphthalamic acid (NPA) acts as a specific IAA efflux inhibitor (Thomson et al., 1973; Bailly

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 84, 827–837

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Figure 2. Screening of YUCCA inhibitors. (a) Effects of inhibitors on primary root length (black bars) and level of free IAA (white bars). To measure primary root length, Arabidopsis seedlings were grown for 7 days on agar medium with 3 lM inhibitors. To measure free IAA, 7-day-old seedlings were treated with 30 lM inhibitors for 3 h in liquid culture medium. Endogenous IAA levels were analyzed by UPLC-MS/MS. Data and error bars represent the means SE of three independent experiments, each using 15 plants. (b) Recovery of the growth defect in the YUC1-overexpressor (CaMV35S:AtYUC1). Wild-type (WT) seedlings and the YUC1-overexpressor were grown with or without (mock) inhibitors (3 lM) for 7 days.


830 Yusuke Kakei et al. et al., 2008; Nagashima et al., 2008). Soeno et al. (2010) reported that aminoethoxyvinylglycine (AVG) and L-aminooxyphenylpropionic acid (AOPP) inhibit IAA biosynthesis, potentially targeting TAA1. AVG and AOPP cause a reduction in endogenous IAA content and inhibit root elongation and gravitropism of roots and shoots. The analysis of inhibitor targets in this work suggested the IPyA-dependent pathway as a main IAA biosynthesis pathway. This conclusion is consistent with later studies, which found that the IPyA pathway is catalyzed by TAA1/TARs and YUC, in this order (Mashiguchi et al., 2011; Won et al., 2011). Another small molecule, L-kynurenine (Kyn), has recently been identified as a potent auxin biosynthesis inhibitor that targets TAA1 (He et al., 2011). We propose a class of auxin biosynthesis inhibitors that effectively inhibit auxin biosynthesis in Arabidopsis. The target is the final step of the major auxin biosynthesis pathway, which is catalyzed by YUC. RESULTS Screening for effective YUC inhibitors We first established the YUC enzyme assay system in vitro and then conducted the first screening of the YUC inhibitor using about 600 diverse compounds from our chemical library. As a result, we identified a lead compound, 3,5dichlorophenylboronic acid, that inhibited YUC activity. To find a more effective YUC inhibitor, we focused on commercially available aromatic boronic acids with molecular weight ranging from 120 to 250 (Figures 1b and S1) and then screened these boronic acids for their ability to inhibit the YUC enzyme. The screening was performed according to the following criteria: (i) inhibition of primary root growth in wild-type Arabidopsis seedlings at 3 lM (Figure S2), (ii) reduction of free IAA content when applied to wild-type Arabidopsis seedlings at 30 lM for 3 h (Figure S3), and (iii) recovery/rescue of the root growth defect in YUC1-overexpressing transgenic plants (Figure 2b). We reduced the list of potential inhibitor candidate compounds during the screening process from 29 (criterion 1) to 7 (criterion 3). For criterion 1, we selected candidates that inhibited primary root elongation but did not cause browning of the leaves. In the following step, 3-pyridylboronic acid was added as a candidate to increase the structural variety of compounds subjected to screening for criterion 2. In the final step of the screening process, we selected 4-chlorophenylboronic acid, 3,5-dichlorophenylboronic acid, 4-biphenylboronic acid [BBo] and 4-phenoxyphenylboronic acid [PPBo] as strong inhibitory candidates that were effective in both short- and long-term treatments. We also selected 4-methyl-4-biphenylboronic acid [MBBo] as a stable inhibitor. To analyze the structural tolerance of YUC on aromatic boronic acids, we included 3,5-dibromophenyl-

boronic acid as an analogue of 3,5-dichlorophenylboronic acid, and 4-bromo-4-biphenylboronic acid as an analogue of MBBo. The screening results for all chemicals are shown in Figures S2 and S3. Figure 2(a) shows the structures of representative candidates, their inhibitory effects on primary root growth (at 3 lM, black bars) and their effects on free IAA content (at 30 lM, white bars). When the inhibitory effects were compared with phenylboronic acid (PBo), the addition of mono- or di-chloride to the structure of PBo at the meta- or para-position (3-chlorophenylboronic acid, 4chlorophenylboronic acid, 3,5-dichlorophenylboronic acid) resulted in stronger inhibitory activity on primary root growth compared with the inhibitory response from PBo alone (Figure 2a, black bars). However, addition of monoor di-chloride to the structure at the ortho-position (2-chlorophenylboronic acid and 2,6-dichlorophenylboronic acid) did not increase inhibition activity. Addition of aromatic ring structures at the para-position (BBo and PPBo) increased inhibition. Addition of further methyl groups to the BBo structure (MBBo) reduced inhibition activity (Figure 2a). The inhibition effects were evaluated by recovery/rescue of the root growth defect in YUC1-overexpressing transgenic plants, which have shorter primary roots and more root hair than the wild-type (Col-0; Figure 2b). Both the short primary roots and rich root hair growth were typical phenotypes that reflected auxin overproduction. BBo and PPBo were more effective in inducing primary root growth recovery in YUC1-overexpressing plants than 3,5dichlorophenylboronic acid, MBBo or their close analogs 3,5-dibromophenylboronic acid and 4-bromo-4-biphenylboronic acid (Figure 2b). BBo and PPBo were effective in recovering growth of YUC overexpressing plants at concentrations of 1 and 3 lM, respectively (Figure S4). Application of the smaller compounds, 4-chlorophenylboronic acid, 3,5-dichlorophenylboronic acid and 3,5-dibromophenylboronic acid, did not recover primary root elongation (Figure 2b). However, these compounds inhibited root hair formation in plants overexpressing YUC. Since an increase in root hair density is a phenotypic expression that typically results from auxin overproduction, we suggest that these compounds reduced auxin levels in plants and had other effects that reduced root growth. Next, we examined the YUC inhibition activity of these compounds in vitro. 2,6-Dichlorophenylboronic acid did not have an inhibitory effect (Figure S5a); it did not inhibit primary root growth or decrease free IAA content (Figure 2a). Thus, 2,6-dichlorophenylboronic acid was consistently inactive in both in vivo and in vitro assays; it ranked with the least active compounds among the boronic acids tested. We also analyzed the expression of the



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Figure 3. Analysis of PPBo and BBo inhibitor activities in Arabidopsis. (a, b) Root growth inhibition in the presence of BBo and PPBo. Plants were grown vertically on agar medium containing BBo or PPBo. (a) Photographs of 9-dayold Arabidopsis WT seedlings. (b) Root growth inhibition by different concentrations of BBo and PPBo. Data and error bars represent the means SE (n ≥ 20). (c) Magnified view of 9-day-old seedling grown in 3 lM PPBo. g indicates direction of gravity. (d) Dose-dependent inhibition of free IAA levels in a whole plant. Seedlings, 7 days old, were treated with BBo and PPBo for 3 h and endogenous IAA levels were analyzed by UPLC-MS/MS. Data and error bars represent the mean SE of three independent experiments, each using 15 seedlings. (e, f) Plant growth inhibition by inhibitors and recovery by auxin treatment. Arabidopsis wild-type seedlings were grown on ½MS agar medium for 7 days in 3 lM of BBo or PPBo, with or without IAA. Data and error bars represent the means SE (n ≥ 20) (e) Shoot growth inhibition and recovery in response to IAA treatment. (f) Root growth inhibition and recovery in response to IAA treatment.

auxin-responsive marker genes Aux/IAA1, Aux/IAA5 and Aux/IAA19. Application of both BBo and PPBo had an inhibitory effect on these marker genes that was stronger than

the effects of application of PBo and MBBo (Figure S5b). Based on these observations, we selected BBo and PPBo as the most effective YUC inhibitors.


832 Yusuke Kakei et al.

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Figure 4. In vitro inhibitory assays for recombinant AtYUC2 and TAA1. (a) AtYUC2 enzyme assay. Activity of YUC2 was measured by HPLC analysis of IAA. The enzyme assay was performed at 30°C for 20 min in a reaction mixture containing 5 lg of ProS2-YUC2, 10 lM IPyA with 30 lM BBo or PPBo. For the mock treatment, the assay was performed without inhibitors. (b) TAA1 enzyme assay. The activity of TAA1 was measured by spectrophotometric detection of 330 nm to quantify the IPyA-borate complex. This assay involved 30 lM Kyn, BBo and PPBo. Data and error bars represent the means SD (n = 3). *P < 0.05 by Student’s t-test. (c) Lineweaver-Burk plot of AtYUC2 activity with 0, 0.25 and 0.35 lM BBo and 0.05, 0.1, 0.2 and 0.3 lM IPyA. (d) Lineweaver–Burk plot of AtYUC2 activity with PPBo.

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To confirm that growth defects were caused by a decrease in IAA, we conducted a more detailed analysis of the growth of Arabidopsis seedlings within the context of BBo and PPBo. Arabidopsis seedlings were grown for 9 days in the presence of various concentrations (0.1–10 lM) of BBo and PPBo, which were found to inhibit primary root growth in a dose-dependent manner (Figure 3a,b). Treatment with 10 lM BBo and PPBo resulted in the shortest roots. Compared with the mock (0 lM) treatment, roots were longer in response to the 0.1 lM treatment. Figure 3(c) shows a typical seedling phenotype following the 3 lM PPBo treatment. The seedling had upwardly curved cotyledons and reduced root response to gravity. We analyzed endogenous free IAA levels in the presence of BBo and PPBo, which reduced free IAA content in seedlings in a dose-dependent manner (Figure 3d). We subsequently analyzed growth recovery after exogenous IAA treatment. Arabidopsis seedlings were grown in BBo or PPBo with a range of IAA concentrations (0, 1, 10 or 100 nM). Co-treatment with 10 nM IAA restored: (i) the weights of aerial tissues, and (ii) primary root growth in the presence of 3 lM BBo or 3 lM PPBo (Figure 3e,f). Thus, growth inhibition by 3 lM concentrations of BBo and PPBo was probably largely due to reductions in endogenous IAA content. Growth recovery was reduced at higher concentrations (10 lM or 30 lM) of BBo or PPBo (Figure S6).

We analyzed inhibitor activity in an in vitro recombinant YUC2 enzyme assay. Treatment with 30 lM BBo and PPBo significantly inhibited the activity of recombinant YUC2, which catalyzes IPyA to IAA (Figure 4a). Boronic acids are known to react with amines and alcohols (Hall, 2012). We tested the possibility that reaction between boronic acid and FAD cofactor might affect the inhibitory activity of BBo and PPBo. YUC2 enzyme activity was analyzed in the presence of normal (40 lM) and higher (80 lM) concentrations of FAD. These concentrations were greater than that (30 lM) of the inhibitors (Figure 4a). Increasing the concentration of FAD did not recover YUC2 activity, suggesting that reaction between FAD and boronic acid do not decrease FAD concentration. Therefore, reaction between FAD and boronic acid was not the main reason for inhibition of enzyme activity. We also confirmed that BBo and PPBo did not form a complex with IPyA (Data S1). The IPyA-boronic acid complex was detected only when the concentration of boronic acid was higher than 10 mM. This concentration was ca. 300–3000-fold higher than the concentrations of boronic acids used for in vitro or in vivo assays in this study. Therefore we conclude that the formation of the IPyA-boronic acid complex is not related to auxin biosynthesis inhibition by PPBo and BBo. To test the specificity of these inhibitors for enzymes in the IPyA pathway, we analyzed their effects on in vitro recombinant TAA1 activity. The combination of 30 lM BBo and 30 lM PPBo did not inhi-



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bit TAA1 activity (Figure 4b), while 3 lM Kyn, an inhibitor of TAA1 (He et al., 2011), reduced TAA1 activity. To understand the mode of action of these inhibitors, velocities of the enzyme reactions were measured at different IPyA concentrations. The Lineweaver-Burk plot revealed that the extent of enzyme inhibition in response to BBo and PPBo treatment was dependent on substrate concentration (Figure 4c,d). The Vmax value was unaffected by the inhibitor, while the Km value was affected, indicating that these inhibitors are competitive. The inhibition constant (Ki) values of BBo and PPBo were 67 and 56 nM, respectively. Analysis of inhibitor target sites using auxin-responsemarker gene expression We analyzed the expression of auxin-marker genes, Aux/ IAA1 and Aux/IAA19, in 7-day-old Arabidopsis seedlings to test the effect of PPBo on auxin sensing and signaling (Figure 5a). A 3-h treatment with 10 lM PPBo decreased the expression of Aux/IAA1 and Aux/IAA19. A 100 nM IAA treatment enhanced expression of the marker genes. A 1 lM IPyA treatment also enhanced the expression of marker genes. Enhancements of gene expression by IAA treatment were not influenced by co-treatment with 10 lM PPBo. Conversely, gene expression induction by IPyA treatment was significantly reduced by co-treatment with 10 lM PPBo. PPBo blocked the expression of auxin markers when cotreated with IPyA, but did not block this expression when co-treated with IAA (Figure 5b). These data suggest that PPBo did not inhibit perception or signaling of IAA, but it inhibited conversion from IPyA to IAA. Effect on IPyA content We then analyzed endogenous levels of IPyA in Arabidopsis seedlings treated with 0, 1, 10 or 30 lM BBo or 0, 1, 10 or 30 lM PPBo. The 30 lM BBo and 30 lM PPBo treatments increased IPyA levels by ca. 40 and 80% of those in the controls, respectively (Figure S7). These relative IPyA con-

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Figure 5. Effect of PPBo on auxin signaling and biosynthesis. (a) Gene expression of auxin-responsive marker genes in response to IAA, IPyA and PPBo treatments. The expression of auxin markers (Aux/IAA1 and Aux/IAA19) was measured by quantitative RTPCR in wild-type Arabidopsis seedlings treated with or without 10 lM PPBo, in combination with 10 nM IAA or 1 lM IPyA. ‘PPBo + IAA’ and ‘PPBo + IPyA’ indicate co-treatment of ‘PPBo and IAA’ and ‘PPBo and IPyA’. The mock treatment was 0.1% DMSO. Data and error bars represent the means SD for three independent experiments, each using 10 seedlings. **P < 0.01 by Student’s t-test. (b) Schematic presentation of BBo and PPBo actions.

IAA receptor complex Aux/IAA1, 19 (Auxin marker genes)

tents in 30 lM BBo- and 30 lM PPBo-treated plants were significantly higher than those in the controls (P < 0.05; Student’s t-test). Effect of BBo and PPBo on Brachypodium distachyon To determine whether BBo and PPBo are effective auxin biosynthesis inhibitors in species other than Arabidopsis, we applied them to a monocot model plant Brachypodium distachyon. The growth of Brachypodium was inhibited when plants were treated with 30 lM BBo and PPBo for 7 days (Figure S8a,b). In comparison with the controls, the endogenous IAA content was reduced in plants treated with the inhibitors (Figure S8c). We had identified auxin-inducible Aux/IAA genes (Bradi2 g04910 and Bradi3 g54610) in Brachypodium distachyon in a previous study (Kakei et al., 2015) to enable monitoring of auxin responses in the presence of inhibitors. The expression levels of these auxin-responsive Aux/IAA genes in Brachypodium were downregulated by a 3 h treatment with 30 lM BBo or 30 lM PPBo (Figure S8d). DISCUSSION Here, we propose a group of IAA biosynthesis inhibitors that comprise PBo and its derivatives. We searched for an effective inhibitor of YUC among commercially available PBo derivatives. Many PBo derivatives had significant inhibitory effects on primary root growth (Figures 2a and S2). They frequently reduced endogenous IAA content (Figures 2a and S3) or inhibited AtYUC2 in in vitro assays (Figure S5a). In contrast, some of the derivatives that had chloro-groups in the ortho-position had weaker inhibitory effects (Figures 2a and S5a). Thus, PBo derivatives with side chains in meta- and para-positions appear to be effective inhibitors of YUC. In particular, treatments with BBo, PPBo, MBBo and 4-bromo-4-biphenylboronic acid recovered the growth defect of a YUC1-overexpressing plant (Figure 2b), indicating that these compounds inhibited


834 Yusuke Kakei et al. overproduction of IAA, without apparent off-target effects that inhibit seedling growth. Among the boronic acids, BBo and PPBo were the strongest inhibitors of primary root growth (Figure 2a). They recovered the primary root growth defect in YUC1-overexpressing plants (Figure 2b) and effectively reduced expression of auxin-marker genes (Figure S5b). Therefore, we selected BBo and PPBo as effective candidate YUC inhibitors for further analysis. The effects of 4-chlorophenylboronic acid, 3,5-dibromophenylboronic acid and 3,5-dichlorophenylboronic acid may not be limited to the inhibition of auxin biosynthesis. These compounds inhibited root hair formation in YUC1overexpressing plants (Figure 2b). As an increase in root hair density is a phenotype that typically results from auxin overproduction, we suggest that these compounds reduced auxin levels in the plants. These results are consistent with our observation of reduced endogenous IAA levels in the presence of these compounds (Figures 2a and S3). However, these compounds could not recover elongation of the primary root in planta, suggesting that they have action sites other than YUC, which is necessary for primary root elongation. A possible explanation is that, since these compounds are smaller than BBo or PPBo, they are less specific to YUC and more likely to inhibit other enzymes. Next, we analyzed the effect of BBo and PPBo in planta in more detail. The 3 lM BBo and PPBo treatments significantly inhibited primary root growth (Figure 3a,b), induced upward curving of cotyledons (Figure 3c), and disturbed root gravitropism (Figure 3c). These phenotypic effects of PPBo and BBo treatment were similar to the phenotypes of auxin-deficient mutants (Muday, 2001; Stepanova et al., 2008; Soeno et al., 2010). A typical phenotype of well characterized auxin mutants is reduced in response to gravity (Muday, 2001). In addition, it was reported that mutants of auxin biosynthesis, such as wei8-1tar2-1, display upwardly curved cotyledons and defects in shoot and root development (Stepanova et al., 2008). Furthermore, the recovery of the growth defect of YUC1-overexpressing plants as a result of BBo and PPBo treatment suggests that BBo and PPBo specifically repressed the YUC enzyme in the IPyA pathway, thereby reducing endogenous IAA levels, with consequent reduction of growth defects (Figure 2b). Free IAA content in BBo- and PPBo-treated plants was reduced compared to the control treatment (Figure 3d). To confirm that the reduction in IAA caused by the inhibitors is the main reason for the phenotypic effect, we treated Arabidopsis seedlings with BBo or PPBo, in combination with IAA, simultaneously (Figures 3e,f). In the presence of IAA, shoot weight and root gravitropism were completely restored, and root elongation was almost restored. These results suggest that BBo and PPBo specifically reduced auxin activity to cause growth defects. Furthermore, plants

treated with 30 lM BBo and 30 lM PPBo have higher concentrations of endogenous IPyA (Figure S7), suggesting that BBo and PPBo inhibit auxin biosynthesis at the step when YUC catalyzes IPyA to IAA. To confirm the enzyme targeted by BBo and PPBo, we conducted a test in vitro to determine their ability to inhibit the AtYUC2 and TAA1 enzymes. The major pathway of the auxin biosynthesis consists of two steps catalyzed by these enzymes (Figure 1a). An in vitro assay with the recombinant AtYUC2 protein demonstrated that 30 lM BBo and PPBo inhibited AtYUC2 activity (Figure 4a). Conversely, 30 lM of BBo and PPBo did not inhibit TAA1 activity (Figure 4b). Therefore, YUC is the only target of BBo and PPBo in the main auxin biosynthesis pathway. At the same time, the FAD concentrations (40 or 80 lM) did not affect the inhibitory effect of BBo or PPBo (Figure 4a). This result suggests that BBo and PPBo inhibit YUC activity and this inhibition is not caused by the interaction/reaction of them with FAD. Kinetic studies of the YUC enzyme in the presence of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA (Figure 4c,d). BBo and PPBo reduced the expression of auxin-responsive marker genes (Figure S5b). Therefore, we analyzed the effect of PPBo on auxin signaling. Enhancements of gene expression by IAA treatment were not affected by co-treatment with 10 lM PPBo (Figure 5a). Conversely, the 1 lM IPyA treatment enhanced expression of the marker genes. The enhancement by IPyA treatment was significantly reduced by co-treatment with 10 lM PPBo. Furthermore, following co-treatment with IAA and BBo or PPBo, 10 nM IAA was sufficient to restore shoot and root growth (Figure 3e,f), although if the compound inhibits the auxin response mediated by the TIR1SCF complex, the concentration required for restoration is more than 100 nM (Hayashi et al., 2012). These results suggest that BBo and PPBo are not inhibitors of the auxin receptor, but auxin biosynthesis inhibitors (Figure 5b). Bassil et al. (2004) reported that 0.1 mM 3-nitrophenylboronic acid (3-NBA) competes with boric acid for binding to cis-diols, and causes the disruption of cytoplasmic strands and cell–cell wall detachment in cultured tobacco (Nicotiana tabacum L.) cells. This disruption was not observed at the lower concentration of 10 lM 3-NBA for 24 h. We have demonstrated that BBo or PPBo treatments at 10 lM for 3 h noticeably decreased free IAA concentration (Figure 3d) and repressed expression of auxin-responsive genes (Figure S5). Although submillimolar levels of boronic acids might function similar to 3-NBA, PBo derivatives at the micromolar level are sufficient to inhibit auxin biosynthesis. Therefore, we concluded that primary BBo and PPBo targets are auxin biosynthesis. Boronic acids have been reported as inhibitors of several microbial enzymes (Minkkila€ et al., 2008; Tondi et al., 2010; Demetriades et al., 2012). However, we are not aware of a PBo derivative that is an inhibitor of plant enzymes.


Phenylboronic acid is a YUCCA inhibitor 835 Recently, Nishimura et al. (2014) reported that yucasin is a potent inhibitor of YUC. We have determined that PBo derivatives are another class of auxin biosynthesis inhibitors that target YUC. Yucasin does not seem to alter the auxin level or plant growth at 100 lM when applied to wildtype Arabidopsis, while BBo and PPBo at 3 lM reduced the auxin level and plant growth (Figure 3). Therefore, the effective dose levels of PPBo and BBo were >30 fold lower than that of yucasin. Furthermore, the inhibitory constants (Ki) of BBo and PPBo (Figure 4c,d) were about 20-fold lower than inhibitory constant of Kyn (11.5 lM) as reported by He et al. (2011). This effective dose level of BBo and PPBo is the lowest among the known auxin biosynthesis inhibitors, including those targeting Trp aminotransferase. Thus, BBo and PPBo are useful biochemical tools for analyses of auxin function. The off-target effect is intrinsic to small-molecule inhibitors; it cannot be avoided or ignored at higher doses. Therefore, avoidance of off-target effects and correct interpretation of inhibitor experiments require establishment of a defined safety dose for specific inhibitors of auxin biosynthesis. However, the effective dose levels of inhibitors differ among organs, application procedures, durations of treatment, plant growth conditions, target plant species, etc. Therefore, general safety dose levels cannot be established. In our study, impairment of growth phenotypes by 3 lM BBo and 3 lM PPBo treatments were fully recovered in shoots (Figure 3e) and 75–90% recovered in roots (Figure 3f) in the presence of 10 nM IAA, suggesting that PPBo and BBo inhibited auxin biosynthesis without apparent off-target effects that inhibited seedling growth. Impaired growth of the primary root in 10 lM BBo and 10 lM PPBo treatments was partially recovered in the presence of 10–100 nM IAA (Figure S6). Thus, the safety dose level for the primary root elongation assay of Arabidopsis seedlings was in the range 1.0–10 lM. Treatments with 0.1 lM BBo and 0.1 lM PPBo effectively reduced the number of lateral roots (Figure 3a), but did not inhibit primary root growth. Therefore, effective and safety dose levels of BBo and PPBo may differ between lateral root formation and primary root growth responses (Figure 3a). The IPyA pathway is reportedly the main pathway in Arabidopsis, but its importance remains unclear for many other plant species. In our study, we applied BBo and PPBo to a monocot plant Brachypodium distachyon and found that both inhibited IAA biosynthesis and growth of seedlings (Figure S8). Thus, the IPyA pathway is likely important for the development of Brachypodium seedling. This example demonstrates that the YUC inhibitors BBo and PPBo are effective chemical tools for the analysis of auxin biosynthesis in plant species other than Arabidopsis. Further application of these chemical tools to a range of plant species and diverse physiological/developmental phenom-

ena will clarify the function of the IPyA pathway across the plant kingdom. EXPERIMENTAL PROCEDURES Chemicals We studied auxin biosynthesis inhibitors in our previous investigations (Ishii et al., 2010; Soeno et al., 2010; Higashide et al., 2014; Ishida et al., 2014), during which we collected about 600 compounds. We synthesized 160 ourselves and others were obtained from commercial sources. Our in-house chemical library comprises these chemicals. We conducted the first screening of YUC inhibitors using this library and tested these 600 in our YUC enzyme assays to discover the first active compound, 3,5dichlorophenylboronic acid. We purchased BBo and PPBo from Wako Pure Chemical Industries, Ltd (http://www.wako-chem.co.jp/ siyaku/index.htm). Other PBo derivatives were also obtained from Wako Pure Chemical Industries, Ltd., except following four chemicals. Phenethylboronic acid was purchased from Sigma (http:// www.sigmaaldrich.com). We purchased 4-bromophenylboronic acid, 2-naphthylboronic acid, and 4-bromo-4-biphenylboronic acid from the Tokyo Chemical Industry Co., Ltd (http://www.tcichemicals.com/).

Plant materials and growing conditions Arabidopsis thaliana accession Col-0 was used as the wild-type. Arabidopsis seedlings were sterilized and grown at 21°C under continuous light and on half-strength Murashige and Skoog (MS) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.8% agar and 1% (w/v) sucrose. To measure primary root length, the wild-type and YUC1-overexpressor (Suzuki et al., 2015) were grown for 7 or 9 days on agar medium containing 3 lM of inhibitors in vertically oriented plastic plates. To measure free IAA, the wild-type seedlings were grown for 6 days on vertically oriented agar medium, transferred to a culture tube containing liquid medium, incubated for 1 day with shaking, and treated with or without inhibitors dissolved in 0.1% (v/v) dimethyl sulfoxide (DMSO) for 3 h. To measure IPyA, the wild-type seedlings were grown for 7 days on agar medium in vertically oriented plates, transferred to a culture tube containing liquid medium, incubated for 1 days with shaking, and treated with or without inhibitors dissolved in 0.1% (v/v) DMSO for 3 h. To assess gene expression of auxin markers, the wild-type seedlings were grown for 6 days on vertically oriented agar medium, transferred to a culture tube containing halfstrength MS liquid medium with 1% sucrose, incubated for 1 day with shaking, and treated with 10 lM inhibitors, 1 lM IPyA and 100 nM IAA for 3 h. To test for recovery from growth defects caused by BBo or PPBo, wild-type seedlings were grown 7 days on half-strength MS plates in the presence of 3, 10 or 30 lM BBo or PPBo in combination with 0, 1, 10 or 100 nM IAA. Seeds of Brachypodium distachyon (diploid inbred line Bd21) were sterilized and placed on ½MS medium supplemented with 1% sucrose, 0.8% agar and 30 lM BBo or 30 lM PPBo. Seeds were then kept at 4°C for 1 week to break dormancy. Germinated plants were grown for 7 days under continuous light at 22°C. In our 3 h inhibitor treatments, we kept seeds of Brachypodium at 4°C for 1 week. Germinated plants were grown on ½MS medium plates for 4 days, after which they were transplanted to ½MS liquid culture medium and cultured for 1 day, during which time they were shaken. After pre-culturing, the seedlings were either treated or not treated over a 3 h period with inhibitors dissolved in 0.1% (v/v) DMSO.


836 Yusuke Kakei et al. Q-RT-PCR

0.3 lM IPyA; 30 lM FAD; and 1 mM NADPH in 50 mM Tris–HCl buffer (pH 8.0). BBo or PPBo were dissolved in 50% ethanol and added to the reaction mixture at a final concentration of 0.25 or 0.35 lM (1/50 volume of the reaction mixture). The mixture was incubated for 10 min at 30°C and the reaction was stopped by adding 1/10 volume of 1 N HCl. Reaction products (IAA) were analyzed by high pressure liquid chromatography (HPLC) (Hitachi LaChrom Elite HPLC system with COSMOSIL 5C18-MS-II column; Hitachi, Tokyo, Japan) with fluorescence detection (kex/kem = 280/ 355 nm).

Quantitative RT-PCR analyses of Arabidopsis genes followed the procedures of Suzuki et al. (2015), with minor modifications. Total RNA was isolated using RNAZol RT (MRC) and treated with DNase I (TaKaRa, Tokyo, Japan), then converted to cDNA using ReverTra Ace with the Oligo dT primer (TOYOBO, Tokyo, Japan). Quantitative RT-PCR was performed in a GeneAce Probe qPCR Mix a Low ROX (Nippon Gene, Tokyo, Japan) using real-time Taq-Man technology (Holland et al., 1991) and a sequence detector (model 7500, Applied Biosystems, Foster City, CA, USA). Gene-specific primers and Taq-Man probes (Table S1) were used to analyze transcript abundances (Soeno et al., 2010). UBQ10 mRNA was analyzed as an internal control and used to normalize the values for transcript abundance. We performed three independent experiments with different plant samples. For RT-PCR analysis of Brachypodium genes, we isolated total RNA with RNAZol RT (MRC), followed by conversion to cDNA using ReverTra Ace (TOYOBO) with the Random primer (Invitrogen). Quantitative RTPCR was performed with GeneAce SYBRâ qPCR Mix a Low ROX (Nippon Gene, Tokyo, Japan) using a Thermal Cycler Dice RealTime System II (TaKaRa). Gene-specific primers (Table S1) were used to analyze transcript abundances. 18S ribosomal RNA abundance was analyzed as an internal control and used to normalize the values of transcript abundance.

The in vitro enzyme assay using recombinant TAA1 was performed according to the previously described borate buffer assay (Matheron and Moore, 1973; Tao et al., 2008) with minor modifications. The reaction mixture was prepared in 500 ll of 500 mM borate buffer (pH 8.5) containing 300 lM L-Trp, 1 mM sodium pyruvate, 10 lM pyridoxal phosphate (PLP), 1 lg of purified TAA1 recombinant protein and the test compound at 30 lM. 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 quantify the IPyA-borate complex using a V-630 spectrophotometer (JASCO Inc., Tokyo, Japan). Reaction mixtures without TAA1 were used as the control and 500 mM borate buffer was used as a blank for the spectrophotometric assay.

Analysis of free IAA and IPyA in plant

ACKNOWLEDGEMENTS

Endogenous IAA was measured according to Soeno et al. (2010), with some modifications. Samples were extracted with modified QuEChERS method (www.quechers.com/) at 4°C and purified with OASIS HLB and MCX cartridge columns (Waters Corporation, Milford, MA, USA), then analyzed by UPLC-MS/MS (ACQUITY UltraPerformance Liquid Chromatography-TQ Detector, Waters) with an ACQUITY UPLC BEH C18 Column (Waters). To analyze total IPyA, extracts were methoximized with methoxiamine–HCl before purification. Method S1 describes the analysis of IAA and IPyA in detail.

We thank Ms Tomoe Tanikawa for the technical assistance. This work was supported by the Program for Promotion of Basic and Applied Researchers for Innovations in Bio-oriented Industry (BRAIN) to Y.S., JSPS KAKENHI Grant Number 26506016 to Y.K. and 26450046 to K.S. and the Scientific Technique Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. This paper is a contribution No. 1013 from the Kihara Institute for Biological Research, Yokohama City University.

Preparation of recombinant YUCCA2 and enzyme assay To generate recombinant YUCCA2 protein, YUCCA2 was cloned into the pColdProS2 DNA expression vector (TaKaRa) using primers 50 -GCCGGAGCTCATGGAGTTTGTTACAGAAACGT-30 and 50 GCCGCTGCAGTTAACAATGTTGAGGACGAG-30 , and transformed into the E. coli strain BL21 (DE3). Transformed cells were grown in LB broth containing carbenicillin (50 lg ml1) at 37°C until the OD600 nm reached 0.5, then cultured for 30 min at 15°C. ProS2YUC2 was induced with 0.4 mM isopropyl-thio-galactosidase (IPTG) for 24 h. Crude protein was extracted using Bugbusterâ protein extraction reagent (Novagen, EMD Chemicals Inc., San Diego, CA) and purified using the HisTrap HP column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Purified proteins were concentrated using the Amicon Ultra-4 10K (Millipore, Billerica, MA, USA) at 4°C. Concentrations of recombinant proteins were determined with a protein assay kit (Bio-Rad, Hercules, CA, USA). Purified protein was frozen in liquid N2, and stored at 80°C until use. The YUCCA2 enzyme assay was performed using the following procedure, except for the kinetic assay. The activity of recombinant YUC2 was measured in a reaction mixture containing 5 lg of ProS2-YUC2, 10 lM IPyA, 40 lM FAD, 1 mM NADPH in PBS buffer. The reaction was performed for 20 min at 30°C and was stopped by adding 1 volume of methanol to the reaction mixture. The inhibitory assay was performed by adding 30 lM inhibitors to the reaction mixture. Inhibitory kinetics was measured in the reaction mixture containing 0.2 lg of ProS2-YUC2; 0.05, 0.1, 0.2 or

Enzyme assay of recombinant TAA1

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Structures of all inhibitor candidates. Figure S2. Screening of inhibitors primary root length. Figure S3. Screening of inhibitors by endogenous free IAA content. Figure S4. Recovery of growth defect in the YUC1-overexpressor in the presence of BBo or PPBo. Figure S5. Effects of inhibitors on in vitro YUC enzyme activity and auxin-marker gene expression. Figure S6. Root growth retardation caused by inhibitors (tested at concentrations of 10 lM and 30 lM) and recovery attributable to auxin treatment. Figure S7. Relative ratio of endogenous IPyA levels in Arabidopsis seedlings treated with BBo or PPBo. Figure S8. Analysis of BBo and PPBo inhibitor activities in Brachypodium. Table S1. Primers used for qPCR. Methods S1. Supporting experimental procedures. Data S1. Reactions between IPyA and inhibitors.

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