Manipulation and Sensing of Auxin Metabolism, Transport and Signaling

Manipulation and Sensing of Auxin Metabolism, Transport and Signaling Abstract The plant hormone auxin is involved in virtually every aspect of plant growth and development. A chemical genetic approach has greatly contributed to the identification of important genes in auxin biosynthesis, transport and signaling. Molecular genetic technologies and structural information for auxin regulatory components have accelerated the identification and characterization of many novel small molecule modulators in auxin biology. These modulators have been widely utilized to dissect auxin responses. Here we provide an overview of the structure, primary target, in planta activity and application of small molecule modulators in auxin biology. Introduction Auxin, a plant hormone, regulates virtually every aspect of plant growth and development. Auxin functions as a master regulator in embryogenesis, apical dominance, lateral root formation, hypocotyl elongation, tropic responses to light and gravity, vascular tissue differentiation and lateral branching of shoots (Woodward and Bartel 2005, Enders and Strader 2015). IAA, a naturally occurring auxin, is a very simple molecule, but it exerts diverse hormonal actions on plant development. The auxin activity on various developmental processes can be spatiotemporally modulated by three major regulatory steps: auxin biosynthesis and inactivation; directional auxin transport; and signaling (Chapman and Estelle 2009, Zazimalova et al. 2010, Ludwig-Muller 2011, Hayashi 2012, Kasahara 2016). The intercellular auxin concentration gradient plays a crucial role in the regulation of auxin action. The auxin gradient is co-ordinately generated by auxin polar transport via auxin influx and efflux transportation and auxin metabolism including auxin biosynthesis, inactivation and degradation of auxin (Korasick et al. 2013, Naramoto 2017). At auxin concentration maxima in the tissue, auxin activates the transcription of auxin-responsive genes via the SCFTIR1/AFB–proteasome pathway, leading to auxin-related developmental responses (Chapman and Estelle 2009). In this transcriptional regulatory process, auxin enhances the proteolysis of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors by promoting the ubiquitination of the Aux/IAA proteins that are recognized as a substrate of the SCFTIR1/AFB-type E3 ubiquitin ligase complex. The Aux/IAA repressors bind to auxin response factor (ARF) transcription factors and block the transcriptional activity of ARFs. Thus, auxin-induced degradation of Aux/IAA results in the activation of ARFs to promote auxin-responsive gene expression (Chapman and Estelle 2009, Hayashi 2012). The conventional forward genetic approach in the model plant Arabidopsis has unveiled the underlying molecular mechanism of auxin action and identified essential genes regarding auxin-related pathways (Woodward and Bartel 2005). In an early work, most of the crucial auxin mutants were identified in a chemical genetic screen using synthetic auxins and small molecule modulators, and those mutations were found in key components in auxin biosynthesis, signaling and transport. Small molecule modulators of auxin biosynthesis, signaling and transport (auxin probes) have been considered as indispensable elements to study particular processes in auxin biology (Ruegger et al. 1997). Synthetic auxins are still used in almost all auxin research. Additionally, auxin probes have made great contributions to complementing genetic approaches in plant biology (De Rybel et al. 2009, Ma and Robert 2014). The functional redundancy of cognate gene families in the auxin regulatory pathway has blocked access to the physiological function of individual genes. Furthermore, the disruption of multiple pivotal genes often results in a lethal impairment (Hicks and Raikhel 2014). Fundamental components in auxin biosynthesis, signaling and catabolism have been suggested to be conserved across diverse land plants such as liverworts, mosses, ferns, gymnosperms and angiosperms (Eklund et al. 2015, Kato et al. 2017). To complement the genetic approach in a range of non-model plants with poor molecular genetic and genomic resources, the use of small molecule modulators to manipulate the auxin action spatiotemporally represents an alternative approach for auxin biology. Here we review natural and synthetic molecules that function as auxins. We also review the synthetic and endogenous small molecule modulators in auxin metabolism, auxin transport and auxin signaling (Fig. 1 and 2; Supplementary Table S1), and we introduce the application of these modulators in plant biology. Fig. 1 View largeDownload slide (A) Natural and synthetic auxins, and small molecule modulators of auxin biosynthesis, transport and signaling. Chemical name, CAS registry number, molecular weight, primary targets and availability are listed in Supplementary Table S1. (B) The binding structures of TIR1–IAA–Aux/IAA (left) and TIR1–auxinole (right). The phenyl ring of auxinole strongly interacts with Phe82 of TIR1 and blocks the access of Aux/IAA to the auxin-binding site. Fig. 1 View largeDownload slide (A) Natural and synthetic auxins, and small molecule modulators of auxin biosynthesis, transport and signaling. Chemical name, CAS registry number, molecular weight, primary targets and availability are listed in Supplementary Table S1. (B) The binding structures of TIR1–IAA–Aux/IAA (left) and TIR1–auxinole (right). The phenyl ring of auxinole strongly interacts with Phe82 of TIR1 and blocks the access of Aux/IAA to the auxin-binding site. Fig. 2 View largeDownload slide Cellular model of auxin biosynthesis, transport and signaling. IAA is biosynthesized from tryptophan (Trp) via indole 3-pyrvic acid (IPyA) by TAA1 and YUCCA in the IPyA pathway. IAA is incorporated by AUX1/LAX1 symporters and exported by PIN and ABCB proteins. ER-localized PIN and PILS carrier proteins regulate the homeostasis of cellular IAA. Auxin blocks the clathrin-mediated endocytosis of PIN from the plasma membrane. The SCFTIR1/AFB, E3 ubiquitin ligase complex is composed of SKP (ASK1), Cullin and F-box protein (TIR1/AFB). Auxin induces the ubiquitination of Aux/IAA proteins via SCFTIR1/AFB. The degradation of Aux/IAA repressor recovers ARF activity to activate the transcription of auxin-responsive genes. The SAUR proteins induced by auxin inhibit PP2C-D phosphatases, resulting in the activation of H+-ATPase to induce a rapid auxin response such as turgor-induced growth. IAA is converted to IAA–aspartate (IAA–Asp) by the auxin-induced GH3 enzyme IAA–amino acid conjugate synthase. IAA is oxidized to 2-oxo-IAA (oxIAA) by DAO1 dioxygenase. Fig. 2 View largeDownload slide Cellular model of auxin biosynthesis, transport and signaling. IAA is biosynthesized from tryptophan (Trp) via indole 3-pyrvic acid (IPyA) by TAA1 and YUCCA in the IPyA pathway. IAA is incorporated by AUX1/LAX1 symporters and exported by PIN and ABCB proteins. ER-localized PIN and PILS carrier proteins regulate the homeostasis of cellular IAA. Auxin blocks the clathrin-mediated endocytosis of PIN from the plasma membrane. The SCFTIR1/AFB, E3 ubiquitin ligase complex is composed of SKP (ASK1), Cullin and F-box protein (TIR1/AFB). Auxin induces the ubiquitination of Aux/IAA proteins via SCFTIR1/AFB. The degradation of Aux/IAA repressor recovers ARF activity to activate the transcription of auxin-responsive genes. The SAUR proteins induced by auxin inhibit PP2C-D phosphatases, resulting in the activation of H+-ATPase to induce a rapid auxin response such as turgor-induced growth. IAA is converted to IAA–aspartate (IAA–Asp) by the auxin-induced GH3 enzyme IAA–amino acid conjugate synthase. IAA is oxidized to 2-oxo-IAA (oxIAA) by DAO1 dioxygenase. Auxin Biosynthesis: Inhibitors of the IPyA Pathway IAA is predominantly biosynthesized via the indole 3-pyruvic acid (IPyA) pathway (Kasahara 2016). In the IPyA pathway, tryptophan is converted to IAA by two sequential enzymatic steps (Mashiguchi et al. 2011, Won et al. 2011). In the initial step, three aminotransferases encoded by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAA1/TAR) gene family produces IPyA from tryptophan in Arabidopsis. Subsequently, the YUCCA (YUC) flavin monooxygenase family catalyzes the oxidative decarboxylation of IPyA to IAA. Additionally, Cyt P450 monooxygenases CYP79B2 and CYP79B3 produce indole-3-acetaldoxime (IAOx) from tryptophan in Arabidopsis (Sugawara et al. 2009). IAOx is then converted to IAA in this auxiliary IAA biosynthesis pathway of the family Brassicaceae. Indole 3-acetaldehyde, indole 3-acetamide and tryptamine have been proposed as IAA precursors, but the roles of these precursors in IAA biosynthesis remain unknown (Kasahara 2016). Auxin biosynthesis inhibitors would be a very powerful tool for plant biology. l-Kynurenine (l-kyn) was identified as an auxin biosynthesis inhibitor from a commercial chemical library (He et al. 2011). l-Kyn was initially screened according to the inhibitory activity of ethylene responses in Arabidopsis root by using a phenotype-based screen of the constitutive ethylene response mutants, ethylene overproducer1-2 (eto1-2) and ctr1-1. l-Kyn is the degradation metabolite of tryptophan in animals, and therefore is regarded as a tryptophan analog. Consistent with the structural similarity of l-kyn and tryptophan, l-kyn was demonstrated to be a competitive inhibitor of TAA1 with a moderate Ki value (11.52 μM). Interestingly, no tryptophan analogs, such as α-methyltryptophan, have been reported to be IAA biosynthesis inhibitors. l-Kyn has been widely utilized to repress endogenous IAA production in various plants including the liverwort Marchantia polymorpha and the moss Physcomitrella patens (Eklund et al. 2015, Tsugafune et al. 2017). Aminoethoxyvinylglycine (AVG) and l-2-aminooxy-3-phenylpropionic acid (AOPP) have been reported to be auxin biosynthesis inhibitors targeting TAA1/TARs. AVG and AOPP were initially identified as inhibitors of pyridoxyl-5-phosphate (PLP)-dependent enzymes, including the aminocyclopropane-1-carboxylic acid (ACC) synthases and phenylalanine ammonia-lyase (Soeno et al. 2010). AVG and AOPP have an aminooxy moiety as a common feature that reacts with PLP of the enzymes via oxime bond formation. AVG and AOPP inhibit TAA1 and PLP enzyme, and reduce free IAA levels in Arabidopsis seedlings. However, these two inhibitors might show off-target effects via the inhibition of various PLP enzymes. To improve the specificity of aminooxy-type TAA1 inhibitors, aminooxy-naphthylpropionic acid (KOK1169/AONP) was designed as a specific inhibitor of TAA1 from AOPP derivatives (Narukawa-Nara et al. 2016). In vitro enzyme kinetic experiments indicated that AONP is a competitive inhibitor with a potent Ki value (76.8 nM) on TAA1, confers typical auxin-deficient phenotypes and suppresses auxin-responsive gene expression, which is reversed by IAA application. Pyruvamine 2031 (KOK2031) has also been identified as a potent aminooxy-type inhibitor of rice OsTAR1 with a potent Ki value (276 nM), and reduces endogenous IAA in rice (Kakei et al. 2017). YUC, a flavin-containing monooxygenase, is the rate-limiting enzyme in the IPyA pathway of Arabidopsis and maize (Kasahara 2016). Recently, two types of YUC inhibitors have been reported. Phenylboronic acid derivatives, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), have been identified as potent and reversible YUC inhibitors (Kakei et al. 2015). These boronic acids restore the extreme high-auxin phenotype of 35S::YUC1 seedlings. BBo and PPBo reduce the endogenous IAA content and exhibit auxin-deficient phenotypes, which are recovered by IAA application in wild-type Arabidopsis seedlings. The kinetic studies showed a competitive effect of BBo and PPBo against the substrate IPyA with a Ki of 50–60 nM. BBo and PPBo inhibit IAA biosynthesis and growth of a monocot plant, Brachypodium distachyon. Yucasin was identified as a YUC inhibitor from a chemical library in a screen for inhibitors of IAA production in maize coleoptiles (Nishimura et al. 2014). Yucasin was identified from derivatives of methimazole, an inhibitor of flavin-containing monooxygenase involving the YUC family. Yucasin has a similar structure to the 1,2,4-triazole-3(4H)-thione moiety of methimazole (Eswaramoorthy et al. 2006). Yucasin is a competitive YUC inhibitor and restores the high-auxin phenotype of YUC overexpression lines. However, in planta, auxin-deficient phenotypes are not observed in wild-type plants treated with yucasin. A structure–activity relationship study of yucasin revealed that its difluorinated analog, yucasin DF (YDF), was a potent YUC inhibitor in planta (Tsugafune et al. 2017). YDF exhibits an auxin-deficient phenotype in wild-type plants, but the in vitro inhibitory activity of YDF toward a recombinant YUC enzyme is similar to that of yucasin. Finally, YDF was found to be a chemically and metabolically stable analog of yucasin, and thereby YDF was maintained at high levels that were sufficient to inhibit auxin biosynthesis fully in planta. YDF was also effective in the liverwort M. polymorpha and moss P. patens, suggesting that YDF functions as a universal tool to modulate auxin biosynthesis in various plants. Off-target effects of compounds are the result of the unspecific effects on non-target proteins. Especially when compounds are used at high concentration, off-target effects are more likely to emerge. Cocktail treatment with multiple inhibitors on the same targeting pathway would be an effective approach to avoid possible off-target effects because a much smaller amount of each inhibitor can be used. The cocktail treatment with the YUC inhibitor, YDF, and two TAA1 inhibitors, l-kyn and AONP, can extensively inhibit IAA synthesis via the IPyA pathway and confers extreme auxin-deficient phenotypes that are fully recovered by IAA application (Tsugafune et al. 2017). Auxin Catabolism: Conjugation and Oxidation of IAA The reversible and irreversible catabolism of IAA plays a crucial role in IAA homeostasis and co-ordinately regulates the local auxin gradient (Ludwig-Muller 2011, Korasick et al. 2013). IAA is inactivated via two major pathways; GRETCHEN HAGEN3 (GH3) is one of the extensively studied early auxin-responsive genes encoding the IAA–amino acid conjugate synthase. The Arabidopsis genome contains 20 GH3 sequences, and the expression of several GH3 genes is regulated by both light and auxin signaling pathways (Ludwig-Muller 2011). Another major inactivation pathway is the oxIAA pathway. Arabidopsis DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1) encodes the 2-oxoglutarate/iron-dependent dioxygenase family and catalyzes the oxidation of the 2-position of IAA to yield 2-oxo-indole 3-acetic acid (oxIAA) (Zhang and Peer 2017). The loss-of-function dao1-1 mutant shows a tremendous accumulation of IAA–Asp and IAA–Glu to compensate for the oxIAA pathway in the dao1-1 mutant, suggesting that AtDAO1 and group II GH3 co-ordinately regulate the catabolism of IAA (Porco et al. 2016). Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), as a mimic of the adenylated reaction intermediate of the GH3 enzyme, has been reported to be a potent competitive inhibitor of recombinant grape GH3 enzymes (Bottcher et al. 2012). AIEP competes for the binding of ATP and IAA in GH3 active sites. However, the in planta effects of AIEP on Arabidopsis phenotypes have not been demonstrated. In contrast, JAR1/GH3-11 catalyzes the formation of a bioactive jasmonoyl–isoleucine conjugate as a genuine hormone. Recently, Jarin-1 was identified as a specific inhibitor of the GH3-11 enzyme (group I) and blocked the JAR1-regulated physiological response in planta (Meesters et al. 2014), implying that specific inhibitors of group II GH3 enzymes can be screened. Auxin Polar Transport: Inhibitors of Auxin Influx and Efflux Transport IAA is biosynthesized and then directionally transported to its responsive site. This auxin polar transport system modulates the local auxin distribution to form an asymmetric auxin concentration gradient that regulates tropic growth, embryo development, lateral root formation, and vascular and root development. The auxin polar stream is regulated by auxin influx and efflux proteins (Adamowski and Friml 2015, Zazimalova et al. 2010). For auxin import, AUXIN-RESISTANT 1/LIKE AUX1 (AUX1/LAX) proton gradient-driven symporters similar to amino acid permeases function as auxin influx transporters. The loss of a functional Arabidopsis aux1 mutant revealed an agravitropic and auxin-resistant phenotype in response to exogenous IAA (Marchant et al. 1999). For auxin efflux transport, PIN-FORMED (PIN) efflux carriers and ATP-BINDING CASSETTE subfamily B/MULTIDRUG RESISTANCE/P-GLYCOPROTEIN (ABCB/MDR/PGP) transporters co-ordinately regulate the direction and rate of intercellular auxin flow (Zazimalova et al. 2010). There are eight PIN family proteins in Arabidopsis, among which five PIN protein members, PIN1–PIN4 and PIN7, are localized at the plasma membrane of specific cells and function as auxin efflux carriers (Adamowski and Friml 2015, Naramoto 2017). In contrast, PIN5, PIN6, PIN8 and PIN-LIKES (PILS) proteins are localized in the endoplasmic reticulum (ER) (Barbez et al. 2012). These ER-localized carrier proteins facilitate intracellular auxin movement between the cytosol and ER to regulate auxin homeostasis (Adamowski and Friml 2015). Thus far, Arabidopsis ABCB proteins ABCB1, ABCB4 and ABCB19 have been characterized as auxin transporters. The single and multiple loss-of-function mutations of these PIN and ABCB proteins show severe defects in embryogenesis, shoot and root tropism, and lateral organ development (Zazimalova et al. 2010). The cell type-specific expression and subcellular localization of AUX/LAX, PIN and ABCB proteins ultimately determine the auxin gradient regulating plant growth and development. Synthetic auxins are very useful diagnostic tools to dissect the physiological responses mediated by auxin transport. The membrane-permeable synthetic auxin naphthalene 1-acetic acid (NAA) rescues the agravitropic phenotype of the aux1 mutant (Marchant et al. 1999). AUX1 recognizes IAA and 2,4-D as substrates and takes them, but not NAA, into the cell (Yang et al. 2006). In contrast, PIN and ABCB transporters export NAA outside cells, but 2,4-D does not (Yang and Murphy 2009). The Arabidopsis yuc 3,5,7,8,9 quintuple mutant (yuc Q) has severe auxin-deficient root phenotypes (Chen et al. 2014), but with the application of exogenous IAA and NAA fully recover from the defects in gravitropism and root growth. In contrast, 2,4-D can restore the defects in root growth only, but not those in root gravitropism, suggesting that 2,4-D is not transported in a polar manner to form the auxin concentration gradient required for the gravitropic response (Chen et al. 2014). Phenylacetic acid (PAA) has also been demonstrated to be a non-polar transport-type auxin (Sugawara et al. 2015). The transport profiles of other synthetic auxins, dicamba and picloram, remain unclear. The auxin transport inhibitor (ATI) has been extensively investigated and widely used in auxin biology. N-1-naphthylphthalamic acid (NPA) is extensively characterized as a classical ATI (Katekar and Geissler 1980). NPA potentially inhibits the directional auxin movement and blocks the plant responses mediated by auxin transport. NPA inhibits auxin efflux in a non-competitive manner. The primary target of NPA has been suggested to be ABCB transporters in planta (Noh et al. 2001). However, NPA blocks auxin transport by both PIN and ABCB in yeast functional assays (Yang and Murphy 2009). Additionally, NPA can bind to the immunophilin-like protein TWD1, and then NPA blocks the interaction of TWD1 with ABCB1 proteins (Bailly et al. 2008). Moreover, at a high concentration, NPA inhibits the subcellular recycling of PIN proteins required for the membrane localization of PIN (Geldner et al. 2001). Recently, NPA has been shown to mediate actin cytoskeleton remodeling via the TWD1–ACTIN7 axis and thereby modulate the membrane localization of the auxin transport proteins PIN and ABCB (Zhu et al. 2016). The effect of NPA on these multiple targets involved in the auxin transport system will result in efficient inhibition of polar auxin movement by NPA in planta. The most successful chemical genetic screen of auxin mutants revealed TRANSPORT INHIBITOR RESPONSE (TIR) mutants that alter the mutant response to NPA (Ruegger et al. 1997). The TIR1 gene has been demonstrated to encode a nuclear auxin receptor (Chapman and Estelle 2009). Later, TIR2 was shown to be a TAA1 in the IPyA auxin biosynthetic pathway (Yamada et al. 2009). Another classical ATI, 2,3,5-triiodobenzoic acid (TIBA), disrupts the membrane localization of PIN by perturbing PIN vesicle trafficking processes (Dhonukshe et al. 2008). In addition to these classical ATIs, a phenotype-based high-throughput screen using Arabidopsis identified two ATIs from a chemical library. BUM, {2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid}, shows similar inhibitory effects on NPA, and its primary target has been elucidated to be ABCB proteins (Kim et al. 2010). BUM has been reported to share its binding site in ABCB with NPA by a competitive binding assay. Gravacin {3-(5-[3, 4- dichlorophenyl]-2-furyl) acrylic acid} shows potent inhibition of gravitropism in Arabidopsis (Rojas-Pierce et al. 2007). A chemical genetic screen of the gravacin-resistant mutant grav-r1 revealed a loss-of-function abcb19 mutant, which suggested that the primary target of gravacin was an ABCB19 transporter. Nishimura et al. reported two groups of ATIs from a chemical library using maize coleoptiles (Nishimura et al. 2012). One (group A) was composed of inhibitors exhibiting the NPA-like activity of the Arabidopsis phenotype. Two active molecules in group A, 37-H4 and 48-F9, are structurally different from NPA. Another (group B) was composed of modulators of subcellular trafficking of PIN proteins. Compound 8-C9 promoted the internalization of PIN2 and PIN1 proteins into compartments, suggesting that this compound exhibited similar activity to brefeldin A (BFA), an inhibitor of subcellular vesicle trafficking (Geldner et al. 2001). Flavonoids such as quercetin inhibit polar auxin transport and are suggested to be endogenous modulators of local auxin distribution. Flavonoid-deficient transparent testa4 mutants show elevated root basipetal auxin transport, suggesting that flavonoids are endogenous negative regulators of auxin transport (Peer and Murphy 2007). cis-Cinnamic acid has been reported as a natural auxin efflux inhibitor (Steenackers et al. 2017); it potentially blocks auxin efflux transport in tobacco BY-2 cells and inhibits the gravitropic response of primary root in Arabidopsis. In contrast to known ATIs (NPA and TIBA), cis-cinnamic acid promotes lateral root formation. cis-Cinnamic acid will inhibit shootward auxin transport by perturbing the redistribution of auxin in the meristem to accumulate endogenous IAA in the root. Another cinnamic acid derivative, 3,4-(methylenedioxy)cinnamic acid (MDCA), has also been reported as an auxin efflux inhibitor (Steenackers et al. 2016). MDCA was originally found in the root of asparagus plants and identified as an allelochemical, showing inhibition of primary root growth and promotion of lateral and adventitious roots. However, the mode of action and primary target of these natural inhibitors have not been elucidated. Alkoxy-auxins, 5-alkoxy-IAA and 7-alkoxy-NAA, are designed as active auxin analogs for auxin transport machinery, but inactive analogs for TIR1/AFB auxin receptors (Tsuda et al. 2011). Alkoxy-auxins competitively inhibit the transport activity of PINs, ABCBs and AUX1 that are expressed heterologously in yeast cells, but alkoxy-auxins are completely inactive in auxin SCFTIR1/AFB signaling in addition to the subcellular membrane relocalization of PIN proteins. Consistent with the transport profiles of IAA and NAA, the 5-alkoxy-IAA, Bz-IAA showed inhibition of PIN, ABCB and AUX1 activities. In contrast, the 7-alkoxy-NAA, Bz-NAA is specific to PINs and ABCBs. Alkoxy-auxins will be reorganized as a substrate for the auxin transport proteins PIN and ABCB, and, therefore, alkoxy-auxin reduces the endogenous IAA transport rate in competition with endogenous IAA, suggesting that alkoxy-auxins are a new class of ATI with an evident mode of action. In contrast to various inhibitors of auxin efflux transport, a few specific inhibitors of auxin influx transport have been reported to date. 1-Naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA) have been identified as specific inhibitors of auxin influx transport by the AUX1/LAX importer (Parry et al. 2001). 1-NOA reduces the root gravitropic response similarly to the aux1 mutant. 1-NOA can inhibit auxin uptake by AUX1 expressed in Xenopus oocytes (Yang et al. 2006). This evidence indicates that 1-NOA will directly target the AUX1 transport protein. 1-NOA has a similar structure to auxins, implying that it can competitively inhibit IAA import by AUX1 like the alkoxy-auxin Bz-IAA. 2,4-D is imported by AUX1 as a substrate, but not by the auxin efflux machinery, which suggests that the inactive 2,4-D analog for auxin signaling functions as a competitive inhibitor of AUX1 (Yang et al. 2006). The 2,4-D analog ethyl 2-[(2-chloro-4-nitrophenyl)thio]acetate shows similar biological activity to 1-NOA and has been identified as an inhibitor of the auxin influx carrier (Suzuki et al. 2014). Recently, the jasmonoyl-l-tryptophan (JA–Trp) conjugate was shown to target AUX1 protein and reduce the root gravitropic response (Staswick et al. 2017); however, the mode of action of the JA–Trp conjugate has not been elucidated. Visualization of the Cellular Auxin Distribution in Planta Visualization of the auxin gradient in planta has been extensively investigated in auxin biology. Visualization of the auxin gradient has been performed using an auxin-responsive reporter line, such as the DR5::GFP and DII-VENUS lines (Friml et al. 2003, Brunoud et al. 2012, Liao et al. 2015). These reporter lines have been widely used as essential tools to monitor the auxin levels in plants. However, the spatiotemporal resolution of these reporter systems is limited because of reporter protein localization within the cell. Fluorescent auxin analogs have been designed based on structure–activity studies of alkoxy-auxin. Alkoxy-auxin analogs are recognized as the substrate of the auxin transporters AUX1, PIN and ABCB, and are transported without interfering with endogenous auxin movement at low concentrations. Therefore, alkoxy-auxin analogs will be distributed like endogenous auxin (Tsuda et al. 2011). Fluorescently tagged alkoxy-auxin analogs function as inactive auxin for auxin signaling, but the analogs show a similar distribution pattern to the endogenous auxin gradient. The fluorescent auxin analogs (NBD-auxins) NBD-IAA and NBD-NAA enabled visualization of the auxin transport site and subcellular auxin gradient in plant cells (Hayashi et al. 2014). Consistent with the localization of some PIN and PILS transport proteins at the ER (Zazimalova et al. 2010, Barbez et al. 2012), NBD-auxins were highly accumulated at the ER in tobacco and Arabidopsis plants. The NBD-auxins can complement molecular biological approaches using auxin reporter lines and provide valuable tools for auxin biology. Recently, another chemical biology approach for the visualization of auxin-binding proteins has been reported (Mravec et al. 2017). The azido derivative of indole-3-propionic acid, (S)-2-azido-3-(3-indolyl)propionic acid (IPA-N3), was used as the tagged auxin analog. IPA-N3 showed similar auxinic activity to IAA in Arabidopsis. After IPA-N3 was applied to seedlings, it was washed and fixed with amino groups of nearby proteins by a coupling reagent. The azido group in IPA-N3 was specifically ligated to alkyne-conjugated fluorescent dye by the click reaction between azido and alkyne. The IPA-N3-binding proteins were visualized by fluorescent imaging. In this approach, IPA-N3-binding protein was found to be localized in the cell walls of elongating root cells (Mravec et al. 2017). Auxin Signaling: Small Molecule Modulator of the SCFTIR1/AFB Pathway The term ‘auxin’, derived from the Greek word ‘auxein’ (to increase or to grow), is associated with a chemical substance that promotes cell elongation activity in plants, originally in Avena (oat) coleoptiles (Enders and Strader 2015). The profound and pleiotropic hormonal effects of IAA on plant growth accelerated the discovery of auxin-like compounds for the development of a potent synthetic auxin. A number of structurally diverse synthetic auxins were reported from the 1940s to 1970s. These synthetic auxins perturb auxin-regulated plant growth and thus have been widely used as herbicides to control weeds. The classical definition of an ‘auxin’ is a molecule that shows similar plant hormone responses to those elicited by IAA. The auxin molecule, in the receptor-based definition, is perceived by the auxin-binding site of the TIR1/AFB family of auxin receptors and then forms the TIR1–auxin–Aux/IAA ternary complex to catalyze the ubiquitination of Aux/IAA repressors (Salehin et al. 2015, Tan et al. 2007). In this molecular model, molecules that bind to the TIR1/AFB receptor and promote the formation of the TIR1–auxin–Aux/IAA ternary complex can be considered ‘auxins’ (Salehin et al. 2015). The molecular structure of the TIR1–auxin–Aux/IAA ternary complex clearly illustrates the essential structural features of the auxin molecule required for the formation of the ternary complex: the planar aromatic ring system and carboxylic acid as a side chain attached to the aromatic ring (Tan et al. 2007). Based on these structural features, synthetic auxins have been classified into five major groups, naphthaleneacetic acid, e.g. NAA; phenoxyacetic acid, e.g. 2,4-D; indole-type auxins, e.g. haloganated IAA; benzoic acid, e.g. dicamba; and picolinate-type, e.g. picloram. The metabolism, transport, distribution and primary target receptors among the TIR1/AFB family differ between natural and synthetic auxins. Simon et al. reported a comprehensive analysis of the biological effects of diverse synthetic auxins on auxin-dependent gene expression, the endocytosis of PIN proteins across the plasma membrane and the phenotypic auxin responses of root and hypocotyl (Simon et al. 2013). These synthetic auxins induce strikingly divergent phenotypes in terms of hypocotyl elongation, lateral root development and primary root growth. This observation indicates that the physiological activities of synthetic auxins are complicated due to the distinct impact of auxins on chemical and metabolic stability, distribution in tissues in a polar manner and affinity towards specific sets of auxin receptors. Auxin-regulated transcription is very rapid, and a large number of genes respond to auxin within minutes. Early auxin-responsive genes are classified into three major families of genes: Aux/IAA, GH3 and SMALL AUXIN UP RNA (SAUR). The SUAR genes are involved in the cell expansion triggered by the phosphorylation of plasma membrane H+-ATPase (Spartz et al. 2012, Takahashi et al. 2012). The SAUR protein inhibits PP2C-D phosphatases to repress dephosphorylation of the H+-ATPase (Takahashi et al. 2012, Spartz et al. 2014). The Aux/IAA family, of which there are 29 members in Arabidopsis, encodes short-lived nuclear proteins that are repressors of auxin-responsive gene expression. The typical Aux/IAA repressor consists of four conserved domains, domains I– IV. Domain III and IV are responsible for heterodimerization with the ARF transcription factor to repress the transcriptional activation of auxin-responsive genes (Hayashi 2012). As the initial step in auxin signaling, auxin is perceived by TIR1, an F-box protein auxin receptor. TIR1 forms the SCFTIR1 E3 ubiquitin ligase complex consisting of Skp1 (ASK1) and Cullin (CUL1) to catalyze the ubiquitination of the F-box target protein. Auxin enhances the ubiquitination of Aux/IAA by promoting the interaction between Aux/IAA and TIR1 receptors. Consequently, in the presence of auxin, Aux/IAA repressors are ubiquitinated and degraded by the 26S proteasome pathway to activate ARF transcriptional activity. Arabidopsis has TIR1, an auxin receptor and five additional TIR1 homolog proteins, AFB1–5 (Auxin F-Box) (Hayashi 2012, Salehin et al. 2015). A structural study of the TIR1–auxin–Aux/IAA complex has demonstrated that auxin nestles on the floor of the surface pocket formed by the leucine-rich repeat (LRR) domain in TIR1, and TIR1-bound auxin enhances the interaction between the Aux/IAA domain II motif (GWPPV) and auxin-bound site of TIR1. The tryptophan (W) and second proline (P) residues in domain II are positioned close to the aromatic ring of auxin via hydrophobic interactions. Aux/IAA works as a co-receptor that forms the small hydrophobic cavity in the auxin-binding site of TIR1 (Tan et al. 2007), which suggests that auxin acts as a ‘molecular glue’ by which the two proteins are tightly bound together. The molecular recognition mechanism of TIR1 or the related AFB proteins for distinct synthetic auxins has been revealed by biochemical and genetic approaches (Calderon Villalobos et al. 2012, Lee et al. 2014). A synthetic auxin-insensitive mutant screen using the novel pyridine-type synthetic auxin DAS534 (an analog of the auxinic herbicide picloram) identified the loss-of-function mutation of AFB5 receptors in Arabidopsis (Walsh et al. 2006). The loss of AFB5 confers resistance to picolinate auxins, but not to 2,4-D or IAA. Additionally, afb4 afb5 double mutants show slightly more resistance to picloram than the afb5 single mutant, suggesting that AFB4 and AFB5 are the primary targets of picloram-type auxinic herbicide (Prigge et al. 2016). A surface plasmon resonance (SPR) binding assay clearly demonstrated that DAS534 and picloram show higher affinity for AFB5 than the TIR1 receptor complex (Lee et al. 2014). The distinct sensitivity of tir1 and afb5 mutants to synthetic auxins indicates significant differences in the molecular recognition of auxins among the auxin receptor family that could be applicable for the design of new auxinic herbicides. Rational Design of the Auxin Antagonist of TIR1 Receptor The molecular mechanism of auxin perception provides an opportunity for the structure-based molecular design of an auxin antagonist. The binding of auxin to the Aux/IAA–TIR1 co-receptor complex does not alter the conformation of the flexible side chains of TIR1 or the Aux/IAA around the auxin-binding site, indicating that the auxin-binding site can be considered as a rigid form. Therefore, the structure of the TIR1-specific ligand can be rationally designed without consideration of flexible side chain conformers determining the cavity shape in the binding site. The rational design of TIR1 probes has led to the generation of tert-butoxycarbonylaminohexyl-IAA (BH-IAA) with potent antagonistic activity toward auxin activity mediated by the SCFTIR1/AFB pathway (Hayashi et al. 2008). The crystal structures of TIR1 in complex with BH-IAA illustrate the molecular mechanism of BH-IAA. In the auxin-binding site, the IAA moiety of BH-IAA is positioned in the same conformation as IAA, and the long alkyl chain is directed toward the Aux/IAA-binding site. This long alkyl chain occupies the Aux/IAA-binding site efficiently to prevent access of the domain II GWPPV motif of Aux/IAA. The long alkyl chain is flexible in the auxin-binding site of TIR1. Therefore, the IAA moiety of BH-IAA is solely responsible for the binding affinity of BH-IAA for TIR1, and Aux/IAA is not involved in the binding of BH-IAA. In contrast, IAA is tightly captured in the small cavity formed by both TIR1 and Aux/IAA, which indicates that the TIR1–Aux/IAA co-receptor complex has a higher affinity for IAA. Thus, the low concentration of exogenous IAA can completely abrogate the antiauxin activity of BH-IAA in planta (Hayashi et al. 2008). Auxinole was designed as an auxin antagonist of the TIR1 receptor based on in silico molecular docking calculations (Hayashi et al. 2012). Auxinole was designed to increase the affinity for TIR1 in the binding site. Auxinole has a 2-oxo-phenylethyl group at the α-position of IAA that can interact with a phenylalanine residue (Phe82) of TIR1 via a strong π–π stacking interaction. Auxinole shows very potent antiauxin activity and is effective in diverse plants, including monocots, rice and the moss P. patens. Auxinole shows reversible inhibition, which can be spatiotemporally controlled, and therefore auxinole has been widely used to study auxin responses mediated by the SCFTIR1 auxin pathway (Hayashi et al. 2012). Non-Transcriptional Auxin Signaling Most auxin responses are transcriptionally regulated by the SCFTIR1/AFB pathway. Auxin triggers rapid cell wall acidification and elongation of hypocotyls. These rapid responses have long been thought to be regulated by non-transcriptional auxin signaling. Fendrych et al. demonstrated that the TIR1/AFB receptor is required for the auxin-induced acidification and subsequent elongation of Arabidopsis etiolated hypocotyls (Fendrych et al. 2016). By using an elegant chemical biology approach, Uchida et al. also revealed that the TIR1 receptor is essential for auxin-induced phosphorylation of H+-ATPase and subsequent hypocotyl elongation (Uchida et al. 2018). The engineered TIR1 mutant (F79G) was designed to recognize specifically 5-aryl IAA (cvxIAA) that could not activate the native TIR1 receptor. Thus, 5-aryl IAA can selectively activate an auxin transcriptional signal and its downstream events via the SCFTIR1(F79G) machinery in then transgenic plants expressing the engineered TIR1 (F79G) receptor. The localization of PIN protein on the plasma membrane determines the direction of auxin flow to modulate the local auxin distribution (Naramoto 2017). Auxin has been reported to inhibit clathrin-mediated endocytosis of PIN protein. ABP1 has been reported to have a positive role in clathrin recruitment to the plasma membrane, leading to the endocytosis of PIN (Robert et al. 2010). Auxin signaling is mediated by ABP1 accompanied by the activation of ROP proteins (Chen et al. 2012). However, abp1 null mutants generated by CRISPR (clustered regularly interspaced short palindromic repeats) technology clearly demonstrate that ABP1 does not play a major role in auxin-regulated developmental processes (Gao et al. 2015). Whether another type of auxin receptor is involved in the non-transcriptional auxin signaling pathway is not known. The molecular actions of non-transcriptional auxin responses remain largely unknown. The chemical probes specific for such non-transcriptional auxin responses are highly anticipated for studies of auxin biology. Concluding Remarks Progress over the past decades in auxin biology has demonstrated fundamental components and molecular mechanisms of auxin biosynthesis, signal transduction and the polar auxin transport system. Small molecule modulators of auxin-related processes, including synthetic auxins, have greatly accelerated auxin research in combination with the molecular genetic approaches in Arabidopsis biology. The most important advance in auxin research was the identification of the function of TIR1 as an auxin receptor and the structural determination of the TIR1–Aux/IAA auxin co-receptor. The structure of TIR1–Aux/IAA proposed the fundamental molecular mechanism of plant hormone perception of the co-receptor system. We now understand the structural information for the basic components of auxin biosynthesis (TAA1) (Tao et al. 2008), metabolism (GH3 and ILL2) (Bitto et al. 2009, Peat et al. 2012) and signaling (TIR1, Aux/IAA and ARF) (Chapman and Estelle 2009, Dinesh et al. 2015, Roosjen et al. 2018). These structural data open the door to the rational design of plant hormone modulators in auxin biology. The auxin concentration gradient is spatiotemporally regulated by auxin biosynthesis, catabolism and transport. The auxin gradient is then transduced to the auxin signaling machinery to alter the developmental output. These auxin-related regulatory processes are tightly interconnected, and so the processes co-ordinately modulate the developmental response to environmental cues. However, the complicated but precise regulatory mechanism underlying pleiotropic auxin effects on plant growth and development remain to be determined. To understand the complicated network in the pleiotropic responses of auxin, a new approach from a different field is highly anticipated. Chemical biology appears to be one promising approach, in combination with new systematic approaches such as proteomics, metabolomics and mathematical modeling. Chemical tools that are specifically designed for auxin biology could be valuable for the modulation of auxin perception, signaling, transport and even biosynthesis and catabolism at a specific developmental stage and in a specific tissue. The development of unique chemical tools for the specific auxin response would uncover new components of auxin-regulated developmental processes. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for Scientific Research (15K01828) to K.H.]. Acknowledgments We are grateful to Professor Hiroyuki Kasahara (Tokyo University of Agriculture and Technology) for critical reading of the manuscript. Disclosures The authors have no conflicts of interest to declare. References Adamowski M., Friml J. ( 2015) PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell  27: 20– 32. Google Scholar CrossRef Search ADS PubMed  Bailly A., Sovero V., Vincenzetti V., Santelia D., Bartnik D., Koenig B.W., et al.   ( 2008) Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins. J. Biol. Chem.  283: 21817– 21826. Google Scholar CrossRef Search ADS PubMed  Barbez E., Kubeš M., Rolčík J., Béziat C., Pěnčík A., Wang B., et al.   ( 2012) A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature  485: 119– 122. Google Scholar CrossRef Search ADS PubMed  Bitto E., Bingman C.A., Bittova L., Houston N.L., Boston R.S., Fox B.G., et al.   ( 2009) X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana. Proteins  74: 61– 71. Google Scholar CrossRef Search ADS PubMed  Bottcher C., Dennis E.G., Booker G.W., Polyak S.W., Boss P.K., Davies C. ( 2012) A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue. PLoS One  7: e37632. Google Scholar CrossRef Search ADS PubMed  Brunoud G., Wells D.M., Oliva M., Larrieu A., Mirabet V., Burrow A.H., et al.   ( 2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature  482: 103– 106. Google Scholar CrossRef Search ADS PubMed  Calderon Villalobos L.I., Lee S., De Oliveira C., Ivetac A., Brandt W., Armitage L., et al.   ( 2012) A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin. Nat. Chem. Biol.  8: 477– 485. Google Scholar CrossRef Search ADS PubMed  Chapman E.J., Estelle M. ( 2009) Mechanism of auxin-regulated gene expression in plants. Annu. Rev. Genet.  43: 265– 285. Google Scholar CrossRef Search ADS PubMed  Chen Q., Dai X., De-Paoli H., Cheng Y., Takebayashi Y., Kasahara H., et al.   ( 2014) Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots. Plant Cell Physiol . 55: 1072– 1079. Google Scholar CrossRef Search ADS PubMed  Chen X., Naramoto S., Robert S., Tejos R., Lofke C., Lin D., et al.   ( 2012) ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots. Curr. Biol . 22: 1326– 1332. Google Scholar CrossRef Search ADS PubMed  De Rybel B., Audenaert D., Beeckman T., Kepinski S. ( 2009) The past, present, and future of chemical biology in auxin research. ACS Chem. Biol.  4: 987– 998. Google Scholar CrossRef Search ADS PubMed  Dhonukshe P., Grigoriev I., Fischer R., Tominaga M., Robinson D.G., Hasek J., et al.   ( 2008) Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes. Proc. Natl. Acad. Sci. USA  105: 4489– 4494. Google Scholar CrossRef Search ADS   Dinesh D.C., Kovermann M., Gopalswamy M., Hellmuth A., Calderon Villalobos L.I., Lilie H., et al.   ( 2015) Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response. Proc. Natl. Acad. Sci. USA  112: 6230– 6235. Google Scholar CrossRef Search ADS   Eklund D.M., Ishizaki K., Flores-Sandoval E., Kikuchi S., Takebayashi Y., Tsukamoto S., et al.   ( 2015) Auxin produced by the indole-3-pyruvic acid pathway regulates development and gemmae dormancy in the liverwort Marchantia polymorpha. Plant Cell  27: 1650– 1669. Google Scholar CrossRef Search ADS PubMed  Enders T.A., Strader L.C. ( 2015) Auxin activity: past, present, and future. Amer. J. Bot.  102: 180– 196. Google Scholar CrossRef Search ADS   Eswaramoorthy S., Bonanno J.B., Burley S.K., Swaminathan S. ( 2006) Mechanism of action of a flavin-containing monooxygenase. Proc. Natl. Acad. Sci. USA  103: 9832– 9837. Google Scholar CrossRef Search ADS   Fendrych M., Leung J., Friml J. ( 2016) TIR1/AFB–Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. eLife  5: e19048. Google Scholar CrossRef Search ADS PubMed  Friml J., Vieten A., Sauer M., Weijers D., Schwarz H., Hamann T., et al.   ( 2003) Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis. Nature  426: 147– 153. Google Scholar CrossRef Search ADS PubMed  Gao Y., Zhang Y., Zhang D., Dai X., Estelle M., Zhao Y. ( 2015) Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc. Natl. Acad. Sci. USA  112: 2275– 2280. Google Scholar CrossRef Search ADS   Geldner N., Friml J., Stierhof Y.D., Jurgens G., Palme K. ( 2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature  413: 425– 428. Google Scholar CrossRef Search ADS PubMed  Hayashi K. ( 2012) The interaction and integration of auxin signaling components. Plant Cell Physiol . 53: 965– 975. Google Scholar CrossRef Search ADS PubMed  Hayashi K., Nakamura S., Fukunaga S., Nishimura T., Jenness M.K., Murphy A.S., et al.   ( 2014) Auxin transport sites are visualized in planta using fluorescent auxin analogs. Proc. Natl. Acad. Sci. USA  111: 11557– 11562. Google Scholar CrossRef Search ADS   Hayashi K., Neve J., Hirose M., Kuboki A., Shimada Y., Kepinski S., et al.   ( 2012) Rational design of an auxin antagonist of the SCF(TIR1) auxin receptor complex. ACS Chem. Biol.  7: 590– 598. Google Scholar CrossRef Search ADS PubMed  Hayashi K., Tan X., Zheng N., Hatate T., Kimura Y., Kepinski S., et al.   ( 2008) Small-molecule agonists and antagonists of F-box protein–substrate interactions in auxin perception and signaling. Proc. Natl. Acad. Sci. USA  105: 5632– 5637. Google Scholar CrossRef Search ADS   He W., Brumos J., Li H., Ji Y., Ke M., Gong X., et al.   ( 2011) A small-molecule screen identifies l-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis. Plant Cell  23: 3944– 3960. Google Scholar CrossRef Search ADS PubMed  Hicks G.R., Raikhel N.V. ( 2014) Plant chemical biology: are we meeting the promise? Front. Plant Sci.  5: 455. Google Scholar CrossRef Search ADS PubMed  Kakei Y., Nakamura A., Yamamoto M., Ishida Y., Yamazaki C., Sato A., et al.   ( 2017) Biochemical and chemical biology study of rice OsTAR1 revealed that tryptophan aminotransferase is involved in auxin biosynthesis: identification of a potent OsTAR1 inhibitor, pyruvamine2031. Plant Cell Physiol . 58: 598– 606. Google Scholar PubMed  Kakei Y., Yamazaki C., Suzuki M., Nakamura A., Sato A., Ishida Y., et al.   ( 2015) Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function. Plant J.  84: 827– 837. Google Scholar CrossRef Search ADS PubMed  Kasahara H. ( 2016) Current aspects of auxin biosynthesis in plants. Biosci. Biotechnol. Biochem . 80: 34– 42. Google Scholar CrossRef Search ADS PubMed  Katekar G.F., Geissler A.E. ( 1980) Auxin transport inhibitors: IV. Evidence of a common mode of action for a proposed class of auxin transport inhibitors: the phytotropins. Plant Physiol . 66: 1190– 1195. Google Scholar CrossRef Search ADS PubMed  Kato H., Nishihama R., Weijers D., Kohchi T. ( 2017) Evolution of nuclear auxin signaling: lessons from genetic studies with basal land plants. J. Exp. Bot . 69: 292– 301. Kim J.Y., Henrichs S., Bailly A., Vincenzetti V., Sovero V., Mancuso S., et al.   ( 2010) Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics. J. Biol. Chem.  285: 23309– 23317. Google Scholar CrossRef Search ADS PubMed  Korasick D.A., Enders T.A., Strader L.C. ( 2013) Auxin biosynthesis and storage forms. J. Exp. Bot . 64: 2541– 2555. Google Scholar CrossRef Search ADS PubMed  Lee S., Sundaram S., Armitage L., Evans J.P., Hawkes T., Kepinski S., et al.   ( 2014) Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)–Aux/IAA co-receptor complex formation. ACS Chem. Biol.  9: 673– 682. Google Scholar CrossRef Search ADS PubMed  Liao C.Y., Smet W., Brunoud G., Yoshida S., Vernoux T., Weijers D. ( 2015) Reporters for sensitive and quantitative measurement of auxin response. Nat. Methods  12: 207– 210. Google Scholar CrossRef Search ADS PubMed  Ludwig-Muller J. ( 2011) Auxin conjugates: their role for plant development and in the evolution of land plants. J. Exp. Bot . 62: 1757– 1773. Google Scholar CrossRef Search ADS PubMed  Ma Q., Robert S. ( 2014) Auxin biology revealed by small molecules. Physiol. Plant.  151: 25– 42. Google Scholar CrossRef Search ADS PubMed  Marchant A., Kargul J., May S.T., Muller P., Delbarre A., Perrot-Rechenmann C., et al.   ( 1999) AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J . 18: 2066– 2073. Google Scholar CrossRef Search ADS PubMed  Mashiguchi K., Tanaka K., Sakai T., Sugawara S., Kawaide H., Natsume M., et al.   ( 2011) The main auxin biosynthesis pathway in Arabidopsis. Proc. Natl. Acad. Sci. USA  108: 18512– 18517. Google Scholar CrossRef Search ADS   Meesters C., Monig T., Oeljeklaus J., Krahn D., Westfall C.S., Hause B., et al.   ( 2014) A chemical inhibitor of jasmonate signaling targets JAR1 in Arabidopsis thaliana. Nat. Chem. Biol.  10: 830– 836. Google Scholar CrossRef Search ADS PubMed  Mravec J., Kracun S.K., Zemlyanskaya E., Rydahl M.G., Guo X., Picmanova M., et al.   ( 2017) Click chemistry-based tracking reveals putative cell wall-located auxin binding sites in expanding cells. Sci. Rep.  7: 15988. Google Scholar CrossRef Search ADS PubMed  Naramoto S. ( 2017) Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport. Curr. Opin. Plant Biol . 40: 8– 14. Google Scholar CrossRef Search ADS PubMed  Narukawa-Nara M., Nakamura A., Kikuzato K., Kakei Y., Sato A., Mitani Y., et al.   ( 2016) Aminooxy-naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l-tryptophan aminotransferase: structure–activity relationships. Plant J.  87: 245– 257. Google Scholar CrossRef Search ADS PubMed  Nishimura T., Hayashi K., Suzuki H., Gyohda A., Takaoka C., Sakaguchi Y., et al.   ( 2014) Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis. Plant J.  77: 352– 366. Google Scholar CrossRef Search ADS PubMed  Nishimura T., Matano N., Morishima T., Kakinuma C., Hayashi K., Komano T., et al.   ( 2012) Identification of IAA transport inhibitors including compounds affecting cellular PIN trafficking by two chemical screening approaches using maize coleoptile systems. Plant Cell Physiol . 53: 1671– 1682. Google Scholar CrossRef Search ADS PubMed  Noh B., Murphy A.S., Spalding E.P. ( 2001) Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell  13: 2441– 2454. Google Scholar CrossRef Search ADS PubMed  Parry G., Delbarre A., Marchant A., Swarup R., Napier R., Perrot-Rechenmann C., et al.   ( 2001) Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1. Plant J . 25: 399– 406. Google Scholar CrossRef Search ADS PubMed  Peat T.S., Bottcher C., Newman J., Lucent D., Cowieson N., Davies C. ( 2012) Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis. Plant Cell  24: 4525– 4538. Google Scholar CrossRef Search ADS PubMed  Peer W.A., Murphy A.S. ( 2007) Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci.  12: 556– 563. Google Scholar CrossRef Search ADS PubMed  Porco S., Pěnčík A., Rashed A., Voß U., Casanova-Sáez R., Bishopp A., et al.   ( 2016) Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis. Proc. Natl. Acad. Sci. USA  113: 11016– 11021. Google Scholar CrossRef Search ADS   Prigge M.J., Greenham K., Zhang Y., Santner A., Castillejo C., Mutka A.M., et al.   ( 2016) The arabidopsis auxin receptor F-box proteins AFB4 and AFB5 are required for response to the synthetic auxin picloram. G3 (Bethesda)  6: 1383– 1390. Google Scholar CrossRef Search ADS PubMed  Robert S., Kleine-Vehn J., Barbez E., Sauer M., Paciorek T., Baster P., et al.   ( 2010) ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis. Cell  143: 111– 121. Google Scholar CrossRef Search ADS PubMed  Rojas-Pierce M., Titapiwatanakun B., Sohn E.J., Fang F., Larive C.K., Blakeslee J., et al.   ( 2007) Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin. Chem. Biol . 14: 1366– 1376. Google Scholar CrossRef Search ADS PubMed  Roosjen M., Paque S., Weijers D. ( 2018) Auxin response factors: output control in auxin biology. J. Exp. Bot.  69: 179– 188. Google Scholar CrossRef Search ADS PubMed  Ruegger M., Dewey E., Hobbie L., Brown D., Bernasconi P., Turner J., et al.   ( 1997) Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell  9: 745– 757. Google Scholar CrossRef Search ADS PubMed  Salehin M., Bagchi R., Estelle M. ( 2015) SCFTIR1/AFB-based auxin perception: mechanism and role in plant growth and development. Plant Cell  27: 9– 19. Google Scholar CrossRef Search ADS PubMed  Simon S., Kubes M., Baster P., Robert S., Dobrev P.I., Friml J., et al.   ( 2013) Defining the selectivity of processes along the auxin response chain: a study using auxin analogues. New Phytol.  200: 1034– 1048. Google Scholar CrossRef Search ADS PubMed  Soeno K., Goda H., Ishii T., Ogura T., Tachikawa T., Sasaki E., et al.   ( 2010) Auxin biosynthesis inhibitors, identified by a genomics-based approach, provide insights into auxin biosynthesis. Plant Cell Physiol . 51: 524– 536. Google Scholar CrossRef Search ADS PubMed  Spartz A.K., Lee S.H., Wenger J.P., Gonzalez N., Itoh H., Inze D., et al.   ( 2012) The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion. Plant J . 70: 978– 990. Google Scholar CrossRef Search ADS PubMed  Spartz A.K., Ren H., Park M.Y., Grandt K.N., Lee S.H., Murphy A.S., et al.   ( 2014) SAUR inhibition of PP2C-D phosphatases activates plasma membrane H+-ATPases to promote cell expansion in Arabidopsis. Plant Cell  26: 2129– 2142. Google Scholar CrossRef Search ADS PubMed  Staswick P., Rowe M., Spalding E.P., Splitt B.L. ( 2017) Jasmonoyl-l-tryptophan disrupts IAA activity through the AUX1 auxin permease. Front. Plant Sci.  8: 736. Google Scholar CrossRef Search ADS PubMed  Steenackers W., Cesarino I., Klima P., Quareshy M., Vanholme R., Corneillie S., et al.   ( 2016) The allelochemical MDCA inhibits lignification and affects auxin homeostasis. Plant Physiol . 172: 874– 888. Google Scholar PubMed  Steenackers W., Klima P., Quareshy M., Cesarino I., Kumpf R.P., Corneillie S., et al.   ( 2017) cis-Cinnamic acid is a novel, natural auxin efflux inhibitor that promotes lateral root formation. Plant Physiol.  173: 552– 565. Google Scholar CrossRef Search ADS PubMed  Sugawara S., Hishiyama S., Jikumaru Y., Hanada A., Nishimura T., Koshiba T., et al.   ( 2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc. Natl. Acad. Sci. USA  106: 5430– 5435. Google Scholar CrossRef Search ADS   Sugawara S., Mashiguchi K., Tanaka K., Hishiyama S., Sakai T., Hanada K., et al.   ( 2015) Distinct characteristics of indole-3-acetic acid and phenylacetic acid, two common auxins in plants. Plant Cell Physiol.  56: 1641– 1654. Google Scholar CrossRef Search ADS PubMed  Suzuki H., Matano N., Nishimura T., Koshiba T. ( 2014) A 2,4-dichlorophenoxyacetic acid analog screened using a maize coleoptile system potentially inhibits indole-3-acetic acid influx in Arabidopsis thaliana. Plant Signal. Behav.  9: e29077. Google Scholar CrossRef Search ADS PubMed  Takahashi K., Hayashi K., Kinoshita T. ( 2012) Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. Plant Physiol . 159: 632– 641. Google Scholar CrossRef Search ADS PubMed  Tan X., Calderon-Villalobos L.I., Sharon M., Zheng C., Robinson C.V., Estelle M., et al.   ( 2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature  446: 640– 645. Google Scholar CrossRef Search ADS PubMed  Tao Y., Ferrer J.L., Ljung K., Pojer F., Hong F., Long J.A., et al.   ( 2008) Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell  133: 164– 176. Google Scholar CrossRef Search ADS PubMed  Tsuda E., Yang H., Nishimura T., Uehara Y., Sakai T., Furutani M., et al.   ( 2011) Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters. J. Biol. Chem.  286: 2354– 2364. Google Scholar CrossRef Search ADS PubMed  Tsugafune S., Mashiguchi K., Fukui K., Takebayashi Y., Nishimura T., Sakai T., et al.   ( 2017) Yucasin DF, a potent and persistent inhibitor of auxin biosynthesis in plants. Sci. Rep.  7: 13992. Google Scholar CrossRef Search ADS PubMed  Uchida N., Takahashi K., Iwasaki R., Yamada R., Yoshimura M., Endo T.A., et al.   ( 2018) Chemical hijacking of auxin signaling with an engineered auxin–TIR1 pair. Nat. Chem. Biol.  14: 299– 305. Google Scholar CrossRef Search ADS PubMed  Walsh T.A., Neal R., Merlo A.O., Honma M., Hicks G.R., Wolff K., et al.   ( 2006) Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. Plant Physiol.  142: 542– 552. Google Scholar CrossRef Search ADS PubMed  Won C., Shen X., Mashiguchi K., Zheng Z., Dai X., Cheng Y., et al.   ( 2011) Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis. Proc. Natl. Acad. Sci. USA  108: 18518– 18523. Google Scholar CrossRef Search ADS   Woodward A.W., Bartel B. ( 2005) Auxin: regulation, action, and interaction. Ann. Bot.  95: 707– 735. Google Scholar CrossRef Search ADS PubMed  Yamada M., Greenham K., Prigge M.J., Jensen P.J., Estelle M. ( 2009) The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development. Plant Physiol . 151: 168– 179. Google Scholar CrossRef Search ADS PubMed  Yang Y., Hammes U.Z., Taylor C.G., Schachtman D.P., Nielsen E. ( 2006) High-affinity auxin transport by the AUX1 influx carrier protein. Curr. Biol . 16: 1123– 1127. Google Scholar CrossRef Search ADS PubMed  Yang H., Murphy A.S. ( 2009) Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. Plant J . 59: 179– 191. Google Scholar CrossRef Search ADS PubMed  Zazimalova E., Murphy A.S., Yang H., Hoyerova K., Hosek P. ( 2010) Auxin transporters—why so many? Cold Spring Harb. Perspect. Biol . 2: a001552. Google Scholar CrossRef Search ADS PubMed  Zhang J., Peer W.A. ( 2017) Auxin homeostasis: the DAO of catabolism. J. Exp. Bot . 68: 3145– 3154. Google Scholar CrossRef Search ADS PubMed  Zhu J., Bailly A., Zwiewka M., Sovero V., Di Donato M., Ge P., et al.   ( 2016) TWISTED DWARF1 mediates the action of auxin transport inhibitors on actin cytoskeleton dynamics. Plant Cell  28: 930– 948. Google Scholar PubMed  Abbreviations Abbreviations ABCB ATP-BINDING CASSETTE subfamily B AOPP L-2-aminooxy-3-phenylpropionic acid ARF auxin response factor ATI auxin transport inhibitor Aux/IAA AUXIN/INDOLE-3-ACETIC ACID AVG aminoethoxyvinylglycine BBo 4-biphenylboronic acid BH-IAA tert-butoxycarbonylaminohexyl-IAA BUM 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid ER endoplasmic reticulum GH3 GRETCHEN HAGEN3 IPA-N3 (S)-2-azido-3-(3-indolyl)propionic acid IPyA indole 3-pyruvic acid L-kyn L-kynurenine NAA naphthalene 1-acetic acid NBD-IAA nitrobenzoxadiazole (NBD)-labeled IAA NBD-NAA nitrobenzoxadiazole (NBD)-labeled naphthalene 1-acetic acid 1-NOA 1-naphthoxyacetic acid NPA N-1-naphthylphthalamic acid PIN PIN-FORMED PLP pyridoxyl-5-phosphate PPBo 4-phenoxyphenylboronic acid TIR TRANSPORT INHIBITOR RESPONSE YDF yucasin DF YUC YUCCA © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Manipulation and Sensing of Auxin Metabolism, Transport and Signaling

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Abstract

Abstract The plant hormone auxin is involved in virtually every aspect of plant growth and development. A chemical genetic approach has greatly contributed to the identification of important genes in auxin biosynthesis, transport and signaling. Molecular genetic technologies and structural information for auxin regulatory components have accelerated the identification and characterization of many novel small molecule modulators in auxin biology. These modulators have been widely utilized to dissect auxin responses. Here we provide an overview of the structure, primary target, in planta activity and application of small molecule modulators in auxin biology. Introduction Auxin, a plant hormone, regulates virtually every aspect of plant growth and development. Auxin functions as a master regulator in embryogenesis, apical dominance, lateral root formation, hypocotyl elongation, tropic responses to light and gravity, vascular tissue differentiation and lateral branching of shoots (Woodward and Bartel 2005, Enders and Strader 2015). IAA, a naturally occurring auxin, is a very simple molecule, but it exerts diverse hormonal actions on plant development. The auxin activity on various developmental processes can be spatiotemporally modulated by three major regulatory steps: auxin biosynthesis and inactivation; directional auxin transport; and signaling (Chapman and Estelle 2009, Zazimalova et al. 2010, Ludwig-Muller 2011, Hayashi 2012, Kasahara 2016). The intercellular auxin concentration gradient plays a crucial role in the regulation of auxin action. The auxin gradient is co-ordinately generated by auxin polar transport via auxin influx and efflux transportation and auxin metabolism including auxin biosynthesis, inactivation and degradation of auxin (Korasick et al. 2013, Naramoto 2017). At auxin concentration maxima in the tissue, auxin activates the transcription of auxin-responsive genes via the SCFTIR1/AFB–proteasome pathway, leading to auxin-related developmental responses (Chapman and Estelle 2009). In this transcriptional regulatory process, auxin enhances the proteolysis of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors by promoting the ubiquitination of the Aux/IAA proteins that are recognized as a substrate of the SCFTIR1/AFB-type E3 ubiquitin ligase complex. The Aux/IAA repressors bind to auxin response factor (ARF) transcription factors and block the transcriptional activity of ARFs. Thus, auxin-induced degradation of Aux/IAA results in the activation of ARFs to promote auxin-responsive gene expression (Chapman and Estelle 2009, Hayashi 2012). The conventional forward genetic approach in the model plant Arabidopsis has unveiled the underlying molecular mechanism of auxin action and identified essential genes regarding auxin-related pathways (Woodward and Bartel 2005). In an early work, most of the crucial auxin mutants were identified in a chemical genetic screen using synthetic auxins and small molecule modulators, and those mutations were found in key components in auxin biosynthesis, signaling and transport. Small molecule modulators of auxin biosynthesis, signaling and transport (auxin probes) have been considered as indispensable elements to study particular processes in auxin biology (Ruegger et al. 1997). Synthetic auxins are still used in almost all auxin research. Additionally, auxin probes have made great contributions to complementing genetic approaches in plant biology (De Rybel et al. 2009, Ma and Robert 2014). The functional redundancy of cognate gene families in the auxin regulatory pathway has blocked access to the physiological function of individual genes. Furthermore, the disruption of multiple pivotal genes often results in a lethal impairment (Hicks and Raikhel 2014). Fundamental components in auxin biosynthesis, signaling and catabolism have been suggested to be conserved across diverse land plants such as liverworts, mosses, ferns, gymnosperms and angiosperms (Eklund et al. 2015, Kato et al. 2017). To complement the genetic approach in a range of non-model plants with poor molecular genetic and genomic resources, the use of small molecule modulators to manipulate the auxin action spatiotemporally represents an alternative approach for auxin biology. Here we review natural and synthetic molecules that function as auxins. We also review the synthetic and endogenous small molecule modulators in auxin metabolism, auxin transport and auxin signaling (Fig. 1 and 2; Supplementary Table S1), and we introduce the application of these modulators in plant biology. Fig. 1 View largeDownload slide (A) Natural and synthetic auxins, and small molecule modulators of auxin biosynthesis, transport and signaling. Chemical name, CAS registry number, molecular weight, primary targets and availability are listed in Supplementary Table S1. (B) The binding structures of TIR1–IAA–Aux/IAA (left) and TIR1–auxinole (right). The phenyl ring of auxinole strongly interacts with Phe82 of TIR1 and blocks the access of Aux/IAA to the auxin-binding site. Fig. 1 View largeDownload slide (A) Natural and synthetic auxins, and small molecule modulators of auxin biosynthesis, transport and signaling. Chemical name, CAS registry number, molecular weight, primary targets and availability are listed in Supplementary Table S1. (B) The binding structures of TIR1–IAA–Aux/IAA (left) and TIR1–auxinole (right). The phenyl ring of auxinole strongly interacts with Phe82 of TIR1 and blocks the access of Aux/IAA to the auxin-binding site. Fig. 2 View largeDownload slide Cellular model of auxin biosynthesis, transport and signaling. IAA is biosynthesized from tryptophan (Trp) via indole 3-pyrvic acid (IPyA) by TAA1 and YUCCA in the IPyA pathway. IAA is incorporated by AUX1/LAX1 symporters and exported by PIN and ABCB proteins. ER-localized PIN and PILS carrier proteins regulate the homeostasis of cellular IAA. Auxin blocks the clathrin-mediated endocytosis of PIN from the plasma membrane. The SCFTIR1/AFB, E3 ubiquitin ligase complex is composed of SKP (ASK1), Cullin and F-box protein (TIR1/AFB). Auxin induces the ubiquitination of Aux/IAA proteins via SCFTIR1/AFB. The degradation of Aux/IAA repressor recovers ARF activity to activate the transcription of auxin-responsive genes. The SAUR proteins induced by auxin inhibit PP2C-D phosphatases, resulting in the activation of H+-ATPase to induce a rapid auxin response such as turgor-induced growth. IAA is converted to IAA–aspartate (IAA–Asp) by the auxin-induced GH3 enzyme IAA–amino acid conjugate synthase. IAA is oxidized to 2-oxo-IAA (oxIAA) by DAO1 dioxygenase. Fig. 2 View largeDownload slide Cellular model of auxin biosynthesis, transport and signaling. IAA is biosynthesized from tryptophan (Trp) via indole 3-pyrvic acid (IPyA) by TAA1 and YUCCA in the IPyA pathway. IAA is incorporated by AUX1/LAX1 symporters and exported by PIN and ABCB proteins. ER-localized PIN and PILS carrier proteins regulate the homeostasis of cellular IAA. Auxin blocks the clathrin-mediated endocytosis of PIN from the plasma membrane. The SCFTIR1/AFB, E3 ubiquitin ligase complex is composed of SKP (ASK1), Cullin and F-box protein (TIR1/AFB). Auxin induces the ubiquitination of Aux/IAA proteins via SCFTIR1/AFB. The degradation of Aux/IAA repressor recovers ARF activity to activate the transcription of auxin-responsive genes. The SAUR proteins induced by auxin inhibit PP2C-D phosphatases, resulting in the activation of H+-ATPase to induce a rapid auxin response such as turgor-induced growth. IAA is converted to IAA–aspartate (IAA–Asp) by the auxin-induced GH3 enzyme IAA–amino acid conjugate synthase. IAA is oxidized to 2-oxo-IAA (oxIAA) by DAO1 dioxygenase. Auxin Biosynthesis: Inhibitors of the IPyA Pathway IAA is predominantly biosynthesized via the indole 3-pyruvic acid (IPyA) pathway (Kasahara 2016). In the IPyA pathway, tryptophan is converted to IAA by two sequential enzymatic steps (Mashiguchi et al. 2011, Won et al. 2011). In the initial step, three aminotransferases encoded by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAA1/TAR) gene family produces IPyA from tryptophan in Arabidopsis. Subsequently, the YUCCA (YUC) flavin monooxygenase family catalyzes the oxidative decarboxylation of IPyA to IAA. Additionally, Cyt P450 monooxygenases CYP79B2 and CYP79B3 produce indole-3-acetaldoxime (IAOx) from tryptophan in Arabidopsis (Sugawara et al. 2009). IAOx is then converted to IAA in this auxiliary IAA biosynthesis pathway of the family Brassicaceae. Indole 3-acetaldehyde, indole 3-acetamide and tryptamine have been proposed as IAA precursors, but the roles of these precursors in IAA biosynthesis remain unknown (Kasahara 2016). Auxin biosynthesis inhibitors would be a very powerful tool for plant biology. l-Kynurenine (l-kyn) was identified as an auxin biosynthesis inhibitor from a commercial chemical library (He et al. 2011). l-Kyn was initially screened according to the inhibitory activity of ethylene responses in Arabidopsis root by using a phenotype-based screen of the constitutive ethylene response mutants, ethylene overproducer1-2 (eto1-2) and ctr1-1. l-Kyn is the degradation metabolite of tryptophan in animals, and therefore is regarded as a tryptophan analog. Consistent with the structural similarity of l-kyn and tryptophan, l-kyn was demonstrated to be a competitive inhibitor of TAA1 with a moderate Ki value (11.52 μM). Interestingly, no tryptophan analogs, such as α-methyltryptophan, have been reported to be IAA biosynthesis inhibitors. l-Kyn has been widely utilized to repress endogenous IAA production in various plants including the liverwort Marchantia polymorpha and the moss Physcomitrella patens (Eklund et al. 2015, Tsugafune et al. 2017). Aminoethoxyvinylglycine (AVG) and l-2-aminooxy-3-phenylpropionic acid (AOPP) have been reported to be auxin biosynthesis inhibitors targeting TAA1/TARs. AVG and AOPP were initially identified as inhibitors of pyridoxyl-5-phosphate (PLP)-dependent enzymes, including the aminocyclopropane-1-carboxylic acid (ACC) synthases and phenylalanine ammonia-lyase (Soeno et al. 2010). AVG and AOPP have an aminooxy moiety as a common feature that reacts with PLP of the enzymes via oxime bond formation. AVG and AOPP inhibit TAA1 and PLP enzyme, and reduce free IAA levels in Arabidopsis seedlings. However, these two inhibitors might show off-target effects via the inhibition of various PLP enzymes. To improve the specificity of aminooxy-type TAA1 inhibitors, aminooxy-naphthylpropionic acid (KOK1169/AONP) was designed as a specific inhibitor of TAA1 from AOPP derivatives (Narukawa-Nara et al. 2016). In vitro enzyme kinetic experiments indicated that AONP is a competitive inhibitor with a potent Ki value (76.8 nM) on TAA1, confers typical auxin-deficient phenotypes and suppresses auxin-responsive gene expression, which is reversed by IAA application. Pyruvamine 2031 (KOK2031) has also been identified as a potent aminooxy-type inhibitor of rice OsTAR1 with a potent Ki value (276 nM), and reduces endogenous IAA in rice (Kakei et al. 2017). YUC, a flavin-containing monooxygenase, is the rate-limiting enzyme in the IPyA pathway of Arabidopsis and maize (Kasahara 2016). Recently, two types of YUC inhibitors have been reported. Phenylboronic acid derivatives, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), have been identified as potent and reversible YUC inhibitors (Kakei et al. 2015). These boronic acids restore the extreme high-auxin phenotype of 35S::YUC1 seedlings. BBo and PPBo reduce the endogenous IAA content and exhibit auxin-deficient phenotypes, which are recovered by IAA application in wild-type Arabidopsis seedlings. The kinetic studies showed a competitive effect of BBo and PPBo against the substrate IPyA with a Ki of 50–60 nM. BBo and PPBo inhibit IAA biosynthesis and growth of a monocot plant, Brachypodium distachyon. Yucasin was identified as a YUC inhibitor from a chemical library in a screen for inhibitors of IAA production in maize coleoptiles (Nishimura et al. 2014). Yucasin was identified from derivatives of methimazole, an inhibitor of flavin-containing monooxygenase involving the YUC family. Yucasin has a similar structure to the 1,2,4-triazole-3(4H)-thione moiety of methimazole (Eswaramoorthy et al. 2006). Yucasin is a competitive YUC inhibitor and restores the high-auxin phenotype of YUC overexpression lines. However, in planta, auxin-deficient phenotypes are not observed in wild-type plants treated with yucasin. A structure–activity relationship study of yucasin revealed that its difluorinated analog, yucasin DF (YDF), was a potent YUC inhibitor in planta (Tsugafune et al. 2017). YDF exhibits an auxin-deficient phenotype in wild-type plants, but the in vitro inhibitory activity of YDF toward a recombinant YUC enzyme is similar to that of yucasin. Finally, YDF was found to be a chemically and metabolically stable analog of yucasin, and thereby YDF was maintained at high levels that were sufficient to inhibit auxin biosynthesis fully in planta. YDF was also effective in the liverwort M. polymorpha and moss P. patens, suggesting that YDF functions as a universal tool to modulate auxin biosynthesis in various plants. Off-target effects of compounds are the result of the unspecific effects on non-target proteins. Especially when compounds are used at high concentration, off-target effects are more likely to emerge. Cocktail treatment with multiple inhibitors on the same targeting pathway would be an effective approach to avoid possible off-target effects because a much smaller amount of each inhibitor can be used. The cocktail treatment with the YUC inhibitor, YDF, and two TAA1 inhibitors, l-kyn and AONP, can extensively inhibit IAA synthesis via the IPyA pathway and confers extreme auxin-deficient phenotypes that are fully recovered by IAA application (Tsugafune et al. 2017). Auxin Catabolism: Conjugation and Oxidation of IAA The reversible and irreversible catabolism of IAA plays a crucial role in IAA homeostasis and co-ordinately regulates the local auxin gradient (Ludwig-Muller 2011, Korasick et al. 2013). IAA is inactivated via two major pathways; GRETCHEN HAGEN3 (GH3) is one of the extensively studied early auxin-responsive genes encoding the IAA–amino acid conjugate synthase. The Arabidopsis genome contains 20 GH3 sequences, and the expression of several GH3 genes is regulated by both light and auxin signaling pathways (Ludwig-Muller 2011). Another major inactivation pathway is the oxIAA pathway. Arabidopsis DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1) encodes the 2-oxoglutarate/iron-dependent dioxygenase family and catalyzes the oxidation of the 2-position of IAA to yield 2-oxo-indole 3-acetic acid (oxIAA) (Zhang and Peer 2017). The loss-of-function dao1-1 mutant shows a tremendous accumulation of IAA–Asp and IAA–Glu to compensate for the oxIAA pathway in the dao1-1 mutant, suggesting that AtDAO1 and group II GH3 co-ordinately regulate the catabolism of IAA (Porco et al. 2016). Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), as a mimic of the adenylated reaction intermediate of the GH3 enzyme, has been reported to be a potent competitive inhibitor of recombinant grape GH3 enzymes (Bottcher et al. 2012). AIEP competes for the binding of ATP and IAA in GH3 active sites. However, the in planta effects of AIEP on Arabidopsis phenotypes have not been demonstrated. In contrast, JAR1/GH3-11 catalyzes the formation of a bioactive jasmonoyl–isoleucine conjugate as a genuine hormone. Recently, Jarin-1 was identified as a specific inhibitor of the GH3-11 enzyme (group I) and blocked the JAR1-regulated physiological response in planta (Meesters et al. 2014), implying that specific inhibitors of group II GH3 enzymes can be screened. Auxin Polar Transport: Inhibitors of Auxin Influx and Efflux Transport IAA is biosynthesized and then directionally transported to its responsive site. This auxin polar transport system modulates the local auxin distribution to form an asymmetric auxin concentration gradient that regulates tropic growth, embryo development, lateral root formation, and vascular and root development. The auxin polar stream is regulated by auxin influx and efflux proteins (Adamowski and Friml 2015, Zazimalova et al. 2010). For auxin import, AUXIN-RESISTANT 1/LIKE AUX1 (AUX1/LAX) proton gradient-driven symporters similar to amino acid permeases function as auxin influx transporters. The loss of a functional Arabidopsis aux1 mutant revealed an agravitropic and auxin-resistant phenotype in response to exogenous IAA (Marchant et al. 1999). For auxin efflux transport, PIN-FORMED (PIN) efflux carriers and ATP-BINDING CASSETTE subfamily B/MULTIDRUG RESISTANCE/P-GLYCOPROTEIN (ABCB/MDR/PGP) transporters co-ordinately regulate the direction and rate of intercellular auxin flow (Zazimalova et al. 2010). There are eight PIN family proteins in Arabidopsis, among which five PIN protein members, PIN1–PIN4 and PIN7, are localized at the plasma membrane of specific cells and function as auxin efflux carriers (Adamowski and Friml 2015, Naramoto 2017). In contrast, PIN5, PIN6, PIN8 and PIN-LIKES (PILS) proteins are localized in the endoplasmic reticulum (ER) (Barbez et al. 2012). These ER-localized carrier proteins facilitate intracellular auxin movement between the cytosol and ER to regulate auxin homeostasis (Adamowski and Friml 2015). Thus far, Arabidopsis ABCB proteins ABCB1, ABCB4 and ABCB19 have been characterized as auxin transporters. The single and multiple loss-of-function mutations of these PIN and ABCB proteins show severe defects in embryogenesis, shoot and root tropism, and lateral organ development (Zazimalova et al. 2010). The cell type-specific expression and subcellular localization of AUX/LAX, PIN and ABCB proteins ultimately determine the auxin gradient regulating plant growth and development. Synthetic auxins are very useful diagnostic tools to dissect the physiological responses mediated by auxin transport. The membrane-permeable synthetic auxin naphthalene 1-acetic acid (NAA) rescues the agravitropic phenotype of the aux1 mutant (Marchant et al. 1999). AUX1 recognizes IAA and 2,4-D as substrates and takes them, but not NAA, into the cell (Yang et al. 2006). In contrast, PIN and ABCB transporters export NAA outside cells, but 2,4-D does not (Yang and Murphy 2009). The Arabidopsis yuc 3,5,7,8,9 quintuple mutant (yuc Q) has severe auxin-deficient root phenotypes (Chen et al. 2014), but with the application of exogenous IAA and NAA fully recover from the defects in gravitropism and root growth. In contrast, 2,4-D can restore the defects in root growth only, but not those in root gravitropism, suggesting that 2,4-D is not transported in a polar manner to form the auxin concentration gradient required for the gravitropic response (Chen et al. 2014). Phenylacetic acid (PAA) has also been demonstrated to be a non-polar transport-type auxin (Sugawara et al. 2015). The transport profiles of other synthetic auxins, dicamba and picloram, remain unclear. The auxin transport inhibitor (ATI) has been extensively investigated and widely used in auxin biology. N-1-naphthylphthalamic acid (NPA) is extensively characterized as a classical ATI (Katekar and Geissler 1980). NPA potentially inhibits the directional auxin movement and blocks the plant responses mediated by auxin transport. NPA inhibits auxin efflux in a non-competitive manner. The primary target of NPA has been suggested to be ABCB transporters in planta (Noh et al. 2001). However, NPA blocks auxin transport by both PIN and ABCB in yeast functional assays (Yang and Murphy 2009). Additionally, NPA can bind to the immunophilin-like protein TWD1, and then NPA blocks the interaction of TWD1 with ABCB1 proteins (Bailly et al. 2008). Moreover, at a high concentration, NPA inhibits the subcellular recycling of PIN proteins required for the membrane localization of PIN (Geldner et al. 2001). Recently, NPA has been shown to mediate actin cytoskeleton remodeling via the TWD1–ACTIN7 axis and thereby modulate the membrane localization of the auxin transport proteins PIN and ABCB (Zhu et al. 2016). The effect of NPA on these multiple targets involved in the auxin transport system will result in efficient inhibition of polar auxin movement by NPA in planta. The most successful chemical genetic screen of auxin mutants revealed TRANSPORT INHIBITOR RESPONSE (TIR) mutants that alter the mutant response to NPA (Ruegger et al. 1997). The TIR1 gene has been demonstrated to encode a nuclear auxin receptor (Chapman and Estelle 2009). Later, TIR2 was shown to be a TAA1 in the IPyA auxin biosynthetic pathway (Yamada et al. 2009). Another classical ATI, 2,3,5-triiodobenzoic acid (TIBA), disrupts the membrane localization of PIN by perturbing PIN vesicle trafficking processes (Dhonukshe et al. 2008). In addition to these classical ATIs, a phenotype-based high-throughput screen using Arabidopsis identified two ATIs from a chemical library. BUM, {2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid}, shows similar inhibitory effects on NPA, and its primary target has been elucidated to be ABCB proteins (Kim et al. 2010). BUM has been reported to share its binding site in ABCB with NPA by a competitive binding assay. Gravacin {3-(5-[3, 4- dichlorophenyl]-2-furyl) acrylic acid} shows potent inhibition of gravitropism in Arabidopsis (Rojas-Pierce et al. 2007). A chemical genetic screen of the gravacin-resistant mutant grav-r1 revealed a loss-of-function abcb19 mutant, which suggested that the primary target of gravacin was an ABCB19 transporter. Nishimura et al. reported two groups of ATIs from a chemical library using maize coleoptiles (Nishimura et al. 2012). One (group A) was composed of inhibitors exhibiting the NPA-like activity of the Arabidopsis phenotype. Two active molecules in group A, 37-H4 and 48-F9, are structurally different from NPA. Another (group B) was composed of modulators of subcellular trafficking of PIN proteins. Compound 8-C9 promoted the internalization of PIN2 and PIN1 proteins into compartments, suggesting that this compound exhibited similar activity to brefeldin A (BFA), an inhibitor of subcellular vesicle trafficking (Geldner et al. 2001). Flavonoids such as quercetin inhibit polar auxin transport and are suggested to be endogenous modulators of local auxin distribution. Flavonoid-deficient transparent testa4 mutants show elevated root basipetal auxin transport, suggesting that flavonoids are endogenous negative regulators of auxin transport (Peer and Murphy 2007). cis-Cinnamic acid has been reported as a natural auxin efflux inhibitor (Steenackers et al. 2017); it potentially blocks auxin efflux transport in tobacco BY-2 cells and inhibits the gravitropic response of primary root in Arabidopsis. In contrast to known ATIs (NPA and TIBA), cis-cinnamic acid promotes lateral root formation. cis-Cinnamic acid will inhibit shootward auxin transport by perturbing the redistribution of auxin in the meristem to accumulate endogenous IAA in the root. Another cinnamic acid derivative, 3,4-(methylenedioxy)cinnamic acid (MDCA), has also been reported as an auxin efflux inhibitor (Steenackers et al. 2016). MDCA was originally found in the root of asparagus plants and identified as an allelochemical, showing inhibition of primary root growth and promotion of lateral and adventitious roots. However, the mode of action and primary target of these natural inhibitors have not been elucidated. Alkoxy-auxins, 5-alkoxy-IAA and 7-alkoxy-NAA, are designed as active auxin analogs for auxin transport machinery, but inactive analogs for TIR1/AFB auxin receptors (Tsuda et al. 2011). Alkoxy-auxins competitively inhibit the transport activity of PINs, ABCBs and AUX1 that are expressed heterologously in yeast cells, but alkoxy-auxins are completely inactive in auxin SCFTIR1/AFB signaling in addition to the subcellular membrane relocalization of PIN proteins. Consistent with the transport profiles of IAA and NAA, the 5-alkoxy-IAA, Bz-IAA showed inhibition of PIN, ABCB and AUX1 activities. In contrast, the 7-alkoxy-NAA, Bz-NAA is specific to PINs and ABCBs. Alkoxy-auxins will be reorganized as a substrate for the auxin transport proteins PIN and ABCB, and, therefore, alkoxy-auxin reduces the endogenous IAA transport rate in competition with endogenous IAA, suggesting that alkoxy-auxins are a new class of ATI with an evident mode of action. In contrast to various inhibitors of auxin efflux transport, a few specific inhibitors of auxin influx transport have been reported to date. 1-Naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA) have been identified as specific inhibitors of auxin influx transport by the AUX1/LAX importer (Parry et al. 2001). 1-NOA reduces the root gravitropic response similarly to the aux1 mutant. 1-NOA can inhibit auxin uptake by AUX1 expressed in Xenopus oocytes (Yang et al. 2006). This evidence indicates that 1-NOA will directly target the AUX1 transport protein. 1-NOA has a similar structure to auxins, implying that it can competitively inhibit IAA import by AUX1 like the alkoxy-auxin Bz-IAA. 2,4-D is imported by AUX1 as a substrate, but not by the auxin efflux machinery, which suggests that the inactive 2,4-D analog for auxin signaling functions as a competitive inhibitor of AUX1 (Yang et al. 2006). The 2,4-D analog ethyl 2-[(2-chloro-4-nitrophenyl)thio]acetate shows similar biological activity to 1-NOA and has been identified as an inhibitor of the auxin influx carrier (Suzuki et al. 2014). Recently, the jasmonoyl-l-tryptophan (JA–Trp) conjugate was shown to target AUX1 protein and reduce the root gravitropic response (Staswick et al. 2017); however, the mode of action of the JA–Trp conjugate has not been elucidated. Visualization of the Cellular Auxin Distribution in Planta Visualization of the auxin gradient in planta has been extensively investigated in auxin biology. Visualization of the auxin gradient has been performed using an auxin-responsive reporter line, such as the DR5::GFP and DII-VENUS lines (Friml et al. 2003, Brunoud et al. 2012, Liao et al. 2015). These reporter lines have been widely used as essential tools to monitor the auxin levels in plants. However, the spatiotemporal resolution of these reporter systems is limited because of reporter protein localization within the cell. Fluorescent auxin analogs have been designed based on structure–activity studies of alkoxy-auxin. Alkoxy-auxin analogs are recognized as the substrate of the auxin transporters AUX1, PIN and ABCB, and are transported without interfering with endogenous auxin movement at low concentrations. Therefore, alkoxy-auxin analogs will be distributed like endogenous auxin (Tsuda et al. 2011). Fluorescently tagged alkoxy-auxin analogs function as inactive auxin for auxin signaling, but the analogs show a similar distribution pattern to the endogenous auxin gradient. The fluorescent auxin analogs (NBD-auxins) NBD-IAA and NBD-NAA enabled visualization of the auxin transport site and subcellular auxin gradient in plant cells (Hayashi et al. 2014). Consistent with the localization of some PIN and PILS transport proteins at the ER (Zazimalova et al. 2010, Barbez et al. 2012), NBD-auxins were highly accumulated at the ER in tobacco and Arabidopsis plants. The NBD-auxins can complement molecular biological approaches using auxin reporter lines and provide valuable tools for auxin biology. Recently, another chemical biology approach for the visualization of auxin-binding proteins has been reported (Mravec et al. 2017). The azido derivative of indole-3-propionic acid, (S)-2-azido-3-(3-indolyl)propionic acid (IPA-N3), was used as the tagged auxin analog. IPA-N3 showed similar auxinic activity to IAA in Arabidopsis. After IPA-N3 was applied to seedlings, it was washed and fixed with amino groups of nearby proteins by a coupling reagent. The azido group in IPA-N3 was specifically ligated to alkyne-conjugated fluorescent dye by the click reaction between azido and alkyne. The IPA-N3-binding proteins were visualized by fluorescent imaging. In this approach, IPA-N3-binding protein was found to be localized in the cell walls of elongating root cells (Mravec et al. 2017). Auxin Signaling: Small Molecule Modulator of the SCFTIR1/AFB Pathway The term ‘auxin’, derived from the Greek word ‘auxein’ (to increase or to grow), is associated with a chemical substance that promotes cell elongation activity in plants, originally in Avena (oat) coleoptiles (Enders and Strader 2015). The profound and pleiotropic hormonal effects of IAA on plant growth accelerated the discovery of auxin-like compounds for the development of a potent synthetic auxin. A number of structurally diverse synthetic auxins were reported from the 1940s to 1970s. These synthetic auxins perturb auxin-regulated plant growth and thus have been widely used as herbicides to control weeds. The classical definition of an ‘auxin’ is a molecule that shows similar plant hormone responses to those elicited by IAA. The auxin molecule, in the receptor-based definition, is perceived by the auxin-binding site of the TIR1/AFB family of auxin receptors and then forms the TIR1–auxin–Aux/IAA ternary complex to catalyze the ubiquitination of Aux/IAA repressors (Salehin et al. 2015, Tan et al. 2007). In this molecular model, molecules that bind to the TIR1/AFB receptor and promote the formation of the TIR1–auxin–Aux/IAA ternary complex can be considered ‘auxins’ (Salehin et al. 2015). The molecular structure of the TIR1–auxin–Aux/IAA ternary complex clearly illustrates the essential structural features of the auxin molecule required for the formation of the ternary complex: the planar aromatic ring system and carboxylic acid as a side chain attached to the aromatic ring (Tan et al. 2007). Based on these structural features, synthetic auxins have been classified into five major groups, naphthaleneacetic acid, e.g. NAA; phenoxyacetic acid, e.g. 2,4-D; indole-type auxins, e.g. haloganated IAA; benzoic acid, e.g. dicamba; and picolinate-type, e.g. picloram. The metabolism, transport, distribution and primary target receptors among the TIR1/AFB family differ between natural and synthetic auxins. Simon et al. reported a comprehensive analysis of the biological effects of diverse synthetic auxins on auxin-dependent gene expression, the endocytosis of PIN proteins across the plasma membrane and the phenotypic auxin responses of root and hypocotyl (Simon et al. 2013). These synthetic auxins induce strikingly divergent phenotypes in terms of hypocotyl elongation, lateral root development and primary root growth. This observation indicates that the physiological activities of synthetic auxins are complicated due to the distinct impact of auxins on chemical and metabolic stability, distribution in tissues in a polar manner and affinity towards specific sets of auxin receptors. Auxin-regulated transcription is very rapid, and a large number of genes respond to auxin within minutes. Early auxin-responsive genes are classified into three major families of genes: Aux/IAA, GH3 and SMALL AUXIN UP RNA (SAUR). The SUAR genes are involved in the cell expansion triggered by the phosphorylation of plasma membrane H+-ATPase (Spartz et al. 2012, Takahashi et al. 2012). The SAUR protein inhibits PP2C-D phosphatases to repress dephosphorylation of the H+-ATPase (Takahashi et al. 2012, Spartz et al. 2014). The Aux/IAA family, of which there are 29 members in Arabidopsis, encodes short-lived nuclear proteins that are repressors of auxin-responsive gene expression. The typical Aux/IAA repressor consists of four conserved domains, domains I– IV. Domain III and IV are responsible for heterodimerization with the ARF transcription factor to repress the transcriptional activation of auxin-responsive genes (Hayashi 2012). As the initial step in auxin signaling, auxin is perceived by TIR1, an F-box protein auxin receptor. TIR1 forms the SCFTIR1 E3 ubiquitin ligase complex consisting of Skp1 (ASK1) and Cullin (CUL1) to catalyze the ubiquitination of the F-box target protein. Auxin enhances the ubiquitination of Aux/IAA by promoting the interaction between Aux/IAA and TIR1 receptors. Consequently, in the presence of auxin, Aux/IAA repressors are ubiquitinated and degraded by the 26S proteasome pathway to activate ARF transcriptional activity. Arabidopsis has TIR1, an auxin receptor and five additional TIR1 homolog proteins, AFB1–5 (Auxin F-Box) (Hayashi 2012, Salehin et al. 2015). A structural study of the TIR1–auxin–Aux/IAA complex has demonstrated that auxin nestles on the floor of the surface pocket formed by the leucine-rich repeat (LRR) domain in TIR1, and TIR1-bound auxin enhances the interaction between the Aux/IAA domain II motif (GWPPV) and auxin-bound site of TIR1. The tryptophan (W) and second proline (P) residues in domain II are positioned close to the aromatic ring of auxin via hydrophobic interactions. Aux/IAA works as a co-receptor that forms the small hydrophobic cavity in the auxin-binding site of TIR1 (Tan et al. 2007), which suggests that auxin acts as a ‘molecular glue’ by which the two proteins are tightly bound together. The molecular recognition mechanism of TIR1 or the related AFB proteins for distinct synthetic auxins has been revealed by biochemical and genetic approaches (Calderon Villalobos et al. 2012, Lee et al. 2014). A synthetic auxin-insensitive mutant screen using the novel pyridine-type synthetic auxin DAS534 (an analog of the auxinic herbicide picloram) identified the loss-of-function mutation of AFB5 receptors in Arabidopsis (Walsh et al. 2006). The loss of AFB5 confers resistance to picolinate auxins, but not to 2,4-D or IAA. Additionally, afb4 afb5 double mutants show slightly more resistance to picloram than the afb5 single mutant, suggesting that AFB4 and AFB5 are the primary targets of picloram-type auxinic herbicide (Prigge et al. 2016). A surface plasmon resonance (SPR) binding assay clearly demonstrated that DAS534 and picloram show higher affinity for AFB5 than the TIR1 receptor complex (Lee et al. 2014). The distinct sensitivity of tir1 and afb5 mutants to synthetic auxins indicates significant differences in the molecular recognition of auxins among the auxin receptor family that could be applicable for the design of new auxinic herbicides. Rational Design of the Auxin Antagonist of TIR1 Receptor The molecular mechanism of auxin perception provides an opportunity for the structure-based molecular design of an auxin antagonist. The binding of auxin to the Aux/IAA–TIR1 co-receptor complex does not alter the conformation of the flexible side chains of TIR1 or the Aux/IAA around the auxin-binding site, indicating that the auxin-binding site can be considered as a rigid form. Therefore, the structure of the TIR1-specific ligand can be rationally designed without consideration of flexible side chain conformers determining the cavity shape in the binding site. The rational design of TIR1 probes has led to the generation of tert-butoxycarbonylaminohexyl-IAA (BH-IAA) with potent antagonistic activity toward auxin activity mediated by the SCFTIR1/AFB pathway (Hayashi et al. 2008). The crystal structures of TIR1 in complex with BH-IAA illustrate the molecular mechanism of BH-IAA. In the auxin-binding site, the IAA moiety of BH-IAA is positioned in the same conformation as IAA, and the long alkyl chain is directed toward the Aux/IAA-binding site. This long alkyl chain occupies the Aux/IAA-binding site efficiently to prevent access of the domain II GWPPV motif of Aux/IAA. The long alkyl chain is flexible in the auxin-binding site of TIR1. Therefore, the IAA moiety of BH-IAA is solely responsible for the binding affinity of BH-IAA for TIR1, and Aux/IAA is not involved in the binding of BH-IAA. In contrast, IAA is tightly captured in the small cavity formed by both TIR1 and Aux/IAA, which indicates that the TIR1–Aux/IAA co-receptor complex has a higher affinity for IAA. Thus, the low concentration of exogenous IAA can completely abrogate the antiauxin activity of BH-IAA in planta (Hayashi et al. 2008). Auxinole was designed as an auxin antagonist of the TIR1 receptor based on in silico molecular docking calculations (Hayashi et al. 2012). Auxinole was designed to increase the affinity for TIR1 in the binding site. Auxinole has a 2-oxo-phenylethyl group at the α-position of IAA that can interact with a phenylalanine residue (Phe82) of TIR1 via a strong π–π stacking interaction. Auxinole shows very potent antiauxin activity and is effective in diverse plants, including monocots, rice and the moss P. patens. Auxinole shows reversible inhibition, which can be spatiotemporally controlled, and therefore auxinole has been widely used to study auxin responses mediated by the SCFTIR1 auxin pathway (Hayashi et al. 2012). Non-Transcriptional Auxin Signaling Most auxin responses are transcriptionally regulated by the SCFTIR1/AFB pathway. Auxin triggers rapid cell wall acidification and elongation of hypocotyls. These rapid responses have long been thought to be regulated by non-transcriptional auxin signaling. Fendrych et al. demonstrated that the TIR1/AFB receptor is required for the auxin-induced acidification and subsequent elongation of Arabidopsis etiolated hypocotyls (Fendrych et al. 2016). By using an elegant chemical biology approach, Uchida et al. also revealed that the TIR1 receptor is essential for auxin-induced phosphorylation of H+-ATPase and subsequent hypocotyl elongation (Uchida et al. 2018). The engineered TIR1 mutant (F79G) was designed to recognize specifically 5-aryl IAA (cvxIAA) that could not activate the native TIR1 receptor. Thus, 5-aryl IAA can selectively activate an auxin transcriptional signal and its downstream events via the SCFTIR1(F79G) machinery in then transgenic plants expressing the engineered TIR1 (F79G) receptor. The localization of PIN protein on the plasma membrane determines the direction of auxin flow to modulate the local auxin distribution (Naramoto 2017). Auxin has been reported to inhibit clathrin-mediated endocytosis of PIN protein. ABP1 has been reported to have a positive role in clathrin recruitment to the plasma membrane, leading to the endocytosis of PIN (Robert et al. 2010). Auxin signaling is mediated by ABP1 accompanied by the activation of ROP proteins (Chen et al. 2012). However, abp1 null mutants generated by CRISPR (clustered regularly interspaced short palindromic repeats) technology clearly demonstrate that ABP1 does not play a major role in auxin-regulated developmental processes (Gao et al. 2015). Whether another type of auxin receptor is involved in the non-transcriptional auxin signaling pathway is not known. The molecular actions of non-transcriptional auxin responses remain largely unknown. The chemical probes specific for such non-transcriptional auxin responses are highly anticipated for studies of auxin biology. Concluding Remarks Progress over the past decades in auxin biology has demonstrated fundamental components and molecular mechanisms of auxin biosynthesis, signal transduction and the polar auxin transport system. Small molecule modulators of auxin-related processes, including synthetic auxins, have greatly accelerated auxin research in combination with the molecular genetic approaches in Arabidopsis biology. The most important advance in auxin research was the identification of the function of TIR1 as an auxin receptor and the structural determination of the TIR1–Aux/IAA auxin co-receptor. The structure of TIR1–Aux/IAA proposed the fundamental molecular mechanism of plant hormone perception of the co-receptor system. We now understand the structural information for the basic components of auxin biosynthesis (TAA1) (Tao et al. 2008), metabolism (GH3 and ILL2) (Bitto et al. 2009, Peat et al. 2012) and signaling (TIR1, Aux/IAA and ARF) (Chapman and Estelle 2009, Dinesh et al. 2015, Roosjen et al. 2018). These structural data open the door to the rational design of plant hormone modulators in auxin biology. The auxin concentration gradient is spatiotemporally regulated by auxin biosynthesis, catabolism and transport. The auxin gradient is then transduced to the auxin signaling machinery to alter the developmental output. These auxin-related regulatory processes are tightly interconnected, and so the processes co-ordinately modulate the developmental response to environmental cues. However, the complicated but precise regulatory mechanism underlying pleiotropic auxin effects on plant growth and development remain to be determined. To understand the complicated network in the pleiotropic responses of auxin, a new approach from a different field is highly anticipated. Chemical biology appears to be one promising approach, in combination with new systematic approaches such as proteomics, metabolomics and mathematical modeling. Chemical tools that are specifically designed for auxin biology could be valuable for the modulation of auxin perception, signaling, transport and even biosynthesis and catabolism at a specific developmental stage and in a specific tissue. The development of unique chemical tools for the specific auxin response would uncover new components of auxin-regulated developmental processes. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for Scientific Research (15K01828) to K.H.]. Acknowledgments We are grateful to Professor Hiroyuki Kasahara (Tokyo University of Agriculture and Technology) for critical reading of the manuscript. Disclosures The authors have no conflicts of interest to declare. References Adamowski M., Friml J. ( 2015) PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell  27: 20– 32. Google Scholar CrossRef Search ADS PubMed  Bailly A., Sovero V., Vincenzetti V., Santelia D., Bartnik D., Koenig B.W., et al.   ( 2008) Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins. J. Biol. Chem.  283: 21817– 21826. Google Scholar CrossRef Search ADS PubMed  Barbez E., Kubeš M., Rolčík J., Béziat C., Pěnčík A., Wang B., et al.   ( 2012) A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature  485: 119– 122. Google Scholar CrossRef Search ADS PubMed  Bitto E., Bingman C.A., Bittova L., Houston N.L., Boston R.S., Fox B.G., et al.   ( 2009) X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana. Proteins  74: 61– 71. Google Scholar CrossRef Search ADS PubMed  Bottcher C., Dennis E.G., Booker G.W., Polyak S.W., Boss P.K., Davies C. ( 2012) A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue. PLoS One  7: e37632. Google Scholar CrossRef Search ADS PubMed  Brunoud G., Wells D.M., Oliva M., Larrieu A., Mirabet V., Burrow A.H., et al.   ( 2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature  482: 103– 106. Google Scholar CrossRef Search ADS PubMed  Calderon Villalobos L.I., Lee S., De Oliveira C., Ivetac A., Brandt W., Armitage L., et al.   ( 2012) A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin. Nat. Chem. Biol.  8: 477– 485. Google Scholar CrossRef Search ADS PubMed  Chapman E.J., Estelle M. ( 2009) Mechanism of auxin-regulated gene expression in plants. Annu. Rev. Genet.  43: 265– 285. Google Scholar CrossRef Search ADS PubMed  Chen Q., Dai X., De-Paoli H., Cheng Y., Takebayashi Y., Kasahara H., et al.   ( 2014) Auxin overproduction in shoots cannot rescue auxin deficiencies in Arabidopsis roots. Plant Cell Physiol . 55: 1072– 1079. Google Scholar CrossRef Search ADS PubMed  Chen X., Naramoto S., Robert S., Tejos R., Lofke C., Lin D., et al.   ( 2012) ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots. Curr. Biol . 22: 1326– 1332. Google Scholar CrossRef Search ADS PubMed  De Rybel B., Audenaert D., Beeckman T., Kepinski S. ( 2009) The past, present, and future of chemical biology in auxin research. ACS Chem. Biol.  4: 987– 998. Google Scholar CrossRef Search ADS PubMed  Dhonukshe P., Grigoriev I., Fischer R., Tominaga M., Robinson D.G., Hasek J., et al.   ( 2008) Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes. Proc. Natl. Acad. Sci. USA  105: 4489– 4494. Google Scholar CrossRef Search ADS   Dinesh D.C., Kovermann M., Gopalswamy M., Hellmuth A., Calderon Villalobos L.I., Lilie H., et al.   ( 2015) Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response. Proc. Natl. Acad. Sci. USA  112: 6230– 6235. Google Scholar CrossRef Search ADS   Eklund D.M., Ishizaki K., Flores-Sandoval E., Kikuchi S., Takebayashi Y., Tsukamoto S., et al.   ( 2015) Auxin produced by the indole-3-pyruvic acid pathway regulates development and gemmae dormancy in the liverwort Marchantia polymorpha. Plant Cell  27: 1650– 1669. Google Scholar CrossRef Search ADS PubMed  Enders T.A., Strader L.C. ( 2015) Auxin activity: past, present, and future. Amer. J. Bot.  102: 180– 196. Google Scholar CrossRef Search ADS   Eswaramoorthy S., Bonanno J.B., Burley S.K., Swaminathan S. ( 2006) Mechanism of action of a flavin-containing monooxygenase. Proc. Natl. Acad. Sci. USA  103: 9832– 9837. Google Scholar CrossRef Search ADS   Fendrych M., Leung J., Friml J. ( 2016) TIR1/AFB–Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. eLife  5: e19048. Google Scholar CrossRef Search ADS PubMed  Friml J., Vieten A., Sauer M., Weijers D., Schwarz H., Hamann T., et al.   ( 2003) Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis. Nature  426: 147– 153. Google Scholar CrossRef Search ADS PubMed  Gao Y., Zhang Y., Zhang D., Dai X., Estelle M., Zhao Y. ( 2015) Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc. Natl. Acad. Sci. USA  112: 2275– 2280. Google Scholar CrossRef Search ADS   Geldner N., Friml J., Stierhof Y.D., Jurgens G., Palme K. ( 2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature  413: 425– 428. Google Scholar CrossRef Search ADS PubMed  Hayashi K. ( 2012) The interaction and integration of auxin signaling components. Plant Cell Physiol . 53: 965– 975. Google Scholar CrossRef Search ADS PubMed  Hayashi K., Nakamura S., Fukunaga S., Nishimura T., Jenness M.K., Murphy A.S., et al.   ( 2014) Auxin transport sites are visualized in planta using fluorescent auxin analogs. Proc. Natl. Acad. Sci. USA  111: 11557– 11562. Google Scholar CrossRef Search ADS   Hayashi K., Neve J., Hirose M., Kuboki A., Shimada Y., Kepinski S., et al.   ( 2012) Rational design of an auxin antagonist of the SCF(TIR1) auxin receptor complex. ACS Chem. Biol.  7: 590– 598. Google Scholar CrossRef Search ADS PubMed  Hayashi K., Tan X., Zheng N., Hatate T., Kimura Y., Kepinski S., et al.   ( 2008) Small-molecule agonists and antagonists of F-box protein–substrate interactions in auxin perception and signaling. Proc. Natl. Acad. Sci. USA  105: 5632– 5637. Google Scholar CrossRef Search ADS   He W., Brumos J., Li H., Ji Y., Ke M., Gong X., et al.   ( 2011) A small-molecule screen identifies l-kynurenine as a competitive inhibitor of TAA1/TAR activity in ethylene-directed auxin biosynthesis and root growth in Arabidopsis. Plant Cell  23: 3944– 3960. Google Scholar CrossRef Search ADS PubMed  Hicks G.R., Raikhel N.V. ( 2014) Plant chemical biology: are we meeting the promise? Front. Plant Sci.  5: 455. Google Scholar CrossRef Search ADS PubMed  Kakei Y., Nakamura A., Yamamoto M., Ishida Y., Yamazaki C., Sato A., et al.   ( 2017) Biochemical and chemical biology study of rice OsTAR1 revealed that tryptophan aminotransferase is involved in auxin biosynthesis: identification of a potent OsTAR1 inhibitor, pyruvamine2031. Plant Cell Physiol . 58: 598– 606. Google Scholar PubMed  Kakei Y., Yamazaki C., Suzuki M., Nakamura A., Sato A., Ishida Y., et al.   ( 2015) Small-molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function. Plant J.  84: 827– 837. Google Scholar CrossRef Search ADS PubMed  Kasahara H. ( 2016) Current aspects of auxin biosynthesis in plants. Biosci. Biotechnol. Biochem . 80: 34– 42. Google Scholar CrossRef Search ADS PubMed  Katekar G.F., Geissler A.E. ( 1980) Auxin transport inhibitors: IV. Evidence of a common mode of action for a proposed class of auxin transport inhibitors: the phytotropins. Plant Physiol . 66: 1190– 1195. Google Scholar CrossRef Search ADS PubMed  Kato H., Nishihama R., Weijers D., Kohchi T. ( 2017) Evolution of nuclear auxin signaling: lessons from genetic studies with basal land plants. J. Exp. Bot . 69: 292– 301. Kim J.Y., Henrichs S., Bailly A., Vincenzetti V., Sovero V., Mancuso S., et al.   ( 2010) Identification of an ABCB/P-glycoprotein-specific inhibitor of auxin transport by chemical genomics. J. Biol. Chem.  285: 23309– 23317. Google Scholar CrossRef Search ADS PubMed  Korasick D.A., Enders T.A., Strader L.C. ( 2013) Auxin biosynthesis and storage forms. J. Exp. Bot . 64: 2541– 2555. Google Scholar CrossRef Search ADS PubMed  Lee S., Sundaram S., Armitage L., Evans J.P., Hawkes T., Kepinski S., et al.   ( 2014) Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)–Aux/IAA co-receptor complex formation. ACS Chem. Biol.  9: 673– 682. Google Scholar CrossRef Search ADS PubMed  Liao C.Y., Smet W., Brunoud G., Yoshida S., Vernoux T., Weijers D. ( 2015) Reporters for sensitive and quantitative measurement of auxin response. Nat. Methods  12: 207– 210. Google Scholar CrossRef Search ADS PubMed  Ludwig-Muller J. ( 2011) Auxin conjugates: their role for plant development and in the evolution of land plants. J. Exp. Bot . 62: 1757– 1773. Google Scholar CrossRef Search ADS PubMed  Ma Q., Robert S. ( 2014) Auxin biology revealed by small molecules. Physiol. Plant.  151: 25– 42. Google Scholar CrossRef Search ADS PubMed  Marchant A., Kargul J., May S.T., Muller P., Delbarre A., Perrot-Rechenmann C., et al.   ( 1999) AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J . 18: 2066– 2073. Google Scholar CrossRef Search ADS PubMed  Mashiguchi K., Tanaka K., Sakai T., Sugawara S., Kawaide H., Natsume M., et al.   ( 2011) The main auxin biosynthesis pathway in Arabidopsis. Proc. Natl. Acad. Sci. USA  108: 18512– 18517. Google Scholar CrossRef Search ADS   Meesters C., Monig T., Oeljeklaus J., Krahn D., Westfall C.S., Hause B., et al.   ( 2014) A chemical inhibitor of jasmonate signaling targets JAR1 in Arabidopsis thaliana. Nat. Chem. Biol.  10: 830– 836. Google Scholar CrossRef Search ADS PubMed  Mravec J., Kracun S.K., Zemlyanskaya E., Rydahl M.G., Guo X., Picmanova M., et al.   ( 2017) Click chemistry-based tracking reveals putative cell wall-located auxin binding sites in expanding cells. Sci. Rep.  7: 15988. Google Scholar CrossRef Search ADS PubMed  Naramoto S. ( 2017) Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport. Curr. Opin. Plant Biol . 40: 8– 14. Google Scholar CrossRef Search ADS PubMed  Narukawa-Nara M., Nakamura A., Kikuzato K., Kakei Y., Sato A., Mitani Y., et al.   ( 2016) Aminooxy-naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l-tryptophan aminotransferase: structure–activity relationships. Plant J.  87: 245– 257. Google Scholar CrossRef Search ADS PubMed  Nishimura T., Hayashi K., Suzuki H., Gyohda A., Takaoka C., Sakaguchi Y., et al.   ( 2014) Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis. Plant J.  77: 352– 366. Google Scholar CrossRef Search ADS PubMed  Nishimura T., Matano N., Morishima T., Kakinuma C., Hayashi K., Komano T., et al.   ( 2012) Identification of IAA transport inhibitors including compounds affecting cellular PIN trafficking by two chemical screening approaches using maize coleoptile systems. Plant Cell Physiol . 53: 1671– 1682. Google Scholar CrossRef Search ADS PubMed  Noh B., Murphy A.S., Spalding E.P. ( 2001) Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell  13: 2441– 2454. Google Scholar CrossRef Search ADS PubMed  Parry G., Delbarre A., Marchant A., Swarup R., Napier R., Perrot-Rechenmann C., et al.   ( 2001) Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1. Plant J . 25: 399– 406. Google Scholar CrossRef Search ADS PubMed  Peat T.S., Bottcher C., Newman J., Lucent D., Cowieson N., Davies C. ( 2012) Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis. Plant Cell  24: 4525– 4538. Google Scholar CrossRef Search ADS PubMed  Peer W.A., Murphy A.S. ( 2007) Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci.  12: 556– 563. Google Scholar CrossRef Search ADS PubMed  Porco S., Pěnčík A., Rashed A., Voß U., Casanova-Sáez R., Bishopp A., et al.   ( 2016) Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis. Proc. Natl. Acad. Sci. USA  113: 11016– 11021. Google Scholar CrossRef Search ADS   Prigge M.J., Greenham K., Zhang Y., Santner A., Castillejo C., Mutka A.M., et al.   ( 2016) The arabidopsis auxin receptor F-box proteins AFB4 and AFB5 are required for response to the synthetic auxin picloram. G3 (Bethesda)  6: 1383– 1390. Google Scholar CrossRef Search ADS PubMed  Robert S., Kleine-Vehn J., Barbez E., Sauer M., Paciorek T., Baster P., et al.   ( 2010) ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis. Cell  143: 111– 121. Google Scholar CrossRef Search ADS PubMed  Rojas-Pierce M., Titapiwatanakun B., Sohn E.J., Fang F., Larive C.K., Blakeslee J., et al.   ( 2007) Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin. Chem. Biol . 14: 1366– 1376. Google Scholar CrossRef Search ADS PubMed  Roosjen M., Paque S., Weijers D. ( 2018) Auxin response factors: output control in auxin biology. J. Exp. Bot.  69: 179– 188. Google Scholar CrossRef Search ADS PubMed  Ruegger M., Dewey E., Hobbie L., Brown D., Bernasconi P., Turner J., et al.   ( 1997) Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects. Plant Cell  9: 745– 757. Google Scholar CrossRef Search ADS PubMed  Salehin M., Bagchi R., Estelle M. ( 2015) SCFTIR1/AFB-based auxin perception: mechanism and role in plant growth and development. Plant Cell  27: 9– 19. Google Scholar CrossRef Search ADS PubMed  Simon S., Kubes M., Baster P., Robert S., Dobrev P.I., Friml J., et al.   ( 2013) Defining the selectivity of processes along the auxin response chain: a study using auxin analogues. New Phytol.  200: 1034– 1048. Google Scholar CrossRef Search ADS PubMed  Soeno K., Goda H., Ishii T., Ogura T., Tachikawa T., Sasaki E., et al.   ( 2010) Auxin biosynthesis inhibitors, identified by a genomics-based approach, provide insights into auxin biosynthesis. Plant Cell Physiol . 51: 524– 536. Google Scholar CrossRef Search ADS PubMed  Spartz A.K., Lee S.H., Wenger J.P., Gonzalez N., Itoh H., Inze D., et al.   ( 2012) The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion. Plant J . 70: 978– 990. Google Scholar CrossRef Search ADS PubMed  Spartz A.K., Ren H., Park M.Y., Grandt K.N., Lee S.H., Murphy A.S., et al.   ( 2014) SAUR inhibition of PP2C-D phosphatases activates plasma membrane H+-ATPases to promote cell expansion in Arabidopsis. Plant Cell  26: 2129– 2142. Google Scholar CrossRef Search ADS PubMed  Staswick P., Rowe M., Spalding E.P., Splitt B.L. ( 2017) Jasmonoyl-l-tryptophan disrupts IAA activity through the AUX1 auxin permease. Front. Plant Sci.  8: 736. Google Scholar CrossRef Search ADS PubMed  Steenackers W., Cesarino I., Klima P., Quareshy M., Vanholme R., Corneillie S., et al.   ( 2016) The allelochemical MDCA inhibits lignification and affects auxin homeostasis. Plant Physiol . 172: 874– 888. Google Scholar PubMed  Steenackers W., Klima P., Quareshy M., Cesarino I., Kumpf R.P., Corneillie S., et al.   ( 2017) cis-Cinnamic acid is a novel, natural auxin efflux inhibitor that promotes lateral root formation. Plant Physiol.  173: 552– 565. Google Scholar CrossRef Search ADS PubMed  Sugawara S., Hishiyama S., Jikumaru Y., Hanada A., Nishimura T., Koshiba T., et al.   ( 2009) Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proc. Natl. Acad. Sci. USA  106: 5430– 5435. Google Scholar CrossRef Search ADS   Sugawara S., Mashiguchi K., Tanaka K., Hishiyama S., Sakai T., Hanada K., et al.   ( 2015) Distinct characteristics of indole-3-acetic acid and phenylacetic acid, two common auxins in plants. Plant Cell Physiol.  56: 1641– 1654. Google Scholar CrossRef Search ADS PubMed  Suzuki H., Matano N., Nishimura T., Koshiba T. ( 2014) A 2,4-dichlorophenoxyacetic acid analog screened using a maize coleoptile system potentially inhibits indole-3-acetic acid influx in Arabidopsis thaliana. Plant Signal. Behav.  9: e29077. Google Scholar CrossRef Search ADS PubMed  Takahashi K., Hayashi K., Kinoshita T. ( 2012) Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. Plant Physiol . 159: 632– 641. Google Scholar CrossRef Search ADS PubMed  Tan X., Calderon-Villalobos L.I., Sharon M., Zheng C., Robinson C.V., Estelle M., et al.   ( 2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature  446: 640– 645. Google Scholar CrossRef Search ADS PubMed  Tao Y., Ferrer J.L., Ljung K., Pojer F., Hong F., Long J.A., et al.   ( 2008) Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell  133: 164– 176. Google Scholar CrossRef Search ADS PubMed  Tsuda E., Yang H., Nishimura T., Uehara Y., Sakai T., Furutani M., et al.   ( 2011) Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters. J. Biol. Chem.  286: 2354– 2364. Google Scholar CrossRef Search ADS PubMed  Tsugafune S., Mashiguchi K., Fukui K., Takebayashi Y., Nishimura T., Sakai T., et al.   ( 2017) Yucasin DF, a potent and persistent inhibitor of auxin biosynthesis in plants. Sci. Rep.  7: 13992. Google Scholar CrossRef Search ADS PubMed  Uchida N., Takahashi K., Iwasaki R., Yamada R., Yoshimura M., Endo T.A., et al.   ( 2018) Chemical hijacking of auxin signaling with an engineered auxin–TIR1 pair. Nat. Chem. Biol.  14: 299– 305. Google Scholar CrossRef Search ADS PubMed  Walsh T.A., Neal R., Merlo A.O., Honma M., Hicks G.R., Wolff K., et al.   ( 2006) Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis. Plant Physiol.  142: 542– 552. Google Scholar CrossRef Search ADS PubMed  Won C., Shen X., Mashiguchi K., Zheng Z., Dai X., Cheng Y., et al.   ( 2011) Conversion of tryptophan to indole-3-acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis. Proc. Natl. Acad. Sci. USA  108: 18518– 18523. Google Scholar CrossRef Search ADS   Woodward A.W., Bartel B. ( 2005) Auxin: regulation, action, and interaction. Ann. Bot.  95: 707– 735. Google Scholar CrossRef Search ADS PubMed  Yamada M., Greenham K., Prigge M.J., Jensen P.J., Estelle M. ( 2009) The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development. Plant Physiol . 151: 168– 179. Google Scholar CrossRef Search ADS PubMed  Yang Y., Hammes U.Z., Taylor C.G., Schachtman D.P., Nielsen E. ( 2006) High-affinity auxin transport by the AUX1 influx carrier protein. Curr. Biol . 16: 1123– 1127. Google Scholar CrossRef Search ADS PubMed  Yang H., Murphy A.S. ( 2009) Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. Plant J . 59: 179– 191. Google Scholar CrossRef Search ADS PubMed  Zazimalova E., Murphy A.S., Yang H., Hoyerova K., Hosek P. ( 2010) Auxin transporters—why so many? Cold Spring Harb. Perspect. Biol . 2: a001552. Google Scholar CrossRef Search ADS PubMed  Zhang J., Peer W.A. ( 2017) Auxin homeostasis: the DAO of catabolism. J. Exp. Bot . 68: 3145– 3154. Google Scholar CrossRef Search ADS PubMed  Zhu J., Bailly A., Zwiewka M., Sovero V., Di Donato M., Ge P., et al.   ( 2016) TWISTED DWARF1 mediates the action of auxin transport inhibitors on actin cytoskeleton dynamics. Plant Cell  28: 930– 948. Google Scholar PubMed  Abbreviations Abbreviations ABCB ATP-BINDING CASSETTE subfamily B AOPP L-2-aminooxy-3-phenylpropionic acid ARF auxin response factor ATI auxin transport inhibitor Aux/IAA AUXIN/INDOLE-3-ACETIC ACID AVG aminoethoxyvinylglycine BBo 4-biphenylboronic acid BH-IAA tert-butoxycarbonylaminohexyl-IAA BUM 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid ER endoplasmic reticulum GH3 GRETCHEN HAGEN3 IPA-N3 (S)-2-azido-3-(3-indolyl)propionic acid IPyA indole 3-pyruvic acid L-kyn L-kynurenine NAA naphthalene 1-acetic acid NBD-IAA nitrobenzoxadiazole (NBD)-labeled IAA NBD-NAA nitrobenzoxadiazole (NBD)-labeled naphthalene 1-acetic acid 1-NOA 1-naphthoxyacetic acid NPA N-1-naphthylphthalamic acid PIN PIN-FORMED PLP pyridoxyl-5-phosphate PPBo 4-phenoxyphenylboronic acid TIR TRANSPORT INHIBITOR RESPONSE YDF yucasin DF YUC YUCCA © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Apr 14, 2018

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