Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.The directed flow of auxin from cell to cell, through tissues and organs, from sites of synthesis to sites of action underlies the coordination of many processes during plant growth and development. Arabidopsis (Arabidopsis thaliana) PIN-FORMED (PIN) genes were the first found to be necessary for the phenomenon known as polar auxin transport (Okada et al., 1991; Chen et al., 1998; Gälweiler et al., 1998). Asymmetric localization of PIN proteins to the downstream ends of each cell in auxin-transporting tissues was correctly suggested to be a molecular component of the efflux mechanisms (Gälweiler et al., 1998) originally hypothesized as necessary for a directionally biased, or polar movement of auxin through tissues (Rubery and Sheldrake, 1974; Raven, 1975; Goldsmith, 1977; Goldsmith et al., 1981). Other members of the eight-gene PIN family in Arabidopsis were subsequently shown to affect auxin distribution in various tissues and stages of development (Křeček et al., 2009).Shortly after the breakthrough work on PIN1, members of the B subfamily of ATP-binding cassette (ABCB) transporters were discovered to be equally necessary for the phenomenon of polar auxin transport. They were originally called P-GLYCOPROTEIN1 (Dudler and Hertig, 1992; Sidler et al., 1998) and MULTIDRUG RESISTANCE1 (Noh et al., 2001) and ultimately renamed AtABCB1 and AtABCB19, respectively (Verrier et al., 2008). The connection between ABCB transporters and auxin transport was first made through the analysis of Arabidopsis knockout mutants. Polar auxin flow through abcb19 mutant stems is impaired by approximately 80% compared with the wild type and further reduced in abcb1 abcb19 double mutants (Noh et al., 2001). Resultant effects on development include abnormal hypocotyl tropisms (Noh et al., 2003) and the photomorphogenic control of hypocotyl elongation (Wu et al., 2010). Import of indole-3-acetic acid (IAA) to cotyledons through the petiole is reduced by 50% in abcb19 mutants, and this is correlated with an equivalent reduction in cotyledon blade expansion (Lewis et al., 2009). In roots, loss of ABCB19 greatly impairs auxin flow toward the tip without any detectable effect on shootward flow (Lewis et al., 2007). Surprisingly, the only defect detected in abcb19 primary roots associated with this major disruption of auxin transport is greater meandering of the tip during elongation down a vertical agar surface; gravitropism is unaffected (Lewis et al., 2007). Outgrowth of lateral roots, although not their initiation, depends significantly on ABCB19-mediated tipward auxin transport (Wu et al., 2007). The emergence of adventitious roots at the base of hypocotyls from which roots have been excised from Arabidopsis seedlings depends strongly on ABCB19-mediated auxin accumulation at the sites of primordium initiation (Sukumar et al., 2013).The ABCB19 protein is present predominantly in the central cylinder and cortex of the root, consistent with its role in rootward auxin transport (Lewis et al., 2007; Mravec et al., 2008), whereas the closely related ABCB4 is restricted to the lateral root cap and epidermis (Cho et al., 2007), where it functions in shootward auxin transport (Lewis et al., 2007). Loss of ABCB4 function alters the timing and spatial pattern of gravitropic curvature development, apparently because the gravity-induced auxin gradient across the root is less rapidly dissipated by normal shootward (basipetal) transport of the hormone through the elongation zone (Lewis et al., 2007). Root hairs are significantly longer in abcb4 mutants, a phenotype attributed to auxin accumulation due to impaired efflux (Cho et al., 2007). ABCB4 is reported to conduct auxin influx or efflux, depending on the prevailing external auxin concentration (Kubeš et al., 2012).Noh et al. (2001) originally isolated ABCB19 in a molecular screen for genes encoding an ion channel activity in Arabidopsis cells shown by patch-clamp electrophysiology to be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). The rationale for the screen was that a plant challenged with a channel blocker would overexpress the gene encoding the blocked activity. A hypothesis emerging from the Noh et al. (2001) study is that ABCB19 encodes such an ion channel, which is required for polar auxin transport. If true, NPPB would be established as a blocker of polar auxin transport.Pharmacological inhibitors, used for decades in auxin transport research, have some advantages over mutations. Mutations can create complicating pleiotropic effects by inhibiting the process throughout development, while inhibitors can be used to impose an effect at a specific time. 1-Naphthylphthalamic acid (NPA) is the most commonly used inhibitor of polar auxin transport (Katekar and Geissler, 1980), but others are being discovered (Rojas-Pierce et al., 2007; Kim et al., 2010; Tsuda et al., 2011). Inhibitors are especially useful when their targets are well defined, which would be the case if NPPB blocked ABCB19 and induced its expression as hypothesized. The experiments reported here were designed to test this hypothesis with electrophysiological measurements of ABCB19 transport activity, radiotracer measurements of polar auxin transport in roots, levels of fluorescently tagged ABCB19 proteins, auxin reporter expression patterns, and machine-vision measurements of a root growth response that depends on auxin redistribution.     

The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.Auxin is known to play an important role in plant development (Davies, 1995). However, many aspects of auxin biology remain poorly understood. Auxin is synthesized primarily in young tissues, such as cotyledons, leaves, and roots (Ljung et al., 2001, 2005), and transported to other tissues where it is perceived by members of the TRANSPORT INHIBITOR RESPONSE1 (TIR1) auxin receptor family. Recent studies have dramatically increased our knowledge of auxin transport and signaling (Quint and Gray, 2006; Vieten et al., 2007). However, the pathways of auxin synthesis and their regulation are still relatively unclear.Several indole-3-acetic acid (IAA) biosynthetic pathways have been proposed in plants based on research in plant-associated bacteria (Patten and Glick, 1996; Woodward and Bartel, 2005; Spaepen et al., 2007). There are two major types of pathways: the Trp-dependent and Trp-independent pathways. It has been hypothesized that plants have four Trp-dependent pathways that are generally named after an intermediate. In bacteria, the indole-3-pyruvic acid (IPA) pathway, one of the Trp-dependent pathways, has been described in detail (Koga, 1995; Spaepen et al., 2007). The current model for the IPA pathway involves a Trp aminotransferase oxidatively transaminating Trp to IPA. Subsequently, an IPA decarboxylase converts IPA to indole-3-acetaldehyde, and indole-3-acetaldehyde is oxidized to IAA. The IPA pathway is considered a major IAA biosynthetic pathway in plants, since potential intermediates have been isolated from different species (Sheldrake, 1973; Cooney and Nonhebel, 1991; Koga, 1995; Tam and Normanly, 1998). In addition, Trp transamination activity has been found in many plants (Gamborg, 1965; Forest and Wightman, 1972; Truelsen, 1973). Recently, two groups reported the identification of a gene called TAA1. This gene encodes an aminotransferase that converts Trp to IPA and functions in IAA biosynthesis (Stepanova et al., 2008; Tao et al., 2008).To identify genes that are required for auxin synthesis, transport, and signaling, we previously screened for Arabidopsis (Arabidopsis thaliana) mutants that are resistant to auxin transport inhibitors, such as N-1-napthylpthalamic (NPA; Ruegger et al., 1997). The treatment of seedlings with NPA results in auxin accumulation in the root tip (Ljung et al., 2005). Thus, mutants that are resistant to NPA may have defects in synthesis, transport, or response because roots of these mutants are expected to have lower levels of IAA or reduced sensitivity to IAA. This screen succeeded in isolating mutations in seven genes with weak NPA-resistant phenotypes, including genes related to auxin signaling (TIR1), auxin transport (TIR3), and auxin synthesis (TIR7; Ruegger et al., 1997, 1998; Ljung et al., 2005).Here, we describe the characterization of TIR2, a gene whose function is required for auxin synthesis. Genetic and physiological analyses of the tir2 mutant suggest that TIR2 is required for the Trp-dependent auxin synthesis pathway and functions as a Trp aminotransferase. Molecular cloning of TIR2 reveals that the gene is identical to TAA1 (Stepanova et al., 2008; Tao et al., 2008). We show that auxin regulates expression of TIR2 in a tissue-specific manner. Furthermore, we show that TIR2 is required for temperature-dependent hypocotyl elongation and that high temperature positively regulates expression of the TIR2 gene, suggesting that temperature regulates hypocotyl elongation directly by stimulating auxin synthesis. Finally, we provide evidence that TIR2 functions in a positive regulatory loop required for root gravitropism.