The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development 1[C][W][OA] Masashi Yamada 2 , Katie Greenham 3 , Michael J. Prigge 3 , Philip J. Jensen 4 , and Mark Estelle 3 * Department of Biology, Indiana University, Bloomington, Indiana 47405 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 coty- ledons, 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 stud- ies 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 path- ways 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 interme- diate. 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 po- tential 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 amino- transferase 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 synthe- sis, 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 ac- cumulation 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 1 This work was supported by the National Institutes of Health (grant no. GM–43644 to M.E.). 2 Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113–0033, Japan. 3 Present address: Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093. 4 Present address: Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802. * Corresponding author; e-mail mestelle@ucsd.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mark Estelle (mestelle@ucsd.edu). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.138859 168 Plant Physiology Ò , September 2009, Vol. 151, pp. 168–179, www.plantphysiol.org Ó 2009 American Society of Plant Biologists