Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response. One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p. Here we report the identification of a new gene called TRANSPORT INHIBITOR RESPONSE 1 (TIR1). The tir1 mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation. These results indicate that TIR1 is also required for normal response to auxin. Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways. The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box. Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2. Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein.[Key Words: F-box protein; Arabidopsis; auxin response; TIR1; human SKP2; yeast Grr1p] Received September 10, 1997; revised version accepted November 14, 1997.Physiological studies have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of diverse developmental processes including stem elongation, apical dominance, photo-and gravitropism, and lateral root initiation (Klee and Estelle 1991). At the cellular level, auxin acts to regulate these processes through changes in cell division and cell expansion (Evans 1984). Although the mechanism or mechanisms of auxin action are largely unknown, the existing evidence suggests that the hormone acts both at the plasma membrane and within the cell (Vesper and Kuss 1990; Venis and Napier 1995). Auxin treatment results in rapid hyperpolarization of the plasma membrane and induction of specific gene expression (Barbier-Brygoo 1995;Abel and Theologis 1996).In Arabidopsis, genetic studies have resulted in the identification of a number of genes that are required for normal auxin response (Hobbie and Estelle 1994, Leyser 1997). For three of the genes, AXR1, AXR4, and AUX1, recessive mutations result in reduced auxin response as well as an array of auxin-related growth defects (Hobbie and Estelle 1994). By genetic criteria, AXR1 and AUX1 appear to act in distinct pathways. AUX1 is a membrane protein with similarity to amino acid permeases from plants and fungi (Bennett et al. 1996). Because IAA is an indolic compound structurally related to tryptophan, AUX1 may function in cellular auxin uptake and not response per se. In contrast, the axr1 and axr4 mutations display a synergistic interaction, suggesting that the wild-type genes function in the same or overlapping pathways (Hobbie and Estelle 1995). Genetic experiments indicate that another gene, called SAR1, can also be placed in this group. The sar1 muta...

The plant hormone auxin has a central role in many aspects of plant growth and development. By screening for mutants of Arabidopsis that are resistant to exogenous auxin, we have identified several genes that are required for normal auxin response. One of these genes, AXR1, is defined by recessive mutations that confer auxin resistance to the roots, rosettes and inflorescences of mutant plants. In addition, axr1 mutants display a variety of morphological defects that are consistent with a reduction in auxin sensitivity. Here we isolate the AXR1 gene using a map-based approach and report that AXR1 encodes a new protein with significant sequence similarity to the ubiquitin-activating enzyme E1. The AXR1 protein is highly diverged from previously characterized E1 enzymes, however, and lacks a key cysteine residue that is essential for E1 activity. AXR1 may therefore define a new class of enzymes in the ubiquitin pathway or it may have a novel function in cellular regulation which is unrelated to ubiquitin conjugation.

We have screened a large population of M2 seeds of Arabidopsis thaliana for plants which are resistant to exogenously applied indole-acetic acid (IAA). One of the resistant lines identified in this screen carries a dominant mutation which we have named axr2. Linkage analysis indicates that the axr2 gene lies on chromosome 3. Plants carrying the axr2 mutation are severe dwarfs and display defects in growth orientation of both the shoot and root suggesting that the mutation affects some aspect of gravitropic growth. In addition, the roots of axr2 plants lack root hairs. Growth inhibition experiments indicate that the roots of axr2 plants are resistant to ethylene and abscisic acid as well as auxin.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.