AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.     

AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling

Arabidopsis root architecture is regulated by shoot-derived signals such as nitrate and auxin. We report that mutations in the putative auxin influx carrier AUX1 modify root architecture as a result of the disruption in hormone transport between indole-3-acetic acid (IAA) source and sink tissues. Gas chromatography-selected reaction monitoring-mass spectrometry measurements revealed that the aux1 mutant exhibited altered IAA distribution in young leaf and root tissues, the major IAA source and sink organs, respectively, in the developing seedling. Expression studies using the auxin-inducible reporter IAA2::uidA revealed that AUX1 facilitates IAA loading into the leaf vascular transport system. AUX1 also facilitates IAA unloading in the primary root apex and developing lateral root primordium. Exogenous application of the synthetic auxin 1-naphthylacetic acid is able to rescue the aux1 lateral root phenotype, implying that root auxin levels are suboptimal for lateral root primordium initiation in the mutant.     

IAR4, a gene required for auxin conjugate sensitivity in Arabidopsis, encodes a pyruvate dehydrogenase E1alpha homolog

The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1alpha-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1alpha-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots

We have found that chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea (Pisum sativum L. cv Alaska), specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Application of 60 microM CSI disrupts the vertically oriented elongation of wild-type roots grown on agar plates but orients the elongation of agravitropic mutant aux1-7 roots toward the gravity. The CSI-induced restoration of gravitropic response in aux1-7 roots was not observed in other agravitropic mutants, axr2 and eir1-1. Because the aux1-7 mutant is reduced in sensitivity to auxin and ethylene, we examined the effects of CSI on another auxin-resistant mutant, axr1-3, and ethylene-insensitive mutant ein2-1. In aux1-7 roots, CSI stimulated the uptake of [(3)H]indole-3-acetic acid (IAA) and induced gravitropic bending. In contrast, in wild-type, axr1-3, and ein2-1 roots, CSI slowed down the rates of gravitropic bending and inhibited IAA uptake. In the null allele of aux1, aux1-22, the agravitropic nature of the roots and IAA uptake were not affected by CSI. This close correlation between auxin uptake and gravitropic bending suggests that CSI may regulate gravitropic response by inhibiting or stimulating the uptake of endogenous auxin in root cells. CSI exhibits selective influence toward IAA versus 1-naphthaleneacetic acid as to auxin-induced inhibition in root growth and auxin uptake. The selective action of CSI toward IAA along with the complete insensitivity of the null mutant aux1-22 toward CSI strongly suggest that CSI specifically interacts with AUX1 protein.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Cloning and characterization of IAR1, a gene required for auxin conjugate sensitivity in Arabidopsis

Most indole-3-acetic acid (IAA) in higher plants is conjugated to amino acids, sugars, or peptides, and these conjugates are implicated in regulating the concentration of the free hormone. We identified iar1 as an Arabidopsis mutant that is resistant to the inhibitory effects of several IAA-amino acid conjugates but remains sensitive to free IAA. iar1 partially suppresses phenotypes of a mutant that overproduces IAA, suggesting that IAR1 participates in auxin metabolism or response. We used positional information to clone IAR1, which encodes a novel protein with seven predicted transmembrane domains and several His-rich regions. IAR1 has homologs in other multicellular organisms, including Drosophila, nematodes, and mammals; in addition, the mouse homolog KE4 can functionally substitute for IAR1 in vivo. IAR1 also structurally resembles and has detectable sequence similarity to a family of metal transporters. We discuss several possible roles for IAR1 in auxin homeostasis.     

The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture

Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.     

The rib1 mutant is resistant to indole-3-butyric acid, an endogenous auxin in Arabidopsis

The presence of indole-3-butyric acid (IBA) as an endogenous auxin in Arabidopsis has been recently demonstrated. However, the in vivo role of IBA remains to be elucidated. We present the characterization of a semi-dominant mutant that is affected in its response to IBA, but shows a wild-type response to indole-3-acetic acid (IAA), the predominant and most studied form of auxin. We have named this mutant rib1 for resistant to IBA. Root elongation assays show that rib1 is specifically resistant to IBA, to the synthetic auxin 2,4-dichlorophenoxyacetic acid, and to auxin transport inhibitors. rib1 does not display increased resistance to IAA, to the synthetic auxin naphthalene acetic acid, or to other classes of plant hormones. rib1 individuals also have other root specific phenotypes including a shortened primary root, an increased number of lateral roots, and a more variable response than wild type to a change in gravitational vector. Adult rib1 plants are morphologically indistinguishable from wild-type plants. These phenotypes suggest that rib1 alters IBA activity in the root, thereby affecting root development and response to environmental stimuli. We propose models in which RIB1 has a function in either IBA transport or response. Our experiments also suggest that IBA does not use the same mechanism to exit cells as does IAA and we propose a model for IBA transport.     

A family of auxin-conjugate hydrolases that contributes to free indole-3-acetic acid levels during Arabidopsis germination

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-beta-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.     

Plant hormone conjugation: A signal decision

Tight regulation of the auxin hormone indole-3-acetic acid (IAA) is crucial for plant development. Newly discovered IAA antagonists are the amide-linked tryptophan conjugates of IAA and jasmonic acid (JA). JA-Trp and IAA-Trp interfered with root gravitropism in Arabidopsis, and inhibited several responses to exogenously supplied IAA. Relatively low concentrations of the inhibitors occurred in Arabidopsis, but Pisum sativum flowers contained over 300 pmole g⁻¹ FW of JA-Trp. DihydroJA was an even more effective inhibitor than JA-Trp, suggesting that Trp conjugates with other JA derivatives may also be functional. JA-Trp and IAA-Trp add to the list of documented bioactive amide hormone conjugates. The only other example is JA-Ile, the recently discovered jasmonate signal. These examples establish that conjugation not only inactivates hormones, but in some cases creates novel compounds that function in hormone signaling.Key words: jasmonic acid, indole-3-acetic acid, auxin, tryptophan, conjugate, plant hormone, signaling, amino acid, antagonistPlants hold an amazing capacity to auto-regulate their growth and respond to a host of environmental challenges. Since the early discovery of the first plant hormone, indole-3-acetic acid (IAA),¹ science has progressively unveiled ever more complex, and sometimes surprising, ways that plants manipulate hormones to optimize their growth and thwart their opponents. Until recently, the covalent coupling of hormones to sugars, amino acids and peptides was thought to be merely a way to dispose of excess hormone.² The amide linkage of IAA to Asp and Glu does indeed result in IAA catabolism, while IAA-Ala and IAA-Leu are inactive stored forms of IAA.³ But the perception that all hormone conjugates are inactive changed abruptly with the discovery that the isoleucine conjugate of jasmonic acid (JA-Ile) is an active hormonal signal.     

Identical amino acid substitutions in the repression domain of auxin/indole-3-acetic acid proteins have contrasting effects on auxin signaling

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-for-leucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.