AOBPreview originally published online on March 4, 2005
Annals of Botany 2005 95(5):707-735; doi:10.1093/aob/mci083
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INVITED REVIEW |
Auxin: Regulation, Action, and Interaction
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77005, USA
* For correspondence. E-mail bartel{at}rice.edu
Received: 5 October 2004 Returned for revision: 1 November 2004 Accepted: 15 December 2004 Published electronically: 4 March 2005
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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION• Background The phytohormone auxin is critical for plant growth and orchestrates many developmental processes.
• Scope This review considers the complex array of mechanisms plants use to control auxin levels, the movement of auxin through the plant, the emerging view of auxin-signalling mechanisms, and several interactions between auxin and other phytohormones. Though many natural and synthetic compounds exhibit auxin-like activity in bioassays, indole-3-acetic acid (IAA) is recognized as the key auxin in most plants. IAA is synthesized both from tryptophan (Trp) using Trp-dependent pathways and from an indolic Trp precursor via Trp-independent pathways; none of these pathways is fully elucidated. Plants can also obtain IAA by ß-oxidation of indole-3-butyric acid (IBA), a second endogenous auxin, or by hydrolysing IAA conjugates, in which IAA is linked to amino acids, sugars or peptides. To permanently inactivate IAA, plants can employ conjugation and direct oxidation. Consistent with its definition as a hormone, IAA can be transported the length of the plant from the shoot to the root; this transport is necessary for normal development, and more localized transport is needed for tropic responses. Auxin signalling is mediated, at least in large part, by an SCFTIR1 E3 ubiquitin ligase complex that accelerates Aux/IAA repressor degradation in response to IAA, thereby altering gene expression. Two classes of auxin-induced genes encode negatively acting products (the Aux/IAA transcriptional repressors and GH3 family of IAA conjugating enzymes), suggesting that timely termination of the auxin signal is crucial. Auxin interaction with other hormone signals adds further challenges to understanding auxin response.
• Conclusions Nearly six decades after the structural elucidation of IAA, many aspects of auxin metabolism, transport and signalling are well established; however, more than a few fundamental questions and innumerable details remain unresolved.
Key words: Auxin, IAA, indole-3-acetic acid, 2,4-D, IBA, phytohormone, hormone signalling, proteasome, auxin biosynthesis, auxin conjugate, auxin transport, Arabidopsis thaliana
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INTRODUCTION |
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To fully understand auxin regulation, action, and interactions will be to understand many aspects of plant growth and development. As a critical plant hormone, auxin modulates such diverse processes as tropic responses to light and gravity, general root and shoot architecture, organ patterning, vascular development and growth in tissue culture (Davies, 1995
). The importance of auxin for human sustenance is both vital and readily apparent: auxin is required for plant growth. Anthropogenic manipulation of auxin physiology has assisted plant propagation, and, through the blind pressure of artificial selection, the development of modern crop varieties (Multani et al., 2003; Salamini, 2003).
Auxin biology is among the oldest fields of experimental plant research. Charles Darwin performed early auxin experiments, observing the effects of a hypothetical substance modulating plant shoot elongation to allow tropic growth toward light (Darwin, 1880). Darwin's experiments expanded upon Theophil Ciesielski's research examining roots bending toward gravity (Ciesielski, 1872). The term auxin was coined by scientists examining plant growth-modulating substances in human urine named auxins A and B (Kögl and Haagen Smit, 1931). A structurally distinct compound with auxin activity isolated from fungi was called heteroauxin; auxins A and B were gradually abandoned for the reproducibly bioactive heteroauxin, which was later determined to be indole-3-acetic acid (IAA) (Thimann, 1977).
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COMPOUNDS WITH AUXIN ACTIVITY |
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Because auxins influence virtually every aspect of plant growth and development, numerous bioassays for auxin response have been described. These assays have proven useful in the isolation of endogenous auxins, the identification of auxin precursors, and the development of synthetic auxin-like compounds (Thimann, 1977
). One of the earliest noted auxin effects was in phototropism, the curvature of stems toward a light source (Darwin, 1880). Application of auxin to decapitated shoots can induce such bending in the absence of a light stimulus (Went, 1926), and several nonphototropic mutants are deficient in auxin signalling components (Harper et al., 2000; Tatematsu et al., 2004).
The pea curvature test also employs auxin-regulated differential growth: dark-grown (etiolated) Pisum sativum stems are decapitated, sliced along part of their length, and floated in solution containing compounds being tested (Wain and Wightman, 1954; Fawcett et al., 1960). In auxin solution, stem segments bend inward, while in water they curl outward (Went and Thimann, 1937). Other tests to establish whether a given compound exerts auxin-like effects include spraying tomato plants and application to wheat coleoptiles, where auxin causes characteristic stem bending and elongation, respectively (Wain and Wightman, 1954; Fawcett et al., 1960).
Another early assay for auxin activity was in tissue culture, where auxins promote rooting from undifferentiated callus (Skoog and Miller, 1957). Along with the phytohormone cytokinin, which induces shoot formation, auxin allows regeneration of plants from cultured callus (Krikorian, 1995).
Current assays for auxin response in the model plant Arabidopsis thaliana often involve growth of seedlings on medium supplemented with the compound of interest. Auxins profoundly influence root morphology, inhibiting root elongation, increasing lateral root production (Fig. 1), and inducing adventitious roots (Zimmerman and Hitchcock, 1942). The relevance of these bioassays to normal plant physiology is supported by the observation that mutants that overproduce auxin tend to have abundant lateral and adventitious roots, along with long hypocotyls and petioles, and epinastic leaves and cotyledons (Boerjan et al., 1995; King et al., 1995; Delarue et al., 1998; Zhao et al., 2001). Conversely, mutants deficient in auxin responses are often characterized by long primary roots, few lateral roots, and short hypocotyls when grown on unsupplemented medium in the light, in addition to reduced auxin responses in the bioassays described above (Estelle and Somerville, 1987; Hobbie and Estelle, 1995; Monroe-Augustus et al., 2003).
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8. All plants were grown at 22 °C under yellow light.Many naturally occurring compounds that exert auxin-like effects have been revealed by these bioassays (Fig. 1). IAA, an extensively studied endogenous auxin, is active in all bioassays described above and is often potent at nanomolar concentrations (Fig. 1). A chlorinated form of IAA with high auxin activity, 4-Cl-IAA, is found in several plants (Slovin et al., 1999
). In addition to the indolic auxins, phenylacetic acid (PAA) has been identified in plants and is an active auxin (Wightman, 1977; Ludwig-Müller and Cohen, 2002).
Certain IAA precursors, such as indole-3-acetonitrile and indole-3-pyruvic acid, are also active in bioassays, presumably because of conversion in the tissue to IAA (Thimann, 1977). Similarly, indole-3-butyric acid (IBA), identical to IAA except for two additional methylene groups in the side chain, is effective in bioassays. Like IAA, exogenous IBA inhibits arabidopsis root elongation (Zolman et al., 2000) and induces lateral (Zolman et al., 2000) and adventitious (King and Stimart, 1998) root formation. IBA, originally classified as a synthetic auxin, is in fact an endogenous plant compound (Epstein and Ludwig-Müller, 1993; Ludwig-Müller, 2000; Bartel et al., 2001). IBA is more effective than IAA at lateral root induction, perhaps because, unlike IAA, IBA efficiently induces lateral roots at concentrations that only minimally inhibit root elongation (Zolman et al., 2000); IBA is employed commercially for this purpose (Hartmann et al., 1990). Biochemical analyses in a variety of plants and genetic studies in arabidopsis indicate that IBA acts primarily through conversion to IAA in a process resembling peroxisomal fatty acid ß-oxidation (Bartel et al., 2001), though roles for IBA independent of conversion to IAA have been proposed (Ludwig-Müller, 2000; Poupart and Waddell, 2000).
Two main types of synthetic plant growth regulators with auxin-like activity have been described: 1-naphthalacetic acid (NAA) and 2,4-D-related compounds. Both compounds exert auxin-like influences, including root elongation inhibition and lateral root promotion (Fig. 1). The NAA isomer 2-NAA has little activity in bioassays (Thimann, 1977) and provides a weak acid control for auxin experiments employing the active 1-NAA. 2,4-Dichlorophenoxybutyric acid (2,4-DB) is a 2,4-D derivative with two additional methylene groups in the side chain (analogous to the structural relationship between IBA and IAA) that elicits similar responses to those observed after 2,4-D treatment. In general, 2,4-dichlorophenoxyacetic acid (2,4-D) and IAA derivatives with even-numbered carbon side chains have more activity than derivatives with odd-numbered carbon side chains (Wain and Wightman, 1954; Fawcett et al., 1960). This result suggests that a process such as ß-oxidation could remove two-carbon units from the side chains, arriving at the active acetate form if the substrate started with an even carbon number (Wain and Wightman, 1954; Fawcett et al., 1960). 2,4,5-Tricholorphenoxybutyric acid (2,4,5-TB) also exerts auxin-like activity; the infamous defoliant herbicide Agent Orange was a mixture of 2,4-D and 2,4,5-TB (Fallon et al., 1994). Agent Orange was particularly toxic because of dioxin produced as a by-product of 2,4,5-TB synthesis (Courtney et al., 1970; Schwetz et al., 1973). Today, 2,4-D alone is a widely used herbicide. In addition to NAA and 2,4-D, several alkylated and halogenated forms of IAA elicit auxin-like growth responses in various bioassays (Antoli
et al., 1996; Nigovi et al., 2000). Though IAA, 2,4-D, NAA, and other synthetic compounds can cause similar physiological responses in bioassays, the molecules cause distinct but overlapping changes in gene expression (Pufky et al., 2003), perhaps reflecting differences in metabolism, transport, or interaction with the signalling machinery.
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IAA BIOSYNTHETIC PATHWAYS |
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Arabidopsis seedlings can synthesize IAA in leaves, cotyledons and roots; young leaves have the highest biosynthetic capacity (Ljung et al., 2001
). Although it is widely accepted that plants use several pathways to synthesize IAA, none of the pathways is yet defined to the level of knowing each relevant gene, enzyme, and intermediate. Plant genes implicated in IAA biosynthesis are listed in Table 1, and the reactions catalysed by the encoded enzymes are illustrated in Fig. 2. Plants use both tryptophan (Trp)-dependent and Trp-independent routes to synthesize IAA; several Trp-dependent pathways have been suggested. Multiple IAA biosynthetic pathways may contribute to regulation of IAA production, but the paucity of informative loss-of-function mutations in IAA biosynthetic enzymes, coupled with functional redundancy, has limited analysis of pathway control and prevented definitive determination of the importance of each pathway. For example, arabidopsis seedlings grown at high temperature accumulate free IAA (Gray et al., 1998) and display high-auxin phenotypes (Gray et al., 1998; Rogg et al., 2001), but the source of the excess IAA is unknown.
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Trp-dependent IAA biosynthesis
Several Trp-dependent pathways, which are generally named after an intermediate, have been proposed: the indole-3-pyruvic acid (IPA) pathway, the indole-3-acetamide (IAM) pathway, the tryptamine pathway, and the indole-3-acetaldoxime (IAOx) pathway. An arabidopsis enzymatic complex that converts Trp to IAA in vitro has been partially purified (Müller and Weiler, 2000b
), and future biochemical and genetic dissection of the process is likely to reveal the relative importance of the pathways discussed below.
The IPA pathway [Trp 
IPA indole-3-acetaldehyde (IAAld) IAA] is important in some IAA-synthesizing microorganisms (Koga, 1995) and may operate in plants as well (Cooney and Nonhebel, 1991). IPA is found in arabidopsis seedlings (Tam and Normanly, 1998), but genes encoding a Trp aminotransferase that oxidatively transaminates Trp to IPA or an IPA decarboxylase that converts IPA to IAAld have not been identified in plants. The final enzyme in the proposed IPA pathway is an IAAld-specific aldehyde oxidase protein (AAO1) that has increased activity in the IAA-overproducing superroot1 (sur1) mutant (Seo et al., 1998). The identification of arabidopsis AAO1 does not verify the existence of the IPA pathway, however, as IAAld may be an intermediate in other IAA biosynthetic pathways (see below).
The IAM pathway [Trp IAM IAA] is a second microbial pathway that also may act in plants. In Agrobacterium tumifaciens and Pseudomonas syringae, for example, Trp monooxygenase (IaaM) converts Trp to IAM, and an IAM hydrolase (IaaH) converts IAM to IAA (Patten and Glick, 1996). IAM lacks auxin activity in arabidopsis, which allows the iaaH gene to be used as a screenable marker that confers IAM sensitivity (Brusslan et al., 1993). Intriguingly, IAM is found in arabidopsis seedlings at levels similar to free IAA (Pollmann et al., 2002), and an arabidopsis amidohydrolase (AMI1) converts IAM to IAA in vitro (Pollmann et al., 2003). It will be interesting to learn whether disruption of AMI1 or AAO1 decreases IAA levels.
YUCCA may catalyse a rate-limiting step in a tryptamine pathway
A tryptamine (TAM) pathway [Trp TAM N-hydroxyl-TAM indole-3-acetaldoxime (IAOx) IAAld IAA] could also convert Trp to IAA (Fig. 2). Trp decarboxylase converts Trp to tryptamine in the first committed step in the biosynthesis of Catharanthus roseus monoterpenoid indole alkaloids (Facchini et al., 2000). The arabidopsis genome contains potential Trp decarboxylase genes, but the encoded enzymes have not been characterized, and tryptamine has not been identified in arabidopsis.
The identification of yucca, an IAA-accumulating mutant with classic high-auxin phenotypes (Zhao et al., 2001), suggests that a tryptamine IAA biosynthetic pathway may operate in some plants. yucca is resistant to toxic Trp analogues, suggesting that the accumulating IAA is Trp-derived (Zhao et al., 2001). The yucca phenotype derives from overexpression of a flavin monooxygenase (FMO)-like enzyme that oxidizes tryptamine to N-hydroxyl-tryptamine in vitro (Zhao et al., 2001). The homologous Petunia x hybrida enzyme FLOOZY is defective in a mutant deficient in leaf venation and apical dominance (Tobeña-Santamaria et al., 2002). Although the loss-of-function floozy mutant has wild-type IAA levels, overexpressing FLOOZY results in increased IAA levels in shoot apices and young leaves (Tobeña-Santamaria et al., 2002). YUCCA may be a rate-limiting enzyme in the tryptamine pathway, but a test of this hypothesis is hampered by genetic redundancy. Arabidopsis has a family of ten YUCCA-like enzymes, and insertional mutations in YUCCA and YUCCA2 confer no morphological phenotypes (Zhao et al., 2001). The N-hydroxyl-tryptamine produced by YUCCA could be dehydrogenated to IAOx or dehydrogenated and hydrolysed to IAAld (Fig. 2). Enzymes that catalyse these conversions have not been identified.
Indole-3-acetaldoxime is a precursor to indolic glucosinolates that can be converted to IAA
The IAOx pathway [Trp IAOx IAN or IAAld IAA] is of particular interest in plants like arabidopsis that make indolic glucosinolate secondary metabolites (Fahey et al., 2001), because IAOx is the branch-point between indole-3-methylglucosinolate and IAA biosynthesis (Fig. 2). Two arabidopsis P450 monooxygenases, CYP79B2 and CYP79B3, oxidize Trp to IAOx in vitro (Hull et al., 2000; Mikkelsen et al., 2000). CYP79B2 overexpressors have increased IAA, IAN (Zhao et al., 2002) and indolic glucosinolate levels (Mikkelsen et al., 2000). Conversely, the cyp79B2 cyp79B3 double mutant has morphological phenotypes suggestive of low auxin, reduced IAA in certain growth conditions, lowered IAN levels, and no detectable indolic glucosinolates (Zhao et al., 2002). Taken together, these results are consistent with IAOx serving as a precursor that can be shunted to either auxin or indolic glucosinolates.
A third P450 monooxygenase, CYP83B1, converts IAOx to its N-oxide, the first committed step in indole-3-methylglucosinolate biosynthesis (Fig. 2; Bak et al., 2001). Loss-of-function cyp83b1 alleles were independently isolated in screens for high-auxin seedling phenotypes (superroot2 or sur2; Delarue et al., 1998), altered resistance to toxic Trp analogues (Smolen and Bender, 2002), defective photomorphogenesis in red light (Hoecker et al., 2004), and P450 monooxygenase insertional disruptions (Winkler et al., 1998). The sur2/cyp83B1 mutant accumulates free IAA (Delarue et al., 1998; Barlier et al., 2000) and the IAA precursor IAAld (Barlier et al., 2000). This phenotypic analysis, along with the nature of the defective gene, suggests that IAOx accumulates in the mutant and is converted to IAAld, which is oxidized to IAA (Fig. 2).
The sur1 mutant (Boerjan et al., 1995), also isolated as rooty (King et al., 1995), alf1 (Celenza et al., 1995) and hookless3 (Lehman et al., 1996), provides another link between high auxin and defects in glucosinolate production. This mutant has high-auxin phenotypes resembling sur2 and yucca, and accumulates free IAA and IAA conjugates (Boerjan et al., 1995; King et al., 1995; Lehman et al., 1996). sur1 is defective in a C-S lyase that apparently cleaves S-(indolylacetohydroximoyl)-L-cysteine to indole-3-thiohydroximate, the third step in glucosinolate production from IAOx (Golparaj et al., 1996; Mikkelsen et al., 2004). Indeed, indolic glucosinolates are undetectable in sur1 (Mikkelsen et al., 2004). Given the multiplicity of available pathways to modulate IAA levels, it is intriguing that arabidopsis plants cannot adequately compensate for the increased IAA precursor levels that result when indolic glucosinolate production is dampened.
Indole-3-acetonitrile and nitrilases in IAA biosynthesis
Nitrilases that can hydrolyse IAN to IAA are found in several plant families, including crucifers and grasses (Thimann and Mahadevan, 1964). These enzymes are encoded by the arabidopsis NIT genes (Bartling et al., 1992, 1994; Bartel and Fink, 1994) and Zea mays (maize) ZmNIT2 (Park et al., 2003). NIT1 and NIT2 can hydrolyse IAN applied to plants (Schmidt et al., 1996; Normanly et al., 1997), and an enzymatic complex with nitrilase immunoreactivity converts Trp to IAA in vitro (Müller and Weiler, 2000b). IAN is present in arabidopsis (Normanly et al., 1993; Ili et al., 1996) and maize (Park et al., 2003), suggesting that this conversion could contribute to IAA homeostasis. In the brassica, IAN is formed following myrosinase-catalysed indole-3-methylglucosinolate hydrolysis, and IAN levels tend to track with indolic glucosinolate levels in arabidopsis mutants (Normanly et al., 1993; Mikkelsen et al., 2000; Müller and Weiler, 2000a; Reintanz et al., 2001; Zhao et al., 2002), consistent with nitrilases acting downstream of glucosinolates in arabidopsis. However, it has also been suggested that IAN is an intermediate in IAOx to IAA conversion (Fig. 2), although enzymes catalysing the conversion of IAOx to IAN have not been isolated, and the source of IAN in maize, which lacks indolic glucosinolates, is unknown.
NIT1 is the most highly expressed of the four arabidopsis NIT genes (Bartel and Fink, 1994). nit1 mutants are resistant to exogenous IAN (Normanly et al., 1997), but lack obvious low-auxin phenotypes, indicating that any role played by NIT1 in IAA biosynthesis is redundant. The NIT2 gene is normally expressed at a low level, but is induced by a bacterial pathogen (Bartel and Fink, 1994), by Plasmodiophora (Grsic-Rausch et al., 2000), during arabidopsis leaf senescence (Quirino et al., 1999), and in response to IAN treatment (Grsic et al., 1998). NIT2 induction correlates with decreased IAN levels and increased IAA levels during senescence (Quirino et al., 1999), increased IAA levels in Plasmodiophora-infected roots (Grsic-Rausch et al., 2000) and higher nitrilase immunoreactivity (Müller and Weiler, 2000a) in the IAN-accumulating trp3 mutant (Normanly et al., 1993). NIT3 expression is induced by sulfur starvation, and is correlated with reduced indolic glucosinolate levels and lateral root proliferation (Kutz et al., 2002). Expression of maize nitrilase ZmNIT2 is elevated in embryonic tissue (Park et al., 2003). Upgrading these correlations between expression and IAA levels to causal relationships awaits the analysis of additional nit family mutants and would be aided by an arabidopsis nit1 nit2 nit3 triple mutant.
Analyses of trp mutants reveal Trp-independent IAA biosynthesis
In addition to the proposed Trp-dependent IAA biosynthetic pathways (Fig. 2), analyses of Trp biosynthetic mutants demonstrate that plants also can synthesize IAA without using a Trp intermediate. The arabidopsis trp3-1 and trp2-1 mutants are defective in Trp synthase 
and ß, respectively (Last et al., 1991; Radwanski et al., 1996). These mutants accumulate amide- and ester-linked IAA conjugates (Normanly et al., 1993; Ouyang et al., 2000), despite having low soluble Trp levels (Müller and Weiler, 2000a; Ouyang et al., 2000). Similarly, the maize orange pericarp Trp synthase ß mutant accumulates IAA conjugates (Wright et al., 1991, 1992). Unlike trp2 and trp3, plants blocked earlier in the Trp pathway, such as trp1 (Last and Fink, 1988) and antisense plants with decreased indole-3-glycerol phosphate synthase (IGS) levels, do not accumulate IAA conjugates (Normanly et al., 1993; Ouyang et al., 2000).
Analyses of the trp mutants imply that a Trp-independent IAA biosynthetic pathway branches from indole-3-glycerol phosphate or indole (Fig. 2). Trp synthase and ß normally channel indole-3-glycerol phosphate to Trp without indole release. In maize, however, Trp synthase -like enzymes can act without ß subunits to produce indole released as a volatile or converted into certain defense compounds (Frey et al., 1997, 2000; Melanson et al., 1997) or possibly IAA. Arabidopsis contains two apparent Trp synthase genes: TSA1, the gene defective in the trp3 mutant (Radwanski et al., 1996), and a second uncharacterized gene (At4g02610).
Because IAA conjugates are hydrolysed under alkaline conditions (Bialek and Cohen, 1986; Baldi et al., 1989), total (free plus conjugated) IAA is often inferred without knowledge of the conjugates present by quantifying free IAA after alkaline hydrolysis. The specificity of the alkaline hydrolysis evidence used to support the importance of the Trp-independent pathway has been questioned (Müller and Weiler, 2000a). Application of this technique requires accommodation for the indolic biochemistry of the plant under study. For example, IAN, which is present in arabidopsis, is hydrolysed to IAA under alkaline conditions, so IAN must be separately quantified and subtracted from apparent total IAA values (Ili et al., 1996). As the individual conjugates of arabidopsis are identified and quantified, it will be interesting to learn the precise conjugate profiles in the various trp mutants, and to reinvestigate alkaline-releasable IAA in mutant plants that lack indolic glucosinolates, for example.
An independent method to clarify biosynthetic pathways involves feeding plants isotopically labelled substrates, which, in a linear pathway, will result in isotopic enrichment of a precursor relative to its product. Intact arabidopsis seedlings do not efficiently convert [2H5]Trp into IAA, but the Trp precursor [15N]anthranilate labels IAA more completely than Trp (Normanly et al., 1993), confirming the importance of Trp-independent IAA biosynthesis during normal growth. Arabidopsis shoot and root explants, however, do efficiently convert [2H5]Trp to IAA (Müller et al., 1998b; Müller and Weiler, 2000a). Because the explant process may damage tissue, this result suggests that Trp-dependent IAA biosynthesis may be wound-induced in arabidopsis, as it is in bean (Sztein et al., 2002). Plants may switch from basal Trp-independent IAA biosynthesis to Trp-dependent pathways during stress, when more IAA may be needed (Ribnicky et al., 2002; Sztein et al., 2002). Studies examining metabolism of a recently synthesized, isotopically labelled indole may allow dissection of Trp-independent IAA biosynthesis (Ili and Cohen, 2004).
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IAA STORAGE: CONJUGATES AND INDOLE-3-BUTYRIC ACID |
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Higher plants can store IAA in the form of IAA conjugates and indole-3-butyric acid (IBA), which can provide free IAA upon hydrolysis or ß-oxidation, respectively (Fig. 3). IAA can be ester-linked to sugars or amide-linked to amino acids and peptides. Proposed functions for these conjugates include storage, transport, compartmentalization, excess IAA detoxification, and protection against peroxidative degradation (Cohen and Bandurski, 1982
). Certain IAA conjugates are active in auxin bioassays, and several plants store IAA conjugates in seeds that are hydrolysed during germination to provide free IAA to developing seedlings. In contrast, biologically inactive conjugates present in plants are probably intermediates in IAA degradation. Analyses of arabidopsis mutants defective in various facets of IAA homeostasis are revealing the roles of the diverse IAA sources during plant growth and development.
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IAA conjugate identification and functions
Different plant species have distinct IAA conjugate profiles (Cohen and Bandurski, 1982
; Slovin et al., 1999). Experiments using alkaline hydrolysis to release free IAA from conjugates indicate that arabidopsis maintains approx. 90 % of IAA in amide linkages, with an additional approx. 10 % as ester-linked conjugates and approx. 1 % as free IAA (Normanly et al., 1993; Tam et al., 2000). Low levels of IAA–Ala, IAA–Asp, IAA–Glu and IAA–Leu are present in arabidopsis seeds (Rampey et al., 2004) and seedlings (Tam et al., 2000; Kowalczyk and Sandberg, 2001; Rampey et al., 2004). However, most of the amide-linked conjugates in arabidopsis seeds are solvent insoluble (Ljung et al., 2002), suggesting that single-amino acid conjugates constitute only part of the amide fraction in this tissue. A 35-kDa IAA–peptide is present in arabidopsis seeds; the large size of this conjugate may contribute to the solvent insolubility of amide conjugates (Ljung et al., 2002). Although genes encoding arabidopsis IAA–peptides have not been identified, an IAA-modified bean protein is similar to a soybean late seed maturation protein (Walz et al., 2002), suggesting that certain seed storage proteins may function in both amino acid and phytohormone storage. In addition to amide conjugates, the ester conjugate IAA–glucose has also been quantified in several dicotyledonous plants (including arabidopsis) and the monocot maize (Tam et al., 2000; Jakubowska and Kowalczyk, 2004).
Among divergent plant phyla, endogenous IAA, IAA–amide and IAA–ester levels are quite variable (Sztein et al., 1999). The lycophyte Selaginella kraussiana accumulates large quantities of conjugates, particularly IAA–amide compounds (Sztein et al., 1999). After feeding labelled IAA to the lycophyte S. kraussiana, the fern Ceratopteris richardii and various mosses and liverworts, varied species-specific conjugate profiles become apparent; the conjugates formed include both previously identified and unknown IAA conjugates (Sztein et al., 1999). These results suggest ancient roles for conjugates in plant biology.
IAA–amino acid conjugates found in plants can be classified into two groups based on bioassay activity and susceptibility to hydrolysis in planta or by plant enzymes. IAA–Ala and IAA–Leu efficiently inhibit arabidopsis root elongation and are substrates of arabidopsis amidohydrolases (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Campanella et al., 2003; Rampey et al., 2004). In arabidopsis, IAA–Ala is present at highest levels in shoots, whereas IAA–Leu accumulates in roots (Kowalczyk and Sandberg, 2001), but neither conjugate is formed at detectable levels following IAA application to seedlings or leaves (Östin et al., 1998; Barratt et al., 1999). These results suggest that IAA–Ala and IAA–Leu function to supply free IAA.
In contrast, although IAA–Asp and IAA–Glu also are present in arabidopsis (Tam et al., 2000; Kowalczyk and Sandberg, 2001), they are not appreciably hydrolysed by arabidopsis seedlings (Östin et al., 1998), and are inefficient inhibitors of root elongation (Campanella et al., 1996; LeClere et al., 2002). Tissues such as expanding leaves and roots that contain the highest free IAA levels also contain the highest levels of IAA–Asp and IAA–Glu (Kowalczyk and Sandberg, 2001). These results are consistent with an IAA catabolic role for IAA–Asp and IAA–Glu (see ‘IAA inactivation’ section).
Genetic analysis of IAA conjugate hydrolysis
Several mutant screens using different bioactive IAA–amino acid conjugates have been conducted. If conjugates with auxin activity function solely through free IAA release, then conjugate-resistant mutants that retain wild-type sensitivity to IAA may have defects in conjugate uptake or hydrolysis. If bioactive conjugates play additional roles, these also may be uncovered through mutant analyses. ilr1 was isolated as an IAA–Leu resistant mutant with reduced sensitivity to root elongation inhibition caused by exogenous IAA–Leu. ilr1 is defective in an amidohydrolase that cleaves IAA–Leu and IAA–Phe (Bartel and Fink, 1995). Similarly, iar3 is IAA–Ala resistant and is defective in an amidohydrolase homologous to ILR1 that specifically hydrolyses IAA–Ala (Davies et al., 1999). The ILR1-like protein ILL2 is the most active IAA amidohydrolase in vitro (LeClere et al., 2002); however, no ill2 alleles were isolated in genetic screens for conjugate-resistant root elongation. Though ILR1 and IAR3 are expressed in seedling roots, ILL2 appears to be expressed predominantly in the shoot (Rampey et al., 2004). An ill2 T-DNA allele is sensitive to IAA–Leu, IAA–Phe and IAA–Ala, but, when combined in double and triple mutants with ilr1 and iar3, ill2 contributes to IAA–Phe resistance in roots and hypocotyls and IAA–Ala resistance in hypocotyls (Rampey et al., 2004).
Interestingly, ilr1 iar3 ill2 triple mutant seedlings display reductions in lateral root number, hypocotyl elongation in the light, sensitivity to exogenous IAA, and free IAA levels (Rampey et al., 2004). These results suggest that the endogenous IAA conjugate substrates of these hydrolases (IAA–Ala and IAA–Leu) are physiologically relevant sources of free IAA. The IAA–Leu insensitivity of the ilr1 iar3 ill2 mutant implies that at least some IAA conjugates with auxin activity act solely via their hydrolysis to free IAA. However, the triple hydrolase mutant retains partial responsiveness to IAA–Ala (Rampey et al., 2004), suggesting that IAA–Ala has some hydrolysis-independent activity or that additional enzymes hydrolysing IAA–Ala remain to be discovered.
The iar1 mutant is resistant to the known substrates of the ILR1 and IAR3 amidohydrolases and is defective in a membrane protein (Lasswell et al., 2000) that weakly resembles the ZIP family of metal transporters (Guerinot, 2000). Although the substrate and membrane localization of IAR1 are unknown, the fact that the amidohydrolases require divalent cations such as Mn2+, Co2+ or Cu2+ for activity in vitro (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002) suggests that metal homeostasis could impact conjugate hydrolysis by modulating amidohydrolase activity. Further supporting a role for metal homeostasis in IAA conjugate metabolism, the IAA–Leu and IAA–Phe resistant ilr2 mutant is also resistant to exogenous Co2+ and Mn2+ (Magidin et al., 2003). Because the novel ILR2 protein appears to influence metal transport and the ilr2 mutant has a resistance profile similar to ilr1, ILR2 may indirectly affect IAA-conjugate metabolism by negatively regulating transport of metals that influence ILR1 activity (Magidin et al., 2003).
The IAA–Ala resistant mutant iar4 harbours a defective mitochondrial-type pyruvate dehydrogenase E1 (LeClere et al., 2004). iar4 is generally defective in root elongation, but is resistant to several IAA–amino acid conjugates. Although a direct role for pyruvate dehydrogenase in IAA-conjugate hydrolysis is difficult to envision, the slight resistance of iar4 to the synthetic auxin 2,4-D implies that the mutant may be generally deficient in auxin metabolism or response. It is possible that pyruvic acid itself, or an anabolic or catabolic product, influences IAA homeostasis. Alternatively, a complex including IAR4 may function directly in IAA biosynthesis, catalysing indole-3-pyruvic acid dehydrogenation to yield IAA–CoA, a hypothetical precursor of IAA or IAA conjugates (LeClere et al., 2004).
The genes defective in the icr1 (IAA-conjugate resistant), icr2 (Campanella et al., 1996), and ilr3 (R. A. Rampey, M. Tierney and B. Bartel, unpubl. res.) mutants have not been reported. Genes currently implicated in IAA-conjugate responses are listed in Table 2. Because ilr2, ilr3, iar4, icr1 and icr2 are each represented by a single allele isolated in forward genetic screens, it is likely that conjugate resistance screens are not yet saturated. Sequence analysis suggests that the IAA–amino acid conjugate hydrolases reside in the ER (endoplasmic reticulum) lumen (Bartel and Fink, 1995; Davies et al., 1999). Interestingly, the essential auxin binding protein ABP1 (Chen et al., 2001) is also predominantly ER-localized (Jones, 1994), reinforcing the possibility of a role for this compartment in auxin biology. Analysis of additional mutants may reveal genes required for conjugate import into or IAA efflux from the ER, amidohydrolase transcript accumulation, or amidohydrolase localization, activity or stability. In theory, conjugate-resistant mutants that fail to import conjugates from the medium might be isolated as well (see ‘Auxin transport’ section).
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The endogenous auxin IBA is converted to IAA in peroxisomes
IBA is a naturally occurring auxin in a variety of plants (Epstein and Ludwig-Müller, 1993
; Ludwig-Müller, 2000; Bartel et al., 2001). Arabidopsis seedlings contain somewhat less free IBA than IAA (Ludwig-Müller et al., 1993), although detailed studies indicating whether this trend holds at all developmental stages have not been completed. Conditions that change IAA levels tend to similarly alter IBA levels (Ludwig-Müller et al., 1993), suggesting that IAA and IBA metabolism are linked. Indeed, arabidopsis seedlings fed labelled IAA make labelled IBA, suggesting that IBA is synthesized from IAA (Ludwig-Müller and Epstein, 1994). Because IBA also acts as an IAA precursor (see below), IBA could function similarly to bioactive IAA conjugates in IAA homeostasis (Bartel et al., 2001).
The auxin activity of IBA results, at least in part, from its conversion to IAA (Fig. 3). Isolation and characterization of arabidopsis mutants with IBA-resistant, IAA-sensitive root elongation are clarifying our understanding of IBA action (Poupart and Waddell, 2000; Zolman et al., 2000, 2001a, b; Zolman and Bartel, 2004; Woodward and Bartel, 2005). Mutants with specific ß-oxidation defects are IBA resistant, suggesting that IBA is converted to IAA in a process paralleling fatty acid ß-oxidation. Because plants ß-oxidize fatty acids solely in peroxisomes (Gerhardt, 1992; Kindl, 1993), and several IBA-response mutants also have peroxisomal defects, IBA to IAA conversion is likely peroxisomal.
Peroxisomal ß-oxidation of seed storage lipids provides energy to germinating seedlings in oil-seed plants like arabidopsis. As a result, arabidopsis fatty acid utilization mutants require supplemental sucrose after germination to prevent developmental arrest (Hayashi et al., 1998). Similarly, many IBA-response mutants are sucrose-dependent during seedling development, have reduced rates of seed storage lipid utilization, and are IBA resistant in both root elongation and lateral root initiation (Zolman et al., 2000, 2001a, b; Zolman and Bartel, 2004; Woodward and Bartel, 2005). These phenotypes suggest defects in the peroxisomal ß-oxidation of long-chain fatty acids and IBA. Other IBA-response mutants appear to metabolize long-chain fatty acids normally during germination (Zolman et al., 2000; Adham et al., 2005), but may still have defects in IBA ß-oxidation, perhaps due to lesions in isozymes specific to short-chain substrates and IBA.
Cloning the genes defective in several IBA-response mutants (Table 3) has substantiated the essential role of peroxisomal ß-oxidation in IBA activity. In addition to the proteins required directly in peroxisomal metabolism, more than 20 proteins are required for peroxisome biogenesis and import of peroxisomal matrix proteins from the cytoplasm (Olsen, 1998; Subramani, 1998; Tabak et al., 1999; Mullen et al., 2001). Mutations in PEX5 or PEX7, receptors that bind and transport proteins into the peroxisomal matrix (Olsen, 1998; Subramani, 1998), confer IBA-response defects (Zolman et al., 2000; Woodward and Bartel, 2005). pex5 and pex7 are likely to have defects importing ß-oxidation enzymes from the cytoplasm, slowing ß-oxidation and causing IBA resistance. Another IBA-response mutant is defective in the peroxisome biogenesis gene PEX6 and has abnormal peroxisome morphology (Zolman and Bartel, 2004). PXA1, a membrane protein that is approx. 30 % identical to human and yeast ATP-binding cassette transporters implicated in importing long-chain fatty acids into peroxisomes (Dubois-Dalcq et al., 1999; Holland and Blight, 1999), is defective in another IBA-response mutant (Zolman et al., 2001b). Because pxa1 is resistant to IBA and is sucrose-dependent during seedling development, PXA1 is probably necessary for the import of both IBA and fatty acids (or the corresponding CoA esters) into peroxisomes (Zolman et al., 2001b; Footitt et al., 2002; Hayashi et al., 2002).
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Defects in ß-oxidation enzymes can also lead to IBA resistance (Table 3). Several arabidopsis peroxisomal ß-oxidation defective mutants have been isolated using resistance to the IBA analogue 2,4-dichlorophenoxybutyric acid (2,4-DB) (Hayashi et al., 1998
), which is converted to the active synthetic auxin 2,4-D similarly to IBA ß-oxidation (Wain and Wightman, 1954). 2,4-DB-resistant mutants include acx3 (Eastmond et al., 2000), acx4 (Rylott et al., 2003), aim1 (Richmond and Bleecker, 1999) and ped1 (Hayashi et al., 1998), which are also IBA resistant (Zolman et al., 2000; Adham et al., 2005). acx mutants have defects in acyl-CoA oxidases catalysing the second step of fatty acid ß-oxidation, aim1 (abnormal inflorescence meristem) is a mutant in a multifunctional protein acting in the third and fourth steps of fatty acid ß-oxidation (Richmond and Bleecker, 1999), and peroxisome defective l (ped1) is defective in a thiolase catalysing the final step of ß-oxidation (Hayashi et al., 1998). Moreover, mutations in the gene encoding PEX14/PED2, which docks PEX5 at the peroxisome membrane, confer resistance to 2,4-DB (Hayashi et al., 1998, 2000) and IBA (Monroe-Augustus, 2004).
Because arabidopsis mutants defective in fatty acid ß-oxidation enzymes and peroxisome biogenesis proteins are IBA resistant, IBA is likely to be converted to IAA in peroxisomes. It remains to be determined whether enzymes that catalyse fatty acid ß-oxidation also directly catalyse IBA ß-oxidation, or whether there are peroxisomal enzymes dedicated to IBA ß-oxidation. At least some fatty acid ß-oxidation enzymes appear not to act on IBA, as evidenced by the normal IBA and 2,4-DB responses of the lacs6 lacs7 double mutant, which is sucrose dependent due to defects in peroxisomal acyl-CoA synthetases catalysing the first step of fatty acid ß-oxidation (Fulda et al., 2004). If IBA to IAA conversion requires dedicated enzymes, one would expect to recover IBA-response mutants defective in these isozymes that retain normal fatty acid ß-oxidation. Moreover, the inferred peroxisomal localization of IBA to IAA conversion implies the existence of a hydrolase that releases IAA from the CoA ester (Fig. 3), unless this thioester is efficiently hydrolysed nonenzymatically, and a transporter that effluxes IAA or IAA–CoA out of the peroxisome. Indeed, several sucrose-independent IBA-response mutants, including ibr1 and ibr3, are candidates for having defects in such functions (Zolman et al., 2000).
Several peroxisome defective IBA-response mutants have reduced lateral root initiation not only following IBA exposure (Zolman et al., 2000), but also in the absence of exogenous auxin (Zolman et al., 2001b; Zolman and Bartel, 2004; Woodward and Bartel, 2005). Similarly, certain Pyrus communis (pear) plants with adventitious root formation defects apparently do not convert IBA to IAA (Baraldi et al., 1993). These defects imply that the IAA formed from endogenous IBA ß-oxidation during seedling development is important for lateral root initiation. The lateral rooting defects in the peroxisome defective IBA-response mutants (Zolman et al., 2001b; Zolman and Bartel, 2004; Woodward and Bartel, 2005) are more severe than those of the conjugate hydrolase triple mutant (Rampey et al., 2004), suggesting that conjugate hydrolysis does not fully compensate for a lack of IBA ß-oxidation, and vice versa.
A few IBA-response mutants with apparently normal fatty acid ß-oxidation are less sensitive than wild type to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and auxin transport inhibitors (Zolman et al., 2000). The rib1 (resistant to IBA) mutant is in this class (Poupart and Waddell, 2000). Moreover, the Lateral rootless (Lrt1) Oryza sativa (rice) mutant is resistant to IAA, IBA and 2,4-D in terms of root elongation, but only IBA can restore lateral root initiation to the mutant (Chhun et al., 2003). Identifying the genes defective in these IBA-response mutants may reveal IAA-independent roles for IBA or unique features of IBA biology, such as factors differentially mediating IBA and IAA transport (Rashotte et al., 2003).
Like IAA, much of the IBA in plants is conjugated to other moieties through amide- and ester-linkages (Epstein and Ludwig-Müller, 1993; Ludwig-Müller, 2000). A wheat homologue of the arabidopsis IAR3 IAA–Ala hydrolase is inactive on IAA conjugates, but rather hydrolyses amino acid conjugates of IBA including IBA–Ala, which is present in wheat extracts (Campanella et al., 2004). It will be interesting to learn whether the other members of the monocot amidohydrolase family have specificity for IAA– or IBA–amino acid conjugates. In arabidopsis, IBA is largely ester linked (Ludwig-Müller et al., 1993), suggesting that a different family of enzymes will catalyse IBA release. Although the complete IAA and IBA conjugate profiles have not been reported for any plant, it is likely that these profiles will be complex and reflect the diversity and specificities of the corresponding conjugate hydrolases and synthases.
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IAA INACTIVATION |
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Pathways that inactivate IAA (Fig. 3) counteract the inputs to the IAA pool. As discussed above, IAA conjugates that accumulate following exposure of arabidopsis to IAA apparently differ from those used for IAA storage, consistent with the conjugated moiety dictating the fate of the attached IAA (Cohen and Bandurski, 1982
). Arabidopsis permanently inactivates applied IAA by ring oxidation to oxIAA (Fig. 3), which can then be conjugated to hexose (Östin et al., 1998). In addition, IAA is conjugated to Asp and Glu after applying 5 µM IAA (Östin et al., 1998), and to Asp, Glu, Gln and glucose in response to 500 µM IAA (Barratt et al., 1999). Arabidopsis seedlings do not appreciably hydrolyse IAA–Asp and IAA–Glu, and IAA–Asp can be further oxidized to oxIAA–Asp (Östin et al., 1998), reinforcing the catabolic nature of Asp conjugation. The catabolic conjugation system is probably present during normal growth, because IAA–Asp and IAA–Glu are present at low levels in arabidopsis seedlings (Tam et al., 2000; Kowalczyk and Sandberg, 2001; Rampey et al., 2004).
In response to elevated IAA levels, catabolic conjugation pathways may be up-regulated and storage conjugation pathways down-regulated. For example, the sur2 mutant accumulates free IAA (see above) and IAA–Asp (Barlier et al., 2000), an intermediate in permanent IAA inactivation (Normanly, 1997; Slovin et al., 1999). However, sur2 plants inefficiently form the putative arabidopsis IAA st