Indole-3-acetic Acid Conjugates Research Articles

Euphorbia abyssinica latex promotes rooting of Boswellia cuttings

Traditional knowledge in northern Ethiopia points towards the root-stimulating properties of the latex found in Euphorbia abyssinica (EAG). Stem cuttings of EAG as well as cuttings of other species treated with EAG latex reportedly root easily in dry soil. This could be attributable to endogenous auxins contained in the latex. We screened EAG latex for auxins and precursor molecules using analytical LC-MS/MS, and tested the effect of EAG latex on the rooting of woody cuttings of Boswellia papyrifera, a threatened multipurpose tree. The EAG latex contained indole acetic acid (IAA) (0.06 μg/g latex), an auxin controlling apical dominance and lateral rooting, as well as IAA metabolites and conjugates. Boswellia cuttings treated with EAG latex rooted significantly sooner and showed higher survival ratios than untreated controls. EAG is widely available and its latex easy to obtain. Therefore it is a promising source of a natural plant growth regulator which may be used for improving low technology vegetative propagation of woody species in East and Northeast Africa, including the threatened Boswellia tree.

Maize nitrilases have a dual role in auxin homeostasis and -cyanoalanine hydrolysis

The auxin indole-3-acetic acid (IAA), which is essential for plant growth and development, is suggested to be synthesized via several redundant pathways. In maize (Zea mays), the nitrilase ZmNIT2 is expressed in auxin-synthesizing tissues and efficiently hydrolyses indole-3-acetonitrile to IAA. Zmnit2 transposon insertion mutants were compromised in root growth in young seedlings and sensitivity to indole-3-acetonitrile, and accumulated lower quantities of IAA conjugates in kernels and root tips, suggesting a substantial contribution of ZmNIT2 to total IAA biosynthesis in maize. An additional enzymatic function, turnover of beta-cyanoalanine, is acquired when ZmNIT2 forms heteromers with the homologue ZmNIT1. In plants carrying an insertion mutation in either nitrilase gene this activity was strongly reduced. A dual role for ZmNIT2 in auxin biosynthesis and in cyanide detoxification as a heteromer with ZmNIT1 is therefore proposed.

Analysis of Four-Way Two-Dimensional Liquid Chromatography-Diode Array Data: Application to Metabolomics

Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of very complex mixtures, including proteomic and metabolomic samples. It provides an effective strategy for separating such samples, because the resolving power of 2D-LC is far superior to that of traditional single-dimension separations. The present work focuses on the development of data analysis methods for the extremely large data sets, on the order of 10 million data points, generated by 2D-LC with diode-array detection (DAD). Specifically, we have applied and adapted chemometric methods to the analysis of maize seedling digests, focusing on compounds related to the biosynthetic pathways of indole-3-acetic acid, the primary growth regulator in plants. The chemometric techniques of window target testing factor analysis (WTTFA), along with parallel factor analysis - alternating least squares (PARAFAC-ALS) were used to analyze 2D-LC-DAD chromatograms of a sample composed of 26 indolic standards, 2 extracts of mutant orange pericarp maize seedlings, 2 extracts of wild-type maize seedlings, and a blank sample. The indolic compounds studied belonged to six spectrally unique groups, and WTTFA was able to specifically identify the presence or absence of any of the 26 indolic standards in the mutant and wild-type samples. A PARAFAC-ALS algorithm and an ALS algorithm with flexible constraints were successfully applied to resolve the spectrally rank deficient data and to demonstrate the quantitative potential of multivariate curve resolution methods. Using this procedure, 95 total peaks were resolved in the data set analyzed. Of those 95 peaks, 45 were found in both the mutant and wild-type maize samples, 16 peaks were unique to the mutant maize samples, 13 peaks were unique to the wild-type maize samples, and 15 peaks were unique to the standard chromatograms. Of the 26 standards included in the data set, several indole acetic acid conjugates were identified and quantified in the maize samples at levels of approximately 0.3-2 microg/g plant material.

Strawberry fruit protein with a novel indole-acyl modification

Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA-glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC-MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue.

Indole-3-Acetic Acid Protein Conjugates: Novel Players in Auxin Homeostasis

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.

Auxin Biosynthesis in Maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.

Auxin and Light Control of Adventitious Rooting in Arabidopsis Require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.

Auxins in the development of an arbuscular mycorrhizal symbiosis in maize

While the levels of free auxins in maize (Zea mays L.) roots during arbuscular mycorrhiza formation have been previously described in detail, conjugates of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with amino acids and sugars were neglected. In this study, we have therefore determined free, ester and amide bound auxins in roots of maize inoculated with Glomus intraradices during early stages of the colonization process. Ester conjugates of IAA and IBA were found only in low amounts and they did not increase in AM colonized roots. The Levels of IAA and IBA amide conjugates increased 20 and 30 days past inoculation (dpi). The formation of free and conjugated IBA but not IAA was systemically induced during AM colonization in leaves of maize plants. This implicated a role for auxin conjugate synthesis and hydrolysis during AM. We have therefore investigated the in vivo metabolism of 3H-labeled IBA by TLC but only slight differences between control and AM-inoculated roots were observed. The activity of auxin conjugate hydrolase activity measured with three different putative substrates showed a decrease in infected roots compared to controls. The fluorinated IBA analog TFIBA inhibited IBA formation in leaves after application to the root system, but was not transported from roots to shoots. AM hyphae were also not able to transport TFIBA. Our results indicate complex control mechanisms to regulate the levels of free and conjugated auxins, which are locally and systemically induced during early stages of the formation of an arbuscular mycorrhizal symbiosis.

Auxin: Regulation, Action, and Interaction

The phytohormone auxin is critical for plant growth and orchestrates many developmental processes. 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 beta-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. 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.

Characterization of an Arabidopsis Enzyme Family That Conjugates Amino Acids to Indole-3-Acetic Acid

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

Flavonoids, NodD1, NodD2, and Nod-Box NB15 Modulate Expression of the y4wEFG Locus That Is Required for Indole-3-Acetic Acid Synthesis in Rhizobium sp. strain NGR234

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.

Daylength affects both free and conjugated indole-3-acetic acid levels in leaves and flowering in Doritis pulcherrima (orchid)

Total indole-3-acetic acid (IAA) levels (free IAA plus IAA conjugates) in Doritis pulcherrima (Doritis pulcherrima Lindley, cv. S 84-335-2) leaves were threefold lower in plants exposed to 30 d of short-day (SD) conditions than in plants exposed to 30 d of long-day (LD) conditions. Free IAA levels were also significantly lower in SD-treated leaves than those in 0 day of photoperiodic treatment and 30 d of LD. Results indicate that plants exposed to 30 d of SD contained significantly higher levels of ester-IAA and reduced amide-IAA concentrations compared to those grown under LD conditions. A high level of ester-IAA and a reduction of amide-IAA in leaves may be related to increased SD floral initiation ability in D. pulcherrima. Key words: Doritis pulcherrima, floral initiation, indole-3-acetic acid, photoperiod

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.

Polar Transport of Indole-3-Acetic Acid in Relation to Rooting in Carnation Cuttings: Influence of Cold Storage Duration and Cultivar

The influence of cold storage of cuttings on the transport and metabolism of indole-3-acetic acid (IAA) and the rooting were studied in two carnation (Dianthus caryophyllus L.) cultivars (Oriana and Elsy), which are known to exhibit very distinct rooting characteristics. The percentage of rooting at 11 d after planting increased with the storage period particularly in Oriana, but the values in Elsy were higher than in Oriana. Auxin transport was measured by applying 3H-IAA to stem sections. Irrespective of the section localization, the oldest node (node) or the basal internode (base), the transport increased as the storage period increased from 2 to 12 weeks in Oriana and from 2 to 8 weeks in Elsy cuttings. The auxin transport rate was higher in bases than in nodes and also in Elsy than in Oriana at a given storage period. IAA oxidation and hydrolyzation of IAA conjugates (determined by extracting the sections with acetonitrile and NaOH once the basipetal IAA movement ceased after a 24 h transport period) showed a negative, highly significant correlation with the amount of IAA transported. Although the rooting percentage and IAA transport were higher in Elsy than in Oriana, the differences in rooting between the cultivars could not be explained solely by differences in IAA transport.

Gravity-induced modification of auxin transport and distribution for peg formation in cucumber seedlings: possible roles for CS-AUX1 and CS-PIN1.

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.

ILR2, a novel gene regulating IAA conjugate sensitivity and metal transport in Arabidopsis thaliana

Plants can regulate levels of the auxin indole-3-acetic acid (IAA) by conjugation to amino acids or sugars, and subsequent hydrolysis of these conjugates to release active IAA. These less active auxin conjugates constitute the majority of IAA in plants. We isolated the Arabidopsis ilr2-1 mutant as a recessive IAA-leucine resistant mutant that retains wild-type sensitivity to free IAA. ilr2-1 is also defective in lateral root formation and primary root elongation. In addition, ilr2-1 is resistant to manganese- and cobalt-mediated inhibition of root elongation, and microsomal preparations from the ilr2-1 mutant exhibit enhanced ATP-dependent manganese transport. We used a map-based positional approach to clone the ILR2 gene, which encodes a novel protein with no predicted membrane-spanning domains that is polymorphic among Arabidopsis accessions. Our results demonstrate that ILR2 modulates a metal transporter, providing a novel link between auxin conjugate metabolism and metal homeostasis.

Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines.

An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg-1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg-1 FW compared to approximately 15 pg mg-1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.

The bushy pea mutant is IAA‐deficient

The bushy mutant of pea (Pisum sativum L.) is characterized by short, thin stems, very small leaves and profuse branching. The bushy phenotype is conferred by a dominant allele, termed bsh. Here we show that bushy plants contain lower levels of free indole‐3‐acetic acid (IAA) than wild‐type (WT) plants. On emergence from the growth medium, bushy seedlings do not immediately display the mutant phenotype, and the effect of bsh on IAA content is small. After 10–14 days, the bushy phenotype begins to develop, and while the IAA content rises in WT plants, it falls in the mutant. The resulting difference in IAA level between WT and mutant can be up to 12‐fold. Although there is a deficiency of free IAA in bushy plants, the total IAA content (including free and conjugated forms) was not reduced in comparison with the WT. Furthermore, in bushy plants, the level of the main IAA conjugate in pea, IAAsp, was not reduced to the same extent as that of IAA, and metabolism studies indicated that faster IAA deactivation might contribute to IAA deficiency in the mutant. Application of IAA to bushy plants did not result in a WT phenocopy. However, the short internodes and profuse branching of bushy plants is consistent with classical views on how IAA affects plant development.

Characterization of a Family of IAA-Amino Acid Conjugate Hydrolases from Arabidopsis

The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-l-amino acid conjugates to inhibit Arabidopsis seedling root growth. We have also determined the ability of purified glutathione S-transferase (GST) fusions of four Arabidopsis IAA-amino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates in vitro, and GST-ILR1, GST-IAR3, and GST-ILL2 have K(m) values that suggest physiological relevance. In vivo inhibition of root elongation correlates with in vitro hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.

Inputs to the Active Indole-3-Acetic Acid Pool: De Novo Synthesis, Conjugate Hydrolysis, and Indole-3-Butyric Acid b-Oxidation

The phytohormone auxin is important in virtually all aspects of plant growth and development, yet our understanding of auxin homeostasis is far from complete. Plants use several mechanisms to control levels of the active auxin, indole-3-acetic acid (IAA). Plants can synthesize IAA both from tryptophan (Trp-dependent pathways) and from a Trp precursor but bypassing Trp (Trp-independent pathways). Despite progress in identifying enzymes in Trp-dependent IAA biosynthesis, no single IAA biosynthetic pathway is yet defined to the level that all of the relevant genes, enzymes, and intermediates are identified. In addition to de novo synthesis, vascular plants can obtain IAA from the hydrolysis of IAA conjugates. IAA can be conjugated to amino acids, sugars, and peptides; endogenous conjugates that are active in bioassays and hydrolyzed in plants are likely to be important free IAA sources. Conjugation is also used to permanently inactivate excess IAA, and these conjugates may be distinct from the hydrolyzable conjugates. The peroxisomal (3-oxidation of endogenous indole-3-butyric acid (IBA) also can supply plants with IAA, which may account for part of the auxin activity of exogenous IBA. Compartmentalization of enzymes and precursors may contribute to the regulation of auxin metabolism. IAA obtained through de novo synthesis, conjugate hydrolysis, or IBA β-oxidation may have different functions in plant development, and possible roles for the IAA derived from the various pathways are discussed.