In vitro binding affinities of 4-chloro-, 2-methyl-, 4-methyl-, and 4-ethylindoleacetic acid to auxin-binding protein 1 (ABP1) correlate with their growth-stimulating activities

4-Chlorindole-3-acetic acid (4-CI-IAA), an endogenous auxin in certain plant species of Fabaceae, has a higher efficiency in stimulating cell elongation of grass coleoptiles compared with indole-3-acetic acid (IAA), particularly at low concentrations. However, some investigations reported a 1,000-fold discrepancy between growth stimulation and binding affinity of 4-CI-IAA to auxin-binding protein 1 (ABP1) from maize. Here we report binding data of 4-CI-IAA and three alkylated IAA derivatives using purified ABP1 in equilibrium dialysis. There is a clear correlation between the growth-promoting effects and the binding affinity to ABP1 of the different IAA analogues measured by competition of [³H]naphthalene-1-acetic acid binding. Our data are consistent with the hypothesis that ABP1 mediates auxin-induced cell elongation.Abbreviations ABP1 auxin-binding protein 1 - 4-CI-IAA 4-chloroindole-3-acetic acid - NAA naphthalene-1-acetic acid - ER endoplasmic reticulum - IAA indole-3-acetic acid - 2-Me-IAA 2-methylindole-3-acetic acid - 4-Me-IAA 4-methylindole-3-acetic acid - 4-Et-IAA 4-ethylindole-3-acetic acid - MES 4-morpholineethanesulfonic acid - PAA phenylacetic acid     

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.

Oncogenes carried by the transferred DNA (T-DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T-DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T-DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28-fold increase in conversion of tryptophan to indole-3-lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis-regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax-1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole-3-acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.     

Membrane-associated binding sites for indoleacetic acid in the rice coleoptile

As described previously, the sensitivity of rice (Oryza sativa L.) coleoptiles to auxin is modulated by oxygen. Under anoxia, coleoptile elongation is insensitive to exogenously applied indole-3-acetic acid (IAA), whereas its sensitivity increases in air in the presence of the exogenous stimulus. Here we report the presence of two independent classes of membrane-bound IAA-binding sites in air-grown coleoptiles. Their binding activity is strictly correlated with the system's sensitivity to IAA. We designate them as site A (high affinity) and site B (low affinity). Site A shows a relatively fast response to anoxia, and is highly specific for auxins. Regulation of site-A binding activity through ATP, whose availability decreases under anoxia, is postulated. A role as auxin carrier is suggested for site B.Abbreviations ABS(s) auxin-binding site(s) - IAA indole-3-acctic acid - NAA 2-naphthaleneacetic acid - ION3 valinomycin, nigericin, carbonylcyanide p-trifluoromethoxyphenyl hydrazone Dedicated to the memory of Professor G. Torti, who passed away on 2 May, 1988     

L'acide indolyl-3-lactique et son métabolisme chez Rhizobium

Summary Among the indole compounds formed when tryptophan 2-¹⁴C is metabolized by Rhizobium, indole-3-lactic acid (ILA) is specially studied. In the course of experiments carried out in the culture medium of growing Rhizobium and in suspensions of washed bacterial cells the amount of ILA formed is compared with that of indole-3-acetic acid (IAA) occurring simulataneously. The formation of ILA and that of IAA directly depend on a transamination reaction. A large quantity of ILA is present in suspensions of washed bacterial cells.When ILA alone, as precursor, is incubated with Rhizobium, several products are identified: IAA, indole-3-acetaldehyde and tryptophol. Tryptophan is also detected in the aqueous fraction and is labelled when ILA 2-¹⁴C is used. The pathway of this metabolism are discussed and a general scheme is suggested.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Identification of indole compounds secreted byFrankia HFPArI3 in defined culture medium

Indole compounds secreted byFrankia sp. HFPArI3 in defined culture medium were identified with gas chromatography-mass spectrometry (GC-MS). WhenFrankia was grown in the presence of¹³C(ring-labelled)-L-tryptophan,¹³C-labelled indole-3-acetic acid (IAA), indole-3-ethanol (IEtOH), indole-3-lactic acid (ILA), and indole-3-methanol (IMeOH) were identified.High performance liquid chromatography (HPLC) and GC-MS with selected ion monitoring were used to quantify levels of IAA and IEtOH inFrankia culture medium. IEtOH was present in greater abundance than IAA in every experiment. When no exogenous trp was supplied, no or only low levels of indole compounds were detected.Seedling roots ofAlnus rubra incubated in axenic conditions in the presence of indole-3-ethanol formed more lateral roots than untreated plants, indicating that IEtOH is utilized by the host plant, with physiological effects that modify patterns of root primordium initiation.     

Solubilized auxin-binding protein

Discontinuous sucrose gradient fractionations indicate that the high-affinity auxin binding protein which can be solubilized from the microsomes of coleoptiles and primary leaves of Zea mays L. seedlings is probably located in the endoplasmic reticulum (ER). Since aromatic hydroxylations are enzymatic activities typical of the ER of plant cells, we have examined the effects of several electron-transport inhibitors on the binding of 1-naphthylacetic acid (NAA). NaN₃ strongly inhibits this binding, but KCN and CO do not. Trans-cinnamic acid and trans-p-coumaric acid, which are the substrates of ER hydroxylase activities in plants (but which are themselves not auxins), also inhibit this binding. Supernatant fractions from corn shoots contain factors inhibitory to the binding of NAA to the intact membranes and solubilized Site I auxin-binding protein. Here we show that these factors are competitive inhibitors of the binding of [¹⁴C]NAA but do not change the apparent affinity of the protein for indoleacetic acid, 2,4-dichlorophenoxyacetic acid or naphthoxyacetic acid. Several tissues were assayed for factors inhibitory to auxin binding to the solubilized protein, but only supernants from corn shoots were markedly inhibitory at low concentrations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - IAA 3-indolylacetic acid - nKP n x 100 x g pellet - NAA 1-naphthylacetic acid C.I.W.-D.P.B. Publication No. 656     

Genetic evidence that the tryptophan 2-mono-oxygenase gene of Pseudomonas savastonoi is functionally equivalent to one of the T-DNA genes involved in plant tumour formation by Agrobacterium tumefaciens

Summary The combined activities of the Agrobacterium tumefaciens T-DNA genes 1 and 2 are sufficient to induce tumorous growth on several plants, by introducing a new auxin biosynthetic pathway in infected cells. We have isolated Nicotiana tabacum plants containing only gene 1 or gene 2. These plants, respectively called rG1 and rG2, grow and develop in a normal fashion, indicating that neither the gene 1 nor the gene 2 activity by itself interferes with the endogenous auxin metabolism in plants. Previous evidence indicated that the auxin biosynthetic pathway of Pseudomonas savastanoi and that proposed to be encoded by the T-DNA of Agrobacterium tumefaciens are similar. When rG2 plants were infected with non-oncogenic A. tumefaciens or Escherichia coli strains that harbour the P. savastanoi iaaM gene (responsible for indole-3-acetamide synthesis) root and callus formation at the infection site was readily observed. This shows that the product of iaaM, indole-3-acetamide, is an in vivo substrate for the gene 2 encoded enzyme and supports the proposal that the gene 1-encoded enzyme is involved in the synthesis of indole-3-acetamide in transformed plants. This result offers new insights in evolution of bacteria and plants involved in pathogenic and symbiotic interactions.Abbreviations IAM indole-3-acetamide - IAA indole-3-acetic acid     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the eukaryotic T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IAM indole-3-acetamide - NAA naphthalene-1-acetic acid - NPT-II neomycin phosphotransferase II - T-DNA transferred DNA     

A protein from maize labeled with azido-IAA has novel β-glucosidase activity

A biologically active and photolabile auxin analog, 5-azido-[7-³H]indole-3-acetic acid ([³H]N₃IAA), was used to search for auxin-binding proteins in cytosolic extracts from maize coleoptiles (Zea mays L.) and identified a protein with a molecular mass of 60 kDa (p60). Binding of [³H]N₃IAA is highly specific as demonstrated by competition analysis with functionally relevant auxin analogs. p60 is found in coleoptiles and roots of etiolated maize seedlings and was detected in cytosolic as well as in microsomal fractions. The protein binds to 1-naphthylacetic acid (1-NAA) sepharose and is eluted with auxins. A purification scheme resulting in homogenous p60 protein was devised and it was shown that p60 has β-d -glucoside glucohydrolase activity (E.C.3.2.1.21). The hydrolytic activity of p60 for the synthetic substrate p-nitro-phenyl-β-d -glucopyranoside is diminished by 1-NAA. p60 shows high substrate specificity since it hydrolyzes indoxyl-O-glucoside, but not β-(1,4)-cellobiose, IAA-inositol or IAA-amino acid conjugates. The present data suggest that p60 might be involved in the hydrolysis of auxin conjugates.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

A soluble auxin-binding protein from mung bean hypocotyls has indole-3-acetaldehyde reductase activity

To clarify the roles of auxin-binding proteins (ABPs) in the action of auxin, soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid (2,4-D)-linked Sepharose 4B. A 39-kDa polypeptide was retained on the affinity column and eluted with a solution containing IAA or 2,4-D, but not with a solution containing benzoic acid. The protein was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 77 kDa by gel filtration and 39 kDa by SDS-PAGE. We designated this protein ABP39. The partial amino acid sequences of ABP39, obtained after chemical cleavage by CNBr, revealed high homology with alcohol dehydrogenase (ADH; EC 1.2.1.1). While the ABP39 was not capable of oxidizing ethanol, it did catalyze the reduction of indole-3-acetaldehyde (IAAld) to indole-3-ethanol (IEt) with an apparent K_m of 22 μ M. The IAAld reductase (EC 1.2.3.1) is specific for NADPH as a cofactor. The ABP39 also catalyzed the reduction of other aldehydes, such as acetaldehyde, benzaldehyde, phenylacetaldehyde and propionealdehyde. Indole-3-aldehyde was a poor substrate. The enzyme activity was inhibited by both indole-3-acetic acid and 2,4-D in a competitive manner. Therefore, the enzyme is considered to be retained on the affinity column by recognition of auxin structure.     

Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

The rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β-glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole-3-acetyl)-β-D-glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole-3-acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild-type and transgenic 35S-rolB tobacco plants and metabolic studies were carried out with [¹³C₆]IAA [2′-¹⁴C]IAA, [¹⁴C]IAGluc, [5-³H]-2-o-(indole-3-acetyl)-myo-inositol and [¹⁴C]indole-3-acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.     

Two isoforms of soluble auxin receptor in rice (Oryza sativa L.) plants: Binding property for auxin and interaction with plasma membrane H+-ATPase

An auxin receptor protein, isolated from the soluble fractionsof rice shoots and roots, was characterised in terms of the affinity andspecificity for IAA and the modulating effect onH⁺-ATPase of plant plasma membrane. The receptor proteingives a biphasic binding isotherm for IAA, indicating the existence ofthe primary and secondary binding sites. The predominant isoform of thereceptor in roots shows much higher affinity to IAA compared with thatin shoots. Being monomeric protein with about the same molecular mass(57–58 kDa) and showing a similar chromatographic behaviour, bothisoforms mediate IAA-induced modulation of the plasma membraneH⁺-ATPase in the respective IAA concentration rangesseparated by ca. 3 orders of magnitudes(10^-10–10^-7 M vs.10^-7–10^-4 M). Analysis of kinetic data ofthe H⁺-ATPase activity revealed that the receptor perse functions as an effector of the enzyme, causing a decrease inK_m and an increase in V_max through protein-proteininteraction at a 1:1 ratio. Further, it appeared that, while IAAdoes not affect by itself the kinetic parameters of theH⁺-ATPase, the auxin exerts its effect via thereceptor, biphasically regulating the efficiency of the effectormolecule probably by inducing two-phase conformational changes thatinvolve IAA binding to two separate binding sites. It was also foundthat other active auxins examined, such as indole-3-propionic acid,1-naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid, do notwork together with the receptor to elicit the same response of theH⁺-ATPase as seen with IAA.     

Auxin levels and auxin binding protein availability inrolB transformedBeta vulgaris cells

The final biological effect of auxin depends both on free auxin levels and on auxin perception capacity.RolB transformedBeta vulgaris L. hairy roots provide a system for studying both factors. Highly purified plasma membrane fractions were prepared with aqueous two-phase partitioning. Individual hairy root clones were assessed for the binding activities of plasma membrane-bound auxin binding proteins and for their free intracellular indole-3-acetic acid levels. The presence of a high affinity auxin binding protein with a dissociation constant of 9.07 x 10^?7 M was detected in the plasma membrane fractions isolated from non-transformed seedling roots and the six clones ofrolB transformed hairy roots. However, the levels of specific IAA binding considerably varied among different hairy root clones and between transformed and non-transformed roots. The levels of the detectable polypeptide in immunoblotting with an antibody against maize 22-kD auxin binding protein subunit were in good agreement to the levels that were detected in auxin binding assays. Differences in the indole-3-acetic acid levels were found between transformed and non-transformed roots and also between different transformed hairy root clones. A negative correlation was observed between free intracellular IAA levels and its specific binding to the plasma membrane-bound auxin binding proteins. A latency study indicated that the binding site for auxin may be located on the exterior face of the plasma membrane     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase.

We have used the photoaffinity label azido-[3H]IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analog of indole-3-acetic acid, to identify auxin-binding proteins (ABPs) in the soluble fraction of Hyoscyamus muticus. A 25-kD polypeptide previously described (H. Macdonald, A. M. Jones, P. J. King [1991] J Biol Chem 266: 7393-7399) has now been purified to homogeneity by conventional methods. Binding of azido-[3H]IAA to the purified protein was reduced by active auxins but not by inactive indoles. Partial amino acid sequences of the purified protein showed high homology to glutathione S-transferase (GST) from tobacco (ParB) and from maize (GT32). The conclusion that the 25-kD ABP is a GST is further supported by high GST activity in fractions highly enriched in the 25-kD polypeptide and recognition of the ABP by antibodies against GST from wheat and maize. Furthermore, purification of a protein from a soluble protein extract from H. muticus by affinity chromatography on glutathione-agarose also yielded a 25-kD polypeptide that was indistinguishable in its N-terminal amino acid sequence and biochemical characteristics from the protein purified by conventional methods. Possible functions of GST in auxin action are discussed.