Auxin-binding Protein Research Articles

Comparison of abp1 primary sequences from monocotyledonous and dicotyledonous species

Summary The cDNA fragments of auxin-binding protein (ABP1) were isolated by degenerated-primer mediated reverse transcription-polymerase chain reaction (RT-PCR) from dicotyledonous plant species, i.e. cress, mung bean, pea, radish and soybean, and monocotyledonous species, i.e. oat and rice. Cloned abp 1 cDNA fragments were sequenced and compared with known abp 1 clones from A. thaliana , maize, strawberry and tobacco at the amino acid level. In all plant species studied, the amino acid sequences of newly isolated abp 1 clones were highly conserved at two regions (region A: Thr-Pro-Ile-His-Arg-His-Ser-Cys-Glu-Glu-Ile/Val-Phe-Ile/Thr/Val-Val-Leu/Pro/Val-Lys-Gly-Xaa-Gly-Thr-LeuAy; region B: His-Glu-Asp-Leu-Gln-Phe/Val-Leu-Asp/Val-Ile/Val-Ile-Ser-Arg-Pro-Pro), which were previously reported to be important for auxin-binding. On the other hand, some putative residues of amino acids in the region between regions A and B could be found to be specific in dicot and monocot species, respectively. Southern-blot analysis indicated a small abp 1 gene family in all species studied. Northern-blot analysis indicated that the size of abp 1 mRNA transcripts in all species studied was conserved at approximately 850bp. The phyloge-netic tree of ABP1 was analyzed by the UPGMA method.

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein.

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.

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

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 Km of 22 μM. 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.

Non-symmetrical cytosine methylation in tobacco pollen DNA.

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants.

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

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

PROTEIN RETENTION IN THE ENDOPLASMIC RETICULUM OF INSECT CELLS IS NOT COMPROMISED BY BACULOVIRUS INFECTION

High level expression of the major auxin-binding protein (ABP1) from maize ( Zea maysL.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immuno-localization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

Identification of a soluble auxin-binding protein as a glutathione-dependent formaldehyde dehydrogenase

Antibodies against the putative auxin-binding site of an ER auxin-binding protein from maize coleoptiles were raised by injecting rabbits with a synthetic oligopeptide (Ile-His-Arg-His-Ser-Cys-Glu) conjugated to hemocyanin from the Keyhole limpet. Soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid-linked (2,4-D-linked) Sepharose 4B. The antiserum against the oligopeptide recognized a specific 40 kDa polypeptide among auxin-binding proteins from mung bean hypocotyls. An auxin-binding protein of 40 kDa was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 80 kDa by gel-filtration and 40 kDa by SDS-PAGE. We designated this protein ABP40. The dissociation constant of purified ABP40 for [ 14C]-2,4-D was calculated to be 1 × 10 −5 M. Binding of [ 14C]-2,4-D was completely inhibited by p-chlorophenoxyisobutyric acid (PCIB) and weakly inhibited by indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA). Benzoic acid and tryptophan had no effect on the binding. The partial amino acid sequences of ABP40, obtained after chemical cleavage by CNBr, revealed high homology to glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1. 2. 1. 1). Moreover, the purified ABP40 had FDH activity.

Synthetic peptides as probes of plant cell signalling

Synthetic peptides have found increasing use in dissecting cell signalling pathways and have been employed as synthetic antigens, protein kinase and protease substrates. Recently, it has become evident that relatively short (10–30mer) peptides are able to mimic that part of the signalling protein to which their sequence corresponds. In particular, peptides corresponding to the C-terminus of Zea mays auxin binding protein, ZmABP1, were able to modulate ion channel function within Vicia guard cells. In this report, GTPγS binding to NaCl-washed Zea microsomal membranes is shown to be stimulated by peptide A6.2, corresponding to the C-terminal 16 residues of ZmABP1, only when the membranes are reconstituted with soluble Zea protein fractions containing GPα1 and Gα0 homologues.

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.

Isolation from cotton shoots of an auxin-binding protein regulating the synthesis of RNA

With the aid of a complex method of purification, including affinity chromatography and HPLC, from a fraction of the water-soluble protons of cotton seeds we have isolated a protein which binds the labeled auxin [3HJIAA with high affinity (Kd = 6 nM) and in the presence of IAA stimulates the synthesis of RNAin vitro in a preparation of cottonplant chromatin. In PAAG electrophoresis with sodium dodecyl sulfate, this protein gave a single band corresponding to a molecular mass of 43 kDa. It is assumed that this protein is a receptor for the auxin phytohormone that is responsible for regulating gene activity.

Auxin-binding Protein 1 Does Not Bind Auxin within the Endoplasmic Reticulum Despite This Being the Predominant Subcellular Location for This Hormone Receptor

Auxin-binding protein 1 (ABP1) is a unique hormone receptor because it resides primarily in the lumen of the endoplasmic reticulum (ER); however, two lines of evidence presented here suggest that ABP1 does not bind auxin within the endoplasmic reticulum, despite its predominant location there. First, ABP1 cannot be photolabeled in intact cells that have accumulated the auxin and photolabeling reagent 5-[7-3H]azidoindole-3-acetic acid, indicating either that auxin is excluded from the ER and is not available for photolabeling to ABP1 or that binding conditions within the ER lumen are insufficient for photolabeling. Second, at the pH of the ER lumen, auxin binding to ABP1 is not detectable. The pH estimate of the ER lumen is based on an indirect assay, which indicates that the pH is closer to pH 7 than to the binding optimum of pH 5.5. These results indicate that ABP1 does not bind auxin within the ER and point to a site of action that is post-ER. The effect of auxin on its trafficking from the ER was tested in an animal expression system. ABP1 expressed at high levels in COS7 cells is efficiently retained in the ER lumen and is not secreted even in the presence of 190 microM indole-3-acetic acid, an auxin concentration that is 40 times above the Kd for indole-3-acetic acid binding to ABP1.

Indole-3-lactic acid is a weak auxin analogue but not an anti-auxin

Indole-3-lactic acid (ILA) is a naturally occurring indole derivative, preferably detected in soil bacteria and fungi and only in low amounts in plants. T-DNA gene 5 of Agrobacterium tumefaciens was found to be involved in the synthesis of ILA in transformed plant tissues, but the physiologic relevance for ILA production in plants is unclear. The related molecular structure of ILA to the natural auxin indole-3-acetic acid (IAA) makes ILA a good candidate for an auxin analogue. We examined the possible auxin activity of ILA on elongation, proliferation, and differentiation in Pisum sativum L. Results presented in this paper indicate that there are no or only weak effects of ILA toward the activity of auxins when used in the physiologic concentration range. Furthermore, no antagonistic effects of ILA were found. Biochemical analysis using the equilibrium dialysis binding system resulted in no high affinity ILA binding to an enriched protein fraction containing auxin-binding protein (ABP44), whereas 1-naphthaleneacetic acid exhibited high affinity auxin binding.

Stable expression of maize auxin-binding protein in insect cell lines

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and car☐y-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

Auxinic Herbicide-Resistant and -Susceptible Wild Mustard ( Sinapis arvensis L.) Biotypes: Effect of Auxinic Herbicides on Seedling Growth and Auxin-Binding Activity

The effect of the auxinic herbicides dicamba, mecoprop, MCPA, and picloram on seedling development of an auxinic herbicide-resistant and -susceptible biotype of wild mustard ( Sinapis arvensis L.) was studied. When treated with dicamba, MCPA, and picloram differences in the seedling dose-response characteristics were observed between the two biotypes. Conversely, following mecoprop treatment there were no differences between the response of the herbicide susceptible and resistant biotypes. Some characteristics of the resistant and susceptible wild mustard biotype auxin-binding protein (ABP) preparations were examined. Although, both ABP preparations appear to have similar substrate ([ 3H]indole-3-acetic acid ([ 3H]IAA) binding) and time course profiles, Scatchard analysis revealed differences between the biotypes; the resistant biotype lacks a high-affinity population of ABP which is present in the susceptible biotype. The effects of the auxinic herbicides on specific [ 3H]IAA binding to ABP preparations from the resistant and susceptible biotypes were also examined. Mecoprop and MCPA were the most potent inhibitors of [ 3H]IAA binding to the susceptible biotype ABP preparations, followed by dicamba and picloram. The same pattern of inhibition was also observed when the effect of these herbicides on seedling growth was examined in the susceptible biotype. When picloram and dicamba were used to inhibit [ 3H]IAA binding and seedling growth, the susceptible biotype was significantly more sensitive to inhibition than the resistant biotype ABP preparations; however, no differences between the two biotypes were observed following MCPA or mecoprop treatment. Our results suggest the effects of various auxinic herbicides on the binding of [ 3H]IAA to ABP preparations and seedling growth correlate with the effect of these herbicides on whole plants. This data suggests there is a relationship between ABP binding activity and sensitivity to auxinic herbicides and this relationship may explain the mechanism of auxinic herbicide resistance in this wild mustard biotype.

Purification and Characterization of an Auxin-Binding Protein from Arabidopsis thaliana Expressed in Baculovirus-Infected Insect Cells

The auxin-binding protein At-ERabpl is of very low abundance in Arabidopsis thaliana; it hinders any study at the protein level as it is difficult to collect large amounts from the plant. We therefore chose to express At-ERabpl in baculovirus-infected insect cells. Recombinant baculoviruses were selected in yeast according to Patel et al. ( Nucleic Acids Res. 20, 97-104, 1991). The recombinant protein was purified to homogeneity by a simple procedure involving an affinity step on a succinyl-concanavalin A column, Labeling with the photoactive auxin 5- N 3-[7- 3H]indole-3-acetic acid demonstrated that the baculovirus-expressed protein belongs to the auxin-binding protein family as deduced from its cDNA homology to a gene previously characterized in maize. The mature polypeptide migrates on SDS-PAGE with an apparent molecular mass of about 23 kDa, its NH 2-leader sequence is properly processed, and it bears a high-mannose-type sugar moiety, All results are in agreement with information derived from the cDNA analysis. The possible role of a functional dimerization is also discussed.

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.

Organ distribution of auxin-binding protein 1 in the etiolated maize seedling

The auxin-binding protein designated ABP1 has been proposed to mediate auxin-induced cellular changes such as cell expansion. Its exact mode of action is unknown, but currently several approaches to elucidate its function are being pursued. One of these approaches, described here, is to determine the organ distribution of this putative auxin receptor in order to correlate spatially the abundance of the protein with some auxin-regulated activity such as cell elongation. The absolute and relative amounts of ABP1 were determined along the entire etiolated shoot, the root, and within the caryopsis of maize. ABP1 can be detected immunologically in all extracts of the etiolated maize seedling except the tip of the primary root and the endosperm. Within the shoot, but excluding the leaf roll, the highest levels compared on a fresh weight basis are in the apical mesocotyl and basal coleoptile regions, the areas of the most rapid cell elongation and the areas where there is the greatest capacity for auxin-induced growth. The relative abundance of ABP1 compared on a fresh weight basis changed more than fivefold in this organ. When compared on a total protein basis, the relative change in ABP1 abundance was approximately two-fold, which is less than the relative change in auxin-induced growth rate along the shoot. Differences in shoot growth rate among varieties of maize were compared with the relative amounts of ABP1 within the apical mesocotyl and basal coleoptile. A statistically significant but not perfect correlation was found between the auxin-induced growth rate of the apical mesocotyl and ABP1 abundance. These results demonstrate a general correlation between the amount of ABP1 and growth along the shoot and within maize hybrid varieties.

Auxin action and auxin-binding proteins.

The plant growth regulator auxin mediates an enormous range of developmental and growth responses, some of which are manifest rapidly and others manifest only after considerable lag periods. The protein that perceives auxin, the auxin receptor, has been sought by many laboratories and the search has identified a good number of candidates. However, a receptor must not only bind auxin, but also transduce the auxin stimulus into the responses we recognize. Finding evidence for this second condition has always proved very demanding. A key requisite is a convenient assay for auxin activity and preferably one involving a rapid response because this is likely to be linked directly to the perception event. For one auxin-binding protein (ABP1) there is growing evidence that it is a functional auxin receptor. The assays used in this work have been rapid auxin-induced changes in protoplast electrophysiology. There are many other responses induced rapidly by auxin for which a link to ABP1 has yet to be established. We have reviewed the whole range of rapid auxin-mediated responses and by doing so we hope to have provided a comprehensive picture of the many events to which a receptor (or receptors) must connect. Against this framework we match the known properties of all putative receptors, including ABP1. Not only have we tried to identify auxin-binding proteins unlikely to be receptors, but we also highlight the remaining gaps in our understanding of the more likely receptor candidates. Contents Summary 167 I. Introduction 168 II. Gene activation 168 III. Mutants 179 IV. Auxin-induced elongation growth 179 V. Other auxin-binding proteins 191 VI. Auxins and signal transduction 192 VII. Overview 194 Acknowledgements 195 References 195.

Auxin Receptors and Auxin Binding Proteins

Abstract In the last few years, a large number of auxin-binding proteins (ABPs) have been reported. Implicitly or explicitly, interest in such proteins resides in their possible role as auxin receptors. Many of these proteins are characterized as ABPs solely by their susceptibility to covalent photolabeling by tritiated azido-indole-3-acetic acid. In most cases where the labeled polypeptides have been identified, they turn out to have roles unconnected with primary auxin perception. It seems likely that auxin is binding to sites of catholic specificity in these cases and the influence of experimental protocols on the data is discussed. Because the term ABP implies that auxin binding affects the function of that protein, the importance of establishing further criteria before photolabeled peptides can be termed ABPs is emphasized. Applying such criteria, only a very few ABPs are currently of interest and only one of these, maize ABP1, has been characterized in detail. This protein is located primarily withi...