Auxin Receptors and Auxin Binding Proteins

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 within the lumen of the endoplasmic reticulum, although an important fraction appears to function on the outside of the plasma membrane. The protein has a wide species distribution and it now seems highly probable that it is a genuine auxin receptor, the only protein for which such a function has yet been established. This conclusion is based on three independent lines of electrophysiological evidence, together with confocal imaging of cytoplasmic pH changes.     

Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling [22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman [7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.     

Do we have the auxin receptor yet?

Several auxin-binding proteins (ABP) have now been identified using a variety of techniques. A 43-kDa glycoprotein thought to be a dimer of 22-kDa subunits has been identified as a strong candidate for the auxin receptor that mediates cell elongation in etiolated maize shoots. The primary sequence has been deduced and several interesting structural features have been discerned. There is indirect evidence that this 22-kDa ABP has a receptor function, the most compelling being that antibodies directed against the ABP can block an auxin-induced response. There is evidence that changes in auxin-induced growth capacity in shoots correlates with changes in the abundance of the 22-kDa ABP suggesting that in some cases the 22-kDa ABP may be limiting growth. Confirmation of receptor function for one of these newly-identified ABP's should open the way for genetic manipulation of crop growth.     

Overexpression of the Auxin Binding PROTEIN1 Modulates PIN-Dependent Auxin Transport in Tobacco Cells

Background

Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive.

Methodology/Principal Findings

Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations.

Conclusions/Significance

The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.     

Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene. Received: 24 February 1999 / Accepted: 25 March 1999     

Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor

The effects of water deficits (WD), heat shock (HS), and both(HSWD) on photosynthetic carbon- and light-use efficienciestogether with leaf ABA content, pigment composition and expressionsof stress- and light harvesting-responsive genes were investigatedin ABP9 [ABA-responsive-element (ABRE) binding protein 9] transgenicArabidopsis (5P2). WD, HS, and HSWD significantly decreasedphotosynthetic rate (A) and stomatal conductance (g_s) in wild-typeplants (WT). A and g_s of 5P2 transgenic plants were slightlyreduced by a single stress and were hardly modified by HSWD.Although A and electron transport rate (ETR) in 5P2 plants weredepressed under optimal growth conditions (control) in relationto WT, they were enhanced under HS and HSWD. These results indicatethat ABP9 transgenic plants are less susceptible to stress thanthe WT. In addition, the increased ABA contents in both WT and5P2 plants in response to WD and/or HS stresses suggest thatdeclines in A and g_s might have been due to ABA-induced stomatalclosure. Moreover, compared with WT, 5P2 plants exhibited higherABA content, instantaneous water use efficiency (IWUE), Chla/b, NPQ, and lower Chl/carotenoid ratios. Finally, alteredexpression of stress-regulated or light harvesting-responsivegenes was observed. Collectively, our results indicate thatconstitutive expression of ABP9 improves the photosyntheticcapacity of plants under stress by adjusting photosyntheticpigment composition, dissipating excess light energy, and elevatingcarbon-use efficiency as well as increasing ABA content, IWUE,and expression of stress-defensive genes, suggesting an importantrole of ABP9 in the regulation of plant photosynthesis understress. Key words: ABP9, ABA, heat shock, photosynthesis, stress tolerance, water deficits Received 1 September 2007; Revised 19 December 2007 Accepted 21 December 2007     

The Plant Oncogene rolB Alters Binding of Auxin to Plant Cell Membranes

We measured auxin-binding capacity of the membrane preparationsfrom tobacco cells transformed by rolB as compared to untransformedcontrols. In the transformed cells, the overall auxin-bindingactivity is severalfold enhanced through an increase in a bindingactivity removable from the membranes at 0.5 M salt, while thebinding activity still attached to the membranes after saltwashes remains unchanged. Antibodies against the 22 kDa maizeauxin binding protein (ABP) depress most of the membrane-attachedbinding activity in both normal and rolB-transformed cells,while they do not affect the salt-washable binding activity.In contrast, antibodies against the RolB protein prevent completelybinding of auxin to the latter activity in both normal and transformedcells, while substantially unaffecting the membrane-associatedbinding. These results point to the presence, in untransformedmembranes, of an auxin-binding activity associated with a proteinimmunologically related to RolB. This activity is much increasedin rolB cells. In contrast, the auxin-binding protein analogousto maize ABP present in tobacco membranes does not increasein the rolB-transformed cells. (Received October 1, 1993; Accepted April 22, 1993)     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

Auxin receptors: recent developments

Rapid advances have been made in the study of auxin binding proteins (ABPs) in the last five years. In particular, an ABP in maize membranes has been cloned, sequenced and both monoclonal and polyclonal antibodies to this ABP have been developed. Structural and functional analysis has begun and there is good electrophysiological evidence that ABP in the plasma membrane functions as a receptor, probably involved in auxin-induced cell expansion. The role of the large amount of ABP in the endoplasmic reticulum is less clear, as is the relationship to soluble ABPs. At present there is only some circumstantial evidence relating any ABP to cell division. Receptors for synthetic inhibitors of auxin transport (phytotropins) are also of interest in relation to auxin action, but are less well characterised. Identification of new naturally-occurring phytotropins could lead to novel plant growth regulators.     

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.Abbreviations ABP1 auxin-binding protein 1 - NAA naphthalene-1-acetic acid - SDS sodium dodecyl sulfate - PAGE poly-acrylamide gel electrophoresis.     

The Olfactory System as a Model for the Analysis of the Contribution of Gene Expression to Programmed Cell Death

The process of programmed cell death is frequently attenuatedby inhibitors of protein and RNA synthesis. This implies thatgene expression is necessary for the active elimination of somecell types. Genes such as bcl-2 and bax have been implicatedin the direct control of cell death, while cellular immediate-earlygenes (clEGs), such as c-fos and c-jun have been repeatedlyassociated with neuronal degeneration. We are using the olfactoryneuroepithelium as a model system to investigate the role thatexpression of such genes might play in cell death. The advantagesof this system is that even in the adult, there is spontaneousdegeneration of olfactory receptor neurons followed by theirreplacement by the division and differentiation of precursors.Futhermore, the receptor neurons can be induced to die synchronouslyby removal of the olfactory bulb or intranasal administrationof toxic agents. We have generated fos-lacZ and jun-lacZ transgenicmice that can be used to assess expression of c-fos and c-junfollowing these various manipulations. In addition, a line oftransgenic mice has been derived that express Bcl-2 under thecontrol of the olfactory receptor protein promoter. These micehave high levels of Bcl-2 selectively in receptor neurons ofthe primary neuro-epithelium and vomeronasal organ. Since insome circumstances, Bcl-2 can protect against programmed celldeath these mice are being assessed for neuronal turnover underbasal conditions and following olfactory bulbectomy.     

The auxin-binding protein 1 is essential for the control of cell cycle

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.     

Auxin binding protein: curiouser and curiouser

Auxin is implicated in a variety of plant developmental processes, yet the molecular mechanism of auxin response remains largely unknown. Auxin binding protein 1 (ABP1) mediates cell expansion and might be involved in cell cycle control. Structural modeling shows that it is a β-barrel dimer, with the C terminus free to interact with other proteins. We do not know where ABP1 performs its receptor function. Most ABP1 is detected within the endoplasmic reticulum but the evidence indicates that it functions at the plasma membrane. ABP1 is established as a crucial component of auxin signaling, but its precise mechanism remains unclear.     

Retention of maize auxin-binding protein in the endoplasmic reticulum: quantifying escape and the role of auxin

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested one proposed model to account for release of ABP1 from the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretion of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions. Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants. Received: 31 October 1996 / Accepted: 16 December 1996     

Androgen binding protein (ABP) in rabbit testis and epididymis

ABP has been measured in 105,000 g supernatants of testis and epididymls from rabbits of different ages and compared with a similar androgen binding protein (TeBG) in rabbit serum. Whereas the concentration of ABP in the caput epididymidis increased markedly from immaturity to adulthood, serum TeBG decreased, indicating that ABP and TeBG are regulated by different hormonal mechanisms.The concentration of ABP (pmoles/mg protein) in sexually mature rabbits was much higher in the epididymis than in the testis. Within the epididymis most of the ABP was concentrated within the caput, and very low amounts were found in the cauda, indicating that binding activity of ABP is destroyed as it passes through the epididymis.In addition to ABP (R_f ~0.7), rabbit epididymal supernatant contains a larger binding protein for 5α-dihydrotestosterone (DHT; 17β-hydroxy-5α-androstan-3-one) with slower electrophoretic mobility (R_f ~0.4) and a more rapid sedimentation rate on sucrose gradients (7S). This component is most probably the intracellular androgen receptor in the rabbit e pididymis.     

Interaction of A-nor A.19-dinor,and A-homo-5α-androstane derivatives with the androgen receptor and the epididymal androgen-binding protein

The equilibrium affinity constant for rat prostate androgen receptor and epididymal androgen binding protein (ABP) has been determined for thirty-four potential progestogens. Three A-nor-, four A,19-dinor-, and one A-homo-5α-androstane derivative bind to the androgen receptor (K_D<0.5 μM). Five of these compounds also bind to ABP with an affinity of the same order of magnitude. “Anordrin” (compound $\text{24}$) and “Dinordrins” (compounds $\text{10, 14, 15, 16, 17}$), which are potential female contraceptives, do not bind with high affinity to the androgen receptor or to ABP. The following modifications in A-nor derivatives favour binding to the receptor as compared to ABP: 19-nor substitution (compound $\text{1}$), C-18 methyl homologation (compound $\text{5}$), 2α-ethinylation (compound $\text{22}$). One 2α-allenyl A-nor derivative (compound $\text{25}$) and one A-homo derivative (compound $\text{34}$) bind almost exclusively to ABP. The interaction with either binding protein is decreased by oxidation or esterification of the hydroxyl group at C-17, and by addition of a 17 α-ethinyl group. The latter modifications are likely to increase the specificity of androstane derivatives for receptors other than androgen binding proteins, such as the progesterone receptor.     

ABP1: An auxin receptor for fast responses at the plasma membrane

Auxin-binding protein 1 (ABP1) is an auxin receptor for responses not primarily regulated by gene regulation. One fast response is protoplast swelling. By using immunological ABP1 tools we showed that the highly conserved box a is not alone important for auxin binding. Box c is another part of the auxin binding domain.¹ Here we present a novel method to analyze auxin-induced, ABP1-mediated effects at the plasma membrane on single cell level in vivo. The fluorescence of FM4-64 in the plasma membrane is reduced by auxin and this response is mediated by ABP1. This method indicates a functional role of ABP1 at the plasma membrane.Key words: Auxin-binding protein 1, auxin, receptor, protoplast, plasma membrane, FM4-64