Points of regulation for auxin action.

There have been few examples of the application of our growing knowledge of hormone action to crop improvement. In this review we discuss what is known about the critical points regulating auxin action. We examine auxin metabolism, transport, perception and signalling and identify genes and proteins that might be keys to regulation, particularly the rate-limiting steps in various pathways. Certain mutants show that substrate flow in biosynthesis can be limiting. To date there is little information available on the genes and proteins of catabolism. There have been several auxin transport proteins and some elegant transport physiology described recently, and the potential for using transport proteins to manage free indole-3-acetic acid (IAA) concentrations is discussed. Free IAA is very mobile, and so while it may be more practical to control auxin action through managing the receptor and signalling pathways, the candidate genes and proteins through which this can be done remain largely unknown. From the available evidence, it is clear that the reason for so few commercial applications arising from the control of auxin action is that knowledge is still limited.

Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin.

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.

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 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 secretions 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 ABP 1 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.

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 mays L.) 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. Immunolocalization 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.

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 carboxy-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.

Regulation of synthesis and turnover of maize auxin-binding protein and observations on its passage to the plasma membrane: comparisons to maize immunoglobulin-binding protein cognate.

Electrophysiological experiments have indicated that a fraction of the major auxin-binding protein (ABP1) of maize (Zea mays L.) might be a receptor on the outer surface of the plasma membrane. The predominant location of ABP1 is in the lumen of the endoplasmic reticulum (ER), in accord with its C-terminal KDEL retention signal. Little is known about the biology of the protein in vivo or the rate at which it might pass to the cell surface. We have examined the turnover of ABP1 by in vivo labelling of maize coleoptile sections. After different chase times, ABP1 was immunoprecipitated from detergent-solubilised membrane preparations. Two polypeptides coprecipitated with ABP1. Neither was recognised by any ABP1 antibodies nor by monoclonals to ER retention sequences. The possible significance of these coprecipitating polypeptides is discussed. In addition, we have used a monoclonal antibody to precipitate HDEL proteins from the same membrane preparations. Two dimensional electrophoresis and N-terminal sequencing showed that the major HDEL protein precipitated was a member of the heat-shock-protein 70 family, a homologue of BiP (immunoglobulin-binding protein). We have investigated the turnover of this BiP homologue for comparison with ABP1 and found that both had extended lifetimes, with half-lives greater than 24 h. Use of cordycepin to inhibit transcription indicated that ABP1 mRNA was also long-lived. Synthesis of ABP1 was strongly reduced by heat stress, was reduced a little in response to dithiothreitol and was not markedly changed by tunicamycin. In contrast, BiP synthesis increased markedly in response to tunicamycin and dithiothreitol and increased a little after heat stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Authentic processing and targeting of active maize auxin-binding protein in the baculovirus expression system.

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.

Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity.

The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.

Epitope mapping reveals conserved regions of an auxin-binding protein.

There is now good evidence that maize (Zea mays) auxin-binding protein (ABP) functions as a receptor. We have synthesized sequential overlapping hexapeptides to map the epitopes recognized by a number of antisera to ABP. Only a few regions of the protein are recognized, and these are shown to be exposed on the surface. Three epitopes predominate, and these are clustered around, but do not include, the glycosylation site. A comparison is made between these maps of sera against purified ABP, maps of sera raised against recombinant maize ABP expressed in Escherichia coli and computer antigenicity predictions. Our anti-(maize ABP) serum recognizes ABP counterparts in other plant species. We have used immunoblotting to affinity-purify the immunoglobulins which cross-react from the antiserum. Epitope mapping of these immunoglobulins suggests that two of the three predominant epitopes may be conserved in both monocotyledonous and dicotyledonous plants. The possible functional significance of these conserved epitopes is discussed.

Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum.

The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxy-peptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.

Rapid reaquisition in conditioning of the rabbit's nictitating membrane response.

Reacquisition after extinction often appears faster than original acquisition. However, data from conditioned suppression studies indicate that this effect may arise from spontaneous recovery and reinstatement of unextinguished contextual stimuli related to the unconditioned stimulus (US). In the present experiments using the rabbit nictitating membrane preparation, spontaneous recovery was eradicated before reaquisition training. US contextual stimuli were controlled by retaining the US during extinction through explicit unpairings of the conditioned stimulus (CS) and US. Attempts were also made to drive the associative strength of the CS into the inhibitory region by differential conditioning and conditioned inhibition procedures. In all cases, reacquisition was very rapid in comparison with a rest control. The results are discussed with respect to their implications for CS and US processing models of conditioning.

In the blink of an eye: real-time stimulus factors in delay and trace conditioning of the rabbit's nictitating membrane response.

Since Pavlov, theories of conditioning have assumed that CR evocation is governed by a series of internal stimuli generated by the CS. This hypothesis was tested in conditioning of the rabbit's nictitating membrane (NM) response by attempting to manipulate the internal sequence through truncating a delay CS and extending a trace CS on test trials. These perturbations of CS duration produced large deficits in CR likelihood and smaller alterations in the CR's time course. As predicted by many models of conditioning, the onset of the CS appeared to play a large but not exclusive role compared to CS duration and CS offset in both evocation and timing of the CR. The results are discussed with respect to their implications for real-time models of conditioning.

From auxin-binding protein to plant hormone receptor?

Plant scientists have long been expecting the description of hormone receptor proteins from plants. A putative auxin receptor has now been purified and sequenced and we are beginning to discover how the protein functions.

Temporal specificity in cross-modal transfer of the rabbit nictitating membrane response.

Two experiments examine cross-modal transfer of response features specific to the interstimulus interval (ISI) between a conditioned stimulus (CS) and an unconditioned stimulus. Rabbits were given initial training with a stimulus (CSA) in one modality (e.g., tone) at a designated ISI (e.g., 600 ms). Training was then shifted to a new stimulus (CSB) in another modality (e.g., light) at a new ISI (e.g., 400 ms). The timing of early conditioned responses (CRs) to CSB reflected the ISI of CSA. Ultimately, CRs to CSB shifted to a temporal location conforming to the ISI of CSB. When the ISI of CSB was shorter than that of CSA, CRs to CSA also shifted to a locus conforming to the ISI of CSB. The present results confirmed previous findings that training in one CS modality accelerates CR acquisition to a CS in another modality. The findings are compared with the transfer of response patterns in instrumental learning sets and are discussed regarding their implications for theories of cross-modal transfer.

Auxin-binding protein--antibodies and genes.

Of several auxin-binding systems that have been characterised the auxin-binding protein (ABP) of maize coleoptile membranes is the best candidate for a true auxin receptor. ABP, which exists as a homodimer of 22 x 10(3) M(r) glycosylated subunits, has been purified, and monoclonal and polyclonal antibodies raised against it. Electrophysiological studies with antibodies indicated the presence of a functional population of auxin receptors on the exterior face of the plasmalemma; electrophysiological experiments with impermeant auxin analogues now reinforce this conclusion. An epitope mapping kit has been used to identify the major epitopes recognised by antibody preparations. Three major epitopes, bracketing the glycosylation site, have been identified in the polyclonal serum. They are also represented in antisera produced in other laboratories and are conserved in ABP prepared from other plants. One monoclonal antibody recognises an epitope close to the amino terminus of ABP and two others recognise the carboxy terminus. The latter antibodies have been used in a sandwich ELISA to demonstrate that auxin binding induces a conformational change in ABP. Maize ABP is encoded by a small gene family and cDNA and genomic clones have been isolated. With a single exception, predicted amino acid sequences indicate remarkably little heterogeneity. The exceptional cDNA sequence predicts 87% amino acid homology with the major class of proteins. Four introns are apparent in the sequence of a complete ABP gene; their sequences are very highly conserved in an incompletely-cloned second gene lacking the first exon. The major difference between the two genes lies in the length of the first intron, which has been estimated to exceed 5.2 kb in the incomplete gene. The site of initiation of transcription has not been unambiguously identified in the complete gene, and some evidence suggests that there may be an additional intron. Homology to maize ABP cDNA has been detected in the genomes of Arabidopsis, spinach and strawberry but not in that of tobacco. A sequence located within the 3'-half of the maize cDNA is highly repeated in the strawberry genome, from which clones with homology to both halves of the maize cDNA (i.e. putative ABP genes) have been isolated.

Characterisation of auxin receptors.

Many auxin-binding systems, both membrane-bound and soluble, have been studied, but most lack credibility as receptors. The exception and best candidate as a valid auxin receptor is the auxin-binding protein of maize coleoptile membranes. This protein can be readily solubilised from the membranes and has been purified to homogeneity. It is a glycosylated homodimer of 22 kDa subunits. Preparations of minimum 50% purity were used to immunise rats and five monoclonal antibodies have been derived. Two of these can be mapped to epitopes within a C-terminal 1 kDa region, while the epitope of a third is within 7 kDa of the N-terminus. Immunotitration of receptor abundance in different tissues of maize seedlings shows that roots contain 40-fold less receptor protein per gram of fresh weight than coleoptiles. Pure receptor was produced by native PAGE and used to generate a high titer polyclonal antiserum in rabbits, capable of detecting receptor protein from as little as 1 mg of coleoptile tissue. The polyclonal specifically precipitates the 22 kDa polypeptide from maize membrane extracts, concomitant with removal of auxinbinding activity. The antiserum also detects homologous polypeptides in maize supernatant and in several other species, both monocots and dicots. In some cases, differences in chromatographic behaviour or size have been found. An auxin-induced conformational change in the receptor has been detected with a sandwich ELISA. The receptor gene has been cloned in four laboratories and the sequence data with knowledge of antibody epitopes can be used to identify parts of the protein involved in conformational changes (and perhaps auxin action). We are currently raising antibodies to a polypeptide thought to be part of the auxin binding site. Most of the auxin-binding protein is found in the endoplasmic reticulum but electrophysiological evidence, using polyclonal antiserum and impermeant auxin conjugates on protoplasts, suggests that a small population of functional receptor is accessible at the exterior face of the plasma membrane. Evidence bearing on the localisation and structure of the receptors is discussed.

Monoclonal antibodies detect an auxin-induced conformational change in the maize auxin-binding protein.

The monoclonal antibody MAC 256 precipitates specifically the auxin-binding protein (ABP) of maize membranes. Auxin-binding activity was recovered from the immunoprecipitate and MAC 256 can, therefore, bind undenatured, native ABP. A sandwich enzyme-linked immunosorbent assay was used to present native ABP to MAC 256 and under these conditions auxins inhibit antibody binding. Millimolar naphthalene-1-acetic acid completely blocks MAC 256 binding and the characteristics of monoclonal antibody MAC 259 are similar. The ability of a range of auxins and related compounds to displace MAC 256 correlates with the known structure-activity relationships of these compounds in vivo and in binding assays. The results are interpreted in terms of an auxin-induced conformational change in ABP, auxin binding leading to a change in, or concealment of, the epitope of the antibody. The epitope for MAC 256 and 259 lies close to the carboxy terminus of the protein, implying that the part of ABP containing the sequence of amino acids responsible for retention within the endoplasmic reticulum is conformationally active.

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein.

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps - anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance.

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.