Planta (2009) 230:917–924

DOI 10.1007/s00425-009-0995-2

ORIGINAL ARTICLE

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c

Renate I. Dahlke · Hartwig Lüthen · Bianka SteVens

Received: 31 March 2009 / Accepted: 17 July 2009 / Published online: 11 August 2009© Springer-Verlag 2009

Abstract The auxin-binding protein 1 (ABP1) hasalready been proved to be an extracellular receptor of auxinin single cell systems. Protoplasts of maize coleoptilesrespond to auxin with an increase in volume. The 2-naph-thaleneacetic acid (2-NAA), an inactive auxin analog, actsas an anti-auxin in protoplast swelling, as it suppresses theeVect of indole-3-acetic acid (IAA). Antibodies raisedagainst box a of ABP1 induce protoplast swelling in theabsence of auxin. This response is inhibited by pre-incuba-tion with 2-NAA. The eVect of 2-NAA on swelling inducedby agonistic antibodies appears to depend on the bindingcharacteristics of the antibody. ScFv12, an antibodydirected against box a, box c and the C-terminal domain ofABP1 also exhibits auxin-agonist activity which is, how-ever, not abolished by 2-NAA. Neither does 2-NAA aVectthe activity of the C-terminal peptide of ABP1, which ispredicted to interact with putative binding proteins ofABP1. These results support the view that box a and box cof ABP1 are auxin-binding domains.

Keywords Auxin-binding protein 1 (ABP1) · Auxin receptor · Protoplast swelling · Zea mays coleoptile

AbbreviationsABP1 Auxin-binding protein 1AFB Auxin-signaling F boxER Endoplasmic reticulumIAA Indole-3-acetic acidNAA Naphthaleneacetic acidTIR1 Transport inhibitor response 1

Introduction

Since the Wrst description of auxin activity by Went duringthe 1920s (Went 1928; Went and Thimann 1937) numerousauxin-regulated processes including tropisms, lateral rootdevelopment and elongation growth have been identiWed.In contrast, our understanding of auxin signal transductionis still patchy.

The recently discovered auxin-binding F-box proteinsTIR1 (transport inhibitor response 1), AFB1, AFB2 andAFB3 (auxin-signaling F box) constitute intracellularreceptors (Dharmasiri et al. 2005a, b; Kepinski and Leyser2005). These F-box proteins are part of the ubiquitin ligasecomplex SCFTIR1 and play a role in degradation of repres-sors of auxin-regulated transcription. Our understanding ofauxin signaling is further complicated by a second type ofauxin receptor candidate, namely, auxin-binding protein 1(ABP1). Although auxin binding to membrane fractionscontaining ABP1 was Wrst demonstrated in the 1970s (Her-tel et al. 1972), the protein was Wrst puriWed and character-ized in the 1980s (Löbler and Klämbt 1985), and itsfunctional role is not yet completely understood. Electro-physiological data (Barbier-Brygoo et al. 1989, 1991;Venis et al. 1992; Rück et al. 1993), protoplast swellingassays (SteVens et al. 2001; Yamagami et al. 2004) and cellexpansion experiments (Chen et al. 2006) suggest the

Electronic supplementary material The online version of this article (doi:10.1007/s00425-009-0995-2) contains supplementary material, which is available to authorized users.

R. I. Dahlke · B. SteVens (&)Botanisches Institut und Botanischer Garten der Christian-Albrechts-Universität zu Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germanye-mail: bsteVens@bot.uni-kiel.de

H. LüthenBiozentrum Klein Flottbek und Botanischer Garten, Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany

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involvement of ABP1 in mediating rapid auxin eVects onion transport and osmoregulation on the plasma membrane.The ABP1 amino acid sequence (Löbler and Klämbt 1985;Hesse et al. 1989; Inohara et al. 1989; Palme et al. 1992)does not possess any predicted transmembrane domains.The C-terminus contains a KDEL-motif known to act as aretention signal for the ER lumen (Munro and Pelham1987; Napier et al. 1992). In fact, the bulk of ABP1 wasfound to be localized in the ER. In addition, minor amountsof ABP1 were detected on the outer surface of the plasmamembrane (Jones and Herman 1993; Diekmann et al. 1995;Bauly et al. 2000). It is this extracellular ABP1 that hasbeen attributed to ABP1-mediated auxin perception.

In order to test whether ABP1 is involved in regulationof cell volume, which may be related to auxin-induced cellelongation (Hertel 1995; Venis 1995), auxin-induced pro-toplasts swelling was employed to facilitate physiologicalstudies of ABP1 action at the single-cell level with a hightime resolution (SteVens and Lüthen 2000). With respect totime course, auxin speciWcity and dose–response resultsobtained with protoplasts show close similarities to thosedescribed for auxin-induced growth responses at the organlevel (SteVens and Lüthen 2000). Immunological studieswith antibodies raised against ABP1 indicated that theauxin signal resulting in protoplast swelling was perceivedby ABP1. Anti-ABP1 antibodies inhibited auxin-inducedswelling. Antibodies raised against box a, part of the auxin-binding domain of ABP1, induced auxin-like protoplastswelling. Synthetic oligopeptides corresponding to theC-terminus of ABP1 likewise induced an auxin-like eVect.Thus, the C-terminus of ABP1 appears to be the site ofinteraction with a putative interacting protein that isinvolved in signal transduction (Klämbt 1990; MacDonald1997). It was concluded that ABP1 may function as anextracellular auxin receptor mediating protoplast swelling(SteVens et al. 2001). These data were conWrmed by usingpea epidermal protoplasts (Yamagami et al. 2004). In addi-tion, Yamagami et al. (2004) found an additional ABP1-independent pathway leading to protoplast swelling.

1-Naphthaleneacetic acid (1-NAA) is an active auxinanalog known to induce growth of maize coleoptiles andprotoplast swelling. Analysis of the crystal structure ofmaize ABP1 with bound 1-NAA showed that the bindingpocket of ABP1 for auxin was predominantly hydrophobic(Woo et al. 2000, 2002; Bertona et al. 2008). ABP1 is a gly-coprotein which exists as a dimer. Each subunit of theABP1 dimer contains a Zn2+-ion coordinated by a gluta-mate and three histidine residues. 1-NAA binds within thispocket. The carboxyl group interacts with the Zn2+-ion,while the aromatic ring system attaches to hydrophobic res-idues including W151 at the C-terminal region of ABP1.

2-NAA has been shown to bind to ABP1 (Löbler andKlämbt 1985) but not to TIR1 (Kepinski and Leyser 2005).

It has a slightly higher aYnity to ABP1 than the activeauxin 1-NAA (Ray et al. 1977) even though 2-NAA activ-ity in auxin bioassays is low or undetectable (Katekar1979). Only at high concentrations, a stimulation of protonpumping in maize coleoptiles was reported (Peters andFelle 1991). In protoplasts, 2-NAA did not induce anyauxin-like swelling (SteVens and Lüthen 2000).

To better explain the discrepancy between the strongbinding of 2-NAA to ABP1 and its weak activity, wedesigned experiments to identify overlapping domainsresponsible for 2-NAA binding and activity of ABP1. Tothat end, we used an antibody raised against box a of ABP1,which is part of the auxin-binding domain. We alsoemployed the antibody fragment scFv12, which was raisedagainst the discontinuous epitopes box a and box c and theC-terminal domain of ABP1. We further used a peptide of15 amino acids identical to the C-terminus of ABP1, whichwas shown to induce protoplast swelling in previously pub-lished work (SteVens et al. 2001).

Materials and methods

Preparation of maize coleoptile protoplasts

Seeds of maize (Zea mays L., cultivar Garant, SaatenUnion, Hannover, Germany) were rinsed in water overnightand grown on well-watered Wlter paper at 26°C in the darkfor 5 days. Eight coleoptiles were harvested and abraded(SiC-powder, 1,200 mesh, Schriever, Hamburg, Germany).One-cm long segments excluding the primary leaf werecollected from each coleoptile starting 3 mm below the tipand cut into very small pieces. The coleoptile pieces weregently shaken for 3 h at 20 § 2°C and a quantum Xow of150 �M photons m¡2 s¡1 in a digestion solution containing1% cellulose ‘Onozuka R-10’ (Serva, Heidelberg), 0.075%Pectolyase Y-23 (Kikkoman Corporation, Tokyo, Japan)and micro- and macronutrients (SteVens and Lüthen 2000).After digestion, protoplasts were sieved successivelythrough 100, 50 and 20 �m meshes. Protoplast isolation andvolume change measurements were done in 1 mMCaCl2·2H2O, 10 mM KCl and 10 mM Mes at pH 6.5 and350 mosmol. Protoplasts were stored in this solution untiluse.

Measurement of protoplast volume changes

Protoplast swelling was measured as described (SteVensand Lüthen 2000; SteVens et al. 2001) with minor changes.BrieXy, single protoplasts were analyzed in microtiterplates with 20 �l protoplast solution per well at a constantosmolarity, 20 § 2°C and a quantum Xow of 150 �Mphotons m¡2 s¡1.

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Single vital protoplasts were observed with an inversemicroscope (objective x32) and pictures were taken at deW-ned time points with a digital camera (Nikon Coolpix 4500,Tokyo, Japan) and analyzed with Image-J (from WayneRasband, http://rsb.info.nih.gov/ij/, National Institute ofHealth, USA, version 1.25). By circumscribing the outlineof the protoplast with a mouse pointer on a monitor the pro-toplast area was recorded. From these data, the relative vol-ume as percentage of the volume at time point zero wascalculated for each protoplast. The relative volumes ofuntreated control protoplasts slowly decreased linearly withtime (r ¸ 0.95). We performed a linear regression up to theaddition of an eVector to correct for this endogenousshrinkage. This value was extrapolated and subtracted frommeasured volumes and termed as net volume as describedpreviously (SteVens and Lüthen 2000). Plots shown indi-cate changes in net volume as a function of time.

Growth substances, antibodies and peptides

An IAA stock solution was freshly prepared from IAApotassium salt (Merck, Darmstadt, Germany) in protoplastsolution. A 2-NAA (Merck) stock solution was made by dis-solving 2-NAA in one drop of KOH and subsequent addi-tion of protoplast solution to the desired volume. Themonoclonal antibody mAb12 and the single chain fragmentvariable antibody scFv12, which is a recombinant antibodyagainst tobacco ABP1 (provided by Dr. Catherine Perrot-Rechenmann, CNRS, Gif sur Yvette, France; David et al.2007) were diluted freshly in protoplast solution before eachexperiment. The C-terminal peptide from maize with thesequence FVWDEDCFEAAKDEL (PSL, Heidelberg, Ger-many) and the control peptide with the sequenceECDAKLFEADFEWVD were stored in protoplast solution.The secondary structure of control peptide was designedaccording to analysis with SSPro (http://www.ics.uci.edu/»baldig/scratch/). The original C-terminal peptide consistsof an 8-amino acid �-helix which is not found in the controlpeptide. Anti-box a antibody from Arabidopsis (provided byDr. Klaus Palme, University of Freiburg, Germany) wasstored in phosphate-buVered saline solution and diluted inprotoplast solution to the Wnal concentration indicated.

Results

IAA but not 2-NAA induces protoplast swelling

As previously described, protoplasts responded to the natu-ral auxin IAA with rapid swelling (Fig. 1a). All protoplastsderived from multiple tissue types of the maize coleoptileshow this eVect. IAA was found to induce protoplast swell-ing at physiological concentrations ranging from 1 to

10 �M IAA. With 1 �M IAA an increase in net volume ofabout 3.9% after 60 min can be observed and 5 �M IAAresulted in a swelling response of about 3% after 60 min(data not shown). Protoplasts increased their volume byabout 4.4% after 60 min and 4.8% after 90 min with 10 �MIAA; 10 �M IAA was shown to be the optimal concentra-tion.

Control protoplasts did not show an increase in net vol-ume (data not shown; see SteVens and Lüthen 2000). Appli-cation of 10 �M 2-NAA resulted in a comparatively weakswelling response after a much longer lag phase than thatobserved with IAA (Fig. 1a). After 60 min, protoplastsincreased their volume by 1.2% and after 90 min by 1.9%with 2-NAA; 1 �M 2-NAA had no eVect on protoplastswelling (data not shown).

2-NAA inhibits IAA-induced protoplast swelling

To test the eVect of 2-NAA and IAA on protoplast swell-ing, we performed competition experiments. When 10 �M2-NAA was applied 10 min before the optimal IAA con-centration of 10 �M was added, no protoplast swellingoccurred (Fig. 1b). When 2-NAA was added 10 min afterapplication of IAA, the IAA-induced swelling response wasreverted (Fig. 1b). In either case, application of bothligands resulted only in a minor increase in net volume ofabout 1% after 60 min.

Fig. 1 EVect of IAA and 2-NAA on protoplast swelling. a Att = 0 min, 10 �M IAA (open circle) or 10 �M 2-NAA (closed circle)was applied to protoplasts. Results are averages §SE from four (opencircle) or Wve (closed circle) protoplasts. b In competition experi-ments, 10 �M IAA was applied at t = 0 min and 10 �M 2-NAA wasadded after 10 min (open circle) or 10 �M 2-NAA was applied att = 0 min and 10 �M IAA was added after 10 min (closed circle). Re-sults are averages § SE from three (open circle) or four (closed circle)protoplasts

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2-NAA strongly inhibits protoplast swelling induced by anti-box a antibodies

Anti-box a antibodies were shown to induce protoplastswelling at concentrations between 10¡10 and 10¡7 M(SteVens et al. 2001). The auxin agonistic eVect of anti-boxa antibodies applied at the optimal concentration of 10¡9 Mis shown in Fig. 2 with an increase in net volume of protop-lasts of 5.1% after 60 min. When protoplasts were pre-treated with 10 �M 2-NAA, antibody-induced swelling wascompletely abolished. In fact, with 2-NAA pretreatmentprotoplasts displayed shrinkage in response to anti-box aantibodies by 7.9% within 60 min.

2-NAA promotes protoplast swelling induced by scFv12 antibody fragments

The antibody fragment scFv12 was derived from the well-characterized monoclonal antibody mAb12, which wasraised against the discontinuous epitopes box a, box c, andthe C-terminal domain of ABP1 (Leblanc et al. 1999;David et al. 2007). As a control, 10¡8 M mAb12 wasapplied to the protoplasts; mAb12 induced an auxin-likeprotoplast swelling response (S1). Application of 10¡8 MscFv12 induced an auxin-like protoplast swelling responsewith an increase in net volume of 3.2% after 60 min, and4.1% after 90 min (Fig. 3). Pre-incubation of protoplastswith 10 �M 2-NAA for 30 min prior to addition of scFv12increased the protoplast volume change to 5.0% after60 min and to 6.6% after 90 min. Pre-incubation withscFv12 prior to 2-NAA resulted in a comparable swellingresponse (data not shown). Protoplasts increased volume to4.1% after 60 min and to 4.5% after 90 min indicating that2-NAA promoted auxin-like swelling induced by the anti-body fragment scFv12.

2-NAA does not aVect swelling induced by the C-terminal peptide

The C-terminal peptide is a 15-amino-acid-long peptidethat corresponds to the C-terminus of ABP1. Application ofC-terminal peptide at 10¡6 M induced a swelling responsein protoplasts conWrming previous results (Fig. 4, SteVenset al. 2001). Protoplasts increased their volume by about2.9% after 60 min and by 3.7% after 90 min. Application of10¡6 M control peptide, consisting of the same amino acids

Fig. 2 Competition experiment with 2-NAA and anti-box a antibody.Anti-box a antibody at 10¡9 M induced protoplast swelling (open cir-cle; modiWed from SteVens et al. 2001). Pre-incubation of protoplastswith 10 �M 2-NAA at t = 0 min and addition of 10¡9 M anti-box aantibody after 4 min resulted in protoplast shrinkage (closed circle).Results are averages § SE from three protoplasts

Fig. 3 Competition experiment with 2-NAA and scFv12. Applicationof 10¡8 M scFv12 induced protoplast swelling (open circle). Pre-incu-bation of protoplasts with 10 �M 2-NAA at t = ¡30 min and additionof 10¡8 M scFv12 at t = 0 min resulted in increased protoplast swelling(closed circle). Results are averages § SE from Wve (open circle) orfour (closed circle) protoplasts

Fig. 4 Competition experiment with 2-NAA and C-terminal peptide.The C-terminal peptide at 10¡6 M induced a swelling response (opencircle). Pre-incubation with 10 �M 2-NAA for 10 min prior to applica-tion of 10¡6 M C-terminal peptide at t = 0 min did not aVect protoplastswelling (closed circle). Results are averages § SE from Wve (closedcircle) or three (open circle) protoplasts

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as the C-terminal peptide, but with another primary andsecondary structure, resulted in a volume change of 0.4%after 60 min (data not shown). When protoplasts were pre-incubated with 10 �M 2-NAA for 10 min prior to applica-tion of C-terminal peptide the net volume change was 3.4%after 60 min and 3.9% after 90 min, which was not statisti-cally diVerent from the response obtained with C-terminalpeptide alone. Thus, 2-NAA did not aVect C-terminal pep-tide-induced protoplast swelling.

Discussion

The auxin receptor ABP1 appears to control a set of auxineVects, some of which are, given their rapid time-course,not likely mediated by gene expression. One of theseresponses is protoplast swelling. Here we show that 2-NAAacts as an auxin antagonist on this response. The data indi-cate that 2-NAA prevents IAA from binding to ABP1 and/or from inducing a conformational change in ABP1 neces-sary for auxin activity.

Auxin enters the binding pocket of ABP1 through theN- and C-terminal extensions (Woo et al. 2002). Detailedanalysis of the crystal structure of ZmABP1 (Woo et al. 2002)indicated that highly conserved amino acids constitute thebinding pocket for the aromatic ring system and that a Zn2+-ion binds to the carboxylic group of auxin by coordinativebinding. Binding of the carboxyl group of 1-NAA wasshown to be strong, because of its symmetric bidentate con-tacts to the Zn2+-ion in the binding pocket (Woo et al.2002). The binding aYnity of 2-NAA is higher than that of1-NAA and this, in turn, exceeds the aYnity of IAA. Thiswould conWrm the prediction of Edgerton et al. (1994). IAAwith its indole amine group has no obvious hydrogen-binding partner as predicted from the crystal structure(Woo et al. 2002). For activation of ABP1 the orientationof the carboxyl group may be important, explaining whythe 2-isomer evokes diVerent activity than the 1-NAA iso-mer. The carboxyl group of 2-NAA is moved one positionin the aromatic ring system and showed in another directionthan that of 1-NAA. This might be one possible reason forthe inactivity of 2-NAA despite strong binding to ABP1.

Another possibility for the inactivity of 2-NAA is thatthe aromatic ring system does not bind to W151. Bindingto W151 is relevant for the conformational change at theC-terminal helix of ABP1 and for auxin activity (Woo et al.2002; Bertona et al. 2008). Because of both advantages of2-NAA, its strong binding to ABP1 and its absence of anauxin-induced swelling response, 2-NAA was used as atool to analyze auxin binding to ABP1 on a single-celllevel.

Four domains of 13–20 amino acids, boxes a, b and cand the C-terminus are highly conserved in ABP1 proteins

from various plants. Five of the amino acids involved inauxin binding are localized in box a. T54 and P55 areinvolved in binding the aromatic ring system and H57, H59and E63 are relevant for interacting with the carboxyl groupthrough the Zn2+-ion (Woo et al. 2002). Membrane hyper-polarization assays in tobacco mesophyll protoplasts(Barbier-Brygoo et al. 1991; Venis et al. 1992) and inpatch-clamp measurements of ATP-driven currents (Rücket al. 1993) showed that anti-box a antibodies had auxin-like activity. Similarly, anti-box a antibodies induced proto-plast swelling in an auxin-like manner (SteVens et al. 2001;Fig. 2 in this study). Our study supports structural andexperimental data, because an allocation of box a with2-NAA inhibited protoplast swelling induced by anti-box aantibody. In conclusion box a seems to be relevant forauxin binding.

As judged from the crystal structure, box c may not takepart in 1-NAA binding (Woo et al. 2002). Warwicker(2001) did note two WBE tripeptides in box c and hypothe-sized that these could occupy the auxin-binding site in theabsence of auxin. Interestingly, box c overlaps with those17 amino acids to which photo aYnity-labeled IAA wasfound to bind (Brown and Jones 1994). Even if box c wasnot involved in 1-NAA binding it may be relevant for thebinding of IAA. The data presented here indicate an inter-action of 2-NAA and an antibody raised against a discon-tinuous epitope of box a and box c (scFv12) are in line withthe idea of box c being a binding site. The antibody detectsbox a with a lower aYnity than box c (Leblanc et al. 1999;David et al. 2007). It is possible that 2-NAA, which hasnearly no eVect by itself binds strongly to box a and that theadditional binding of the antibody to box c leads to a stron-ger response than the antibody alone. The striking diVer-ence between anti-box a antibodies and scFv12 indicatesthat 2-NAA binding to ABP1 may somehow involve theparticipation of box c.

Strikingly, mAb12, the antibody where scFv12 wasderived from, while being an auxin agonist in the protoplastswelling assay (S1), appears to be an auxin antagonist in theelectrical response of tobacco protoplasts (Leblanc et al.1999). In addition, ABP1 was shown to be involved indevelopmental processes. With Arabidopsis transgenicsexpressing scFv12 to inhibit ABP1 in planta it was shownthat scFv12 is an antagonistic antibody that inhibits ABP1function at the cell cycle (David et al. 2007) and cell expan-sion in older Arabidopsis plants (Braun et al. 2008). This isthe Wrst time antibodies were found to act diVerently in thethree test systems, and these two discrepancies have to beaddressed: (1) in the electrophysiological system, mAb12inhibits the 1-NAA response (Leblanc et al. 1999). 1-NAAand 2-NAA show a diVerent binding aYnity to the boxes aand c which may result in the opposed response. (2) In thetransgenics, the expressed scFv12 speciWcally interacts with

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ABP1 in the ER (pH 7), whereas the interaction in the proto-plast swelling system occurs in the apoplast (pH 5). Theinteraction in the ER possibly leads to a degradation of theABP1 protein. Thus, less ABP1 is transported to the plasmamembrane, and it has been shown that the sensitivity of themembrane hyperpolarization test system strongly dependson the apoplasmic abundance of ABP1 (Barbier-Brygooet al. 1991). In the protoplast swelling assay ABP1 is postu-lated to be at the plasma membrane. Adding scFv12 or theeven larger IgG mAb12 directly to protoplasts derived fromgrowing organs might therefore lead to the eVect observedfor the swelling response. Analysis with transgenics knock-ing down ABP1 in an induced system using the protoplastswelling response and additional anti-box c antibody experi-ments could help to solve this problem.

The present data also show that 2-NAA does not aVectprotoplast swelling induced by the ABP1 C-terminal pep-tide. These data are important as they demonstrate that2-NAA does not impair the general ability of protoplasts toundergo swelling by some unspeciWc mechanism. Activityof C-terminal peptide was thought to indicate that the C-ter-minus of ABP1 is the site at which the protein binds to atransmembrane docking partner. Other evidence indicatingconformational changes of the C-terminus of ABP1 camefrom experiments which showed that auxin bindinginduced a change in the circular dichroic spectrum

(Shimomura et al. 1986) and from experiments using anti-bodies to detect the C-terminus. The detection failed afterauxin binding (Napier and Venis 1990). One candidate foran interacting protein was identiWed as CBP1, which is aGPI-anchored protein and is homologous to SKU5, an Ara-bidopsis GPI-anchored glycoprotein (Shimomura 2006). Itwas also speculated that the C-terminus may play a rolein auxin binding. The 2-NAA does not interfere with theC-terminal auxin binding as it does not disturb the activityof ABP1 C-terminal peptides.

In summary, we propose that the fast protoplast swellingis induced by binding of IAA to both boxes a and c. Bind-ing of IAA might alter the position of W151 and causes theC-terminus to move (Woo et al. 2002; Bertona et al. 2008).The 2-NAA binds to box a, but not to box c (Fig. 5).Although both agonistic antibodies target preliminary box aor box c, respectively, the bulky molecules probably alsosterically aVect neighboring domains and therefore induceconformational changes triggering a swelling response;2-NAA, adding a high aYnity binding partner to box a, willincrease the action of scFv12 antibodies (Fig. 5).

Studying auxin-induced ABP1-mediated protoplastswelling will help to shed some light on the conclusionsdrawn from structural data. Finally, the goal is to character-ize ABP1 and understand the signal transduction chainleading to the observed early responses.

Fig. 5 Summarized results of protoplast assay. a ABP1 consists of theauxin-binding boxes a and c. W151 of the C-terminal domain might beinvolved in auxin signaling. ABP1 alone does not induce protoplastswelling. IAA and scFv12 bind to boxes a and c. Binding to box cmight induce W151 to move into the binding pocket. Pulling the C-ter-minal domain with it, induced protoplast swelling. 2-NAA binds onlyto box a and does not induce protoplast swelling. Anti-box a antibody

binds to box a and induces protoplast swelling. The C-terminal peptideinduces a swelling response while binding directly to an interactingprotein. b Competition of 2-NAA with IAA or anti-box a antibodyblocked swelling. Application of 2-NAA and scFv12 induced proto-plast swelling, because they bind to boxes a and c. 2-NAA does notinhibit protoplast swelling induced by C-terminal peptide, becausebinding of the peptide is independent of ABP1

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Acknowledgments Funding by the Deutsche Forschungsgemeins-chaft is gratefully acknowledged. We are very indebted to CatherinePerrot-Rechenmann (CNRS, Gif sur Yvette, France) and Klaus Palme(University of Freiburg, Germany) for their generous gifts of antibod-ies and Margret Sauter (University of Kiel, Germany), Daniel Schenck(University of Hamburg, Germany) and Rainer Hertel (University ofFreiburg, Germany) for helpful discussions.

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