TRENDS in Plant Science Vol.6 No.12 December 2001

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586 ReviewReview

Candace Timpte

Dept Biological Sciences,University of NewOrleans, New Orleans,LA 70148, USA.e-mail: ctimpte@uno.edu

‘Curiouser and curiouser!’ cried Alice. (As her body

grew after swallowing a potion, she realized that she

should give her now-distant feet a new pair of boots

for Christmas). Alice went on planning to herself how

she would manage it. ‘They must go by the carrier’,

she thought; ‘and how funny it will seem, sending

presents to one’s own feet! And how odd the

directions will look!’

Through the Looking Glass (Lewis Carrol)

The plant hormone auxin (indole-3-acetic acid, or

IAA) is central to diverse plant growth and

developmental responses. Some of the best-

characterized examples are tropic growth responses

(such as to gravity or light), stem elongation, lateral

branching of roots and shoots, and vascular

development1. These whole-plant responses are the

result of changes at the cellular level that include

elongation, division or differentiation. However, the

mechanisms of auxin perception and response are

understood poorly. Some responses are rapid and

others occur after a lag period, complicating the

situation further.

The first step in a classic hormone response

pathway is a receptor binding a hormone. Many

investigators have sought auxin receptors and several

good candidates have been isolated2. However, as well

as binding auxin, the receptor must also transduce the

auxin stimulus into the known responses. Collecting

evidence that the auxin–receptor interaction causes

direct changes in the cell has been difficult. The

immediate short-term auxin responses include

changes in protoplast electrophysiology, guard-cell

gating and early-response-gene induction. Longer-

term responses include cell elongation, cell division

and phenotypic changes in the whole plant. The choice

of assay is the key to establishing an auxin–receptor

interaction; one must remember that more than one

pathway might be activated by one receptor, and

direct cause-and-effect relations must be established.

Auxin-binding protein

Twenty years ago, an auxin-binding activity was

purified from maize coleoptiles by several groups2,3.

This auxin-binding protein, ABP1, was shown

by photoaffinity labeling to bind auxin4 (its

characterization is summarized in Ref. 2). The maize

ABP1 cDNA encodes a 201 amino acid protein, with a

38 residue signal sequence. The unglycosylated

protein is 20 kDa, whereas the mature protein is

22 kDa, containing a high-mannose-type

oligosaccharide2. ABP1 was the first plant protein

discovered with a C-terminal KDEL sequence, which

is an endoplasmic reticulum (ER) retention signal5.

ABP1 has no hydrophobic regions. Thus, to function

as a receptor, it probably associates with a

membrane-bound ‘docking’protein. ABP1 bears no

resemblance to well-known hormone receptors from

animal systems and does not have substantial

similarity to any mammalian gene. Yet, ABP1 has

been identified from many plant species including

maize, Arabidopsis, tobacco and radish2.

In spite of excellent research efforts, important

questions need to be answered if ABP1 is to be

established as the auxin receptor. First, does ABP1

ligand binding have biological relevance? The auxin

receptor must bind auxin but also must evoke

changes in the cell. Second, what is the structure of

this protein, and how does this structure relate to its

signaling mechanism? Third, where does ABP1

reside in the cell? Typically, a mammalian hormone

binds the target ligand at the plasma membrane,

although one exception is the steroid hormone

receptor. Paradoxically, the KDEL sequence of ABP1

suggests an ER, not a plasma membrane, location.

Could it be elsewhere in the cell?

ABP1 is crucial for embryogenesis

Recent genetic studies provide strong evidence for

ABP1 mediating responses leading to cell elongation

and embryogenesis. Arabidopsis has a single gene

encoding ABP1 (Ref. 6) and disruption of this gene is

expected to affect auxin signaling processes and to

reveal ABP1’s role in plant development. A ‘knockout’

plant harboring a T-DNA insertion in the first exon of

the ABP1 gene has been identified7. Homozygous

individuals were not recovered from this plant line,

strongly indicating that disruption of the ABP1 gene

is lethal. About 25% of the seeds in transgenic siliques

were white and nonviable, clear evidence of

segregation of a lethal homozygous phenotype.

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 ββ-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.

Auxin binding protein: curiouser and

curiouser

Candace Timpte

Addition of a transgenic, functional copy of ABP1

rescued the embryonic-lethal phenotype, suggesting

that normal embryo development requires at least

one copy of ABP1.

Examination of the nonviable embryos revealed

that ABP1 is required early in plant development:

embryos arrested after the globular stage. Newly

formed cross walls between cells were wrongly

oriented and cells failed to elongate, leading to

embryo death7. These results provide direct evidence

that ABP1 plays a crucial role in embryonic

morphogenesis. Whether the role is in cell elongation,

embryo polarity establishment or individual cell

polarity could not be determined. To address the

polarity versus elongation issue, antisense

suppression was used to create an ABP1 loss-of-

function mutation in the BY-2 tobacco cell line. The

results enabled the two types of expansion commonly

observed in cultured plant cells to be differentiated8.

Auxin-induced elongation to increase cell volume

beyond that of the divided cell was abolished in these

transgenic cells7. However, cell expansion to replace

cell volume following division was not affected in the

ABP1-antisense lines. Thus, elongation growth is the

crucial auxin response in cultured cells and failure to

elongate is the probable cause of embryo lethality in

the transgenic knockout plants.

ABP1 mediates cell expansion

The complementary approach, overproducing ABP1,

confirms the role of ABP1 in auxin-mediated cell

expansion. Tobacco was transformed with ABP1

under the control of an inducible promoter9. In control

plants, auxin only induced growth at the leaf tips,

whereas, in overproducing transgenic plants, it

induced growth throughout the leaf. Regions that are

not normally auxin responsive acquired inducible

growth that was strictly dependent on the presence

of auxin; a structurally similar inactive auxin did not

stimulate growth. Thus, overproducing ABP1

extended the range of auxin sensitivity in mature

leaf tissue9. A meticulous analysis of individual cells

from ABP1-overproducing plants reveals that

auxin-inducible cell expansion is a component of

this growth10. The abundance of ABP1 in each cell

correlates with the extent of auxin-induced cell

expansion and with cell size in transgenic plants.

Evidence from cultured cells supports a role for

ABP1 in cell expansion. Cultured maize cells

overproducing ABP1 expanded in an auxin-

dependent fashion and were greater in volume than

control cells9. Antisense-suppressed ABP1 tobacco

BY-2 cells had undetectable levels of ABP1 protein

and lacked auxin-induced cell expansion when

compared with wild-type cells10.

Auxin-induced cell division might involve ABP1

Auxin-mediated growth might also have a division

component. Cells from ABP1-overproducing tobacco

leaves were examined for nuclear division stage10.

The proportion of nuclei in G2 stage was double that

of the wild type. By sequential analysis of cells in

developing leaves, cell expansion was found to

precede the G2 advance in the cycle. The premature

G2 advance is probably an indirect effect of the

increased cell volume of transgenic plants10.

A conditional ABP1 knockout mutation has

been constructed by producing a transgenic

ABP1 antibody in the tobacco BY2 cell line

(C. Perrot-Rechenmann, pers. commun.). This

transgenic antibody presumably binds ABP1

in planta and limits its activity within the cell.

These knockout cells showed no significant change in

cell volume but arrested at the G1 phase of the cell

cycle. Thus, ABP1 might play a crucial role in the

regulation G1 and G2/M phases of the cell cycle.

Although the conclusions from these two

transgenic studies differ, the results indicate a

crucial role for ABP1 in plant cells. Furthermore,

either knocking out or overproducing ABP1 provides

crucial evidence that ABP1 mediates perception of

auxin in cultured cells and that disruption of this

signal causes changes in the cell cycle.

ABP1 triggers a plasma membrane electrical response

Hyperpolarization of the cell membrane occurs

within minutes after applying biologically active

auxin, providing a convenient assay for evaluating

auxin response at the outer face of the plasma

membrane. ABP1 has been implicated in this

response in many studies11. Synthetic peptides

corresponding to the C terminus of ABP1 were tested

in the hyperpolarization assay12,13 (Table 1). Peptide

Pz152-163 is a maize-derived sequence. Two others

are tobacco-derived peptides: the first, Nt-C15, is

most similar to the wild-type sequence whereas the

second, Nt-C12, lacks three conserved residues.

The maize and Nt-C15 peptides all induce

hyperpolarization, much as auxin does when applied

to tobacco protoplasts. The truncated Nt-C12 peptide

fails to induce the hyperpolarization response. This

study confirms previous results that exogenous

peptides derived from ABP1 can elicit an electrical

response. These results confirm that the homologous

system is more efficient than the heterologous

system, because peptides and membranes were

derived from the same species13.

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Table 1. C-terminal sequences tested for hyperpolarization

Peptide Sequence Hyperpolarization?

Consensus WDE.C......KEDL Not known

Maize ABP1 WDEDCFEAA..KDEL Yes

Nicotiana tabacum ABP1 WDEECYQTTSWKDEL Yes

Pz152–163 .DEDCFEAA..KDEL Yes

Nt-C15 WDEECYQTTSWKDEL Yes

Nt-C12 ..ECYQTTSW.KDEL No

Mutation targets WDEECYQTTSWKDELa No

Deletion WDEECYQTTSW.... NoaText in bold indicates target residues for mutation.

The C-terminal charged residues in ABP1 were

mutagenized and the entire protein was tested in the

hyperpolarization assay14. The charged residues were

mutated to the cognate amine residues, singly and

paired, and a KDEL deletion mutant protein was

constructed (Table 1). The KDEL deletion evoked the

same hyperpolarization response as the wild type when

applied to cells. None of the charge-substituted mutant

proteins evoked a hyperpolarization response14. Thus,

the substitution of charged residues causes ABP1 to

fail to interact with the plasma membrane protein

that affects hyperpolarization, implicating a

charge–charge interaction between the proteins.

Alternatively, the mutated ABP1 might simply misfold

and fail to interact with the plasma membrane protein.

The electrical response of plant cells was affected by

antibodies directed against ABP1. Several monoclonal

antibodies induce hyperpolarization in tobacco cell

protoplasts and act as auxin agonists13. Three other

monoclonal antibodies act as antagonists and block

auxin action, either by recognizing the auxin-binding

site as the epitope13 or by immobilizing ABP1 in a non-

functional conformation. Similarly, antibodies affected

orchid cell stomatal opening. Both the D16 monoclonal

antibody, raised against the putative auxin binding

site of ABP1 (Ref. 15), and a monoclonal antibody

against an ABP1 peptide, induced stomatal opening

and acidification, similar to the effects of auxin16. A

monoclonal antibody that targets the C terminus of

ABP1 and the peptide Pz152-163 stimulated stomatal

closure and increased pH, similar to the mode of

abscisic acid. A Pz152-163 peptide lacking the KDEL

sequence had no effect. This result is curious because

other data suggests that the KDEL is not required

for hyperpolarization stimulation. However, small

changes in a peptide can cause great changes in

peptide structure. These immunological results

indicate that ABP1 transduces the auxin signal to

the plasma membrane to effect hyperpolarization,

perhaps by interacting with another protein.

The amount of ABP1 might be tightly regulated

in the cell. As the evidence above indicates,

increased production of ABP1 enhances auxin

sensitivity9,10,17,18. Examination at the molecular level

reveals that transgenic overexpression of wild-type

ABP1 generated a ~100-fold increase in expression by

RNA blotting but only a 30% increase in detectable

protein by immunoblotting18. In antisense transgenic

plants, maximal inhibition of ABP1 protein was

merely 50%, indicating that complete inhibition of

ABP1 might be detrimental to the plant18.

Structure of ABP1

The three-dimensional structure of ABP1 could give

clues about the mechanism of signaling or potential

protein–protein interactions. The first model for the

auxin-binding site of ABP1 was based on the

structure and interaction of 45 different auxin

analogs19. This model proposed a planar, indole ring-

binding platform, a charged carboxylic acid-binding

site and a hydrophobic transition region. By

photoaffinity labeling with azido IAA (Ref. 20) and

immunology21, two regions were implicated in auxin

binding. Structure mapping studies using a panel of

monoclonal antibodies further defined the identity

of residues forming the auxin-binding platform and

the carboxylic acid-binding site13.

β-Barrel dimerComparisons of amino acid sequences show that there

are several highly conserved residues between auxin

binding proteins in monocots and dicots2,22. An

augmented model has been proposed based on these

and additional comparisons with the cupin and vicilin

superfamily of proteins23. The structural basis of this

model relies on conserved residues corresponding to

β-barrel turn anchors in the germin protein structure24.

The proposed structure is a β-barrel homodimer,

containing β-sheets and no α-helix, consistent with

circular dichroism spectra25, and resembles the

pseudodimer symmetry of a vicilin monomer23.

Recently, ABP1 was crystallized, and X-ray diffraction

analysis to 1.9 Å resolution shows two glycosylated

homodimers in asymmetric units26. These crystal

structure data are consistent with a β-barrel (Fig. 1).

This level of resolution cannot confirm the auxin-

binding site. A conserved region might be analogous to

the metal-binding site of oxalate oxidase23 and thus

indicate that ABP1 has some unknown enzyme

function. This speculation is intriguing, because no

enzymatic activity has been reported for ABP1.

Mobile C-terminusExperimental evidence suggests that binding auxin

causes a conformational change involving the

C-terminus27. Interference mapping studies suggest

that the C-terminus interacts with the auxin-binding

site, perhaps through disulfide bonds13. Two

antibodies map to overlapping ABP1 regions but have

opposite electrochemical effects, one agonistic and the

other antagonistic to auxin action14. In the presence of

auxin, binding by one agonist antibody is completely

abolished and the other antibody has a weaker

interaction with ABP1 (Ref. 14). This result suggests

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N-term

N-term

C-term

C-term

Fig. 1. Conceptual modelof ABP1. ABP1 is a β-barrelstructure modeled onsimilarity with cupinfamilies and concavalin Afor dimerization. ResidueW44 (red) and the clusterof residues forming theputative metal-bindingsite (blue) might form theplatform for auxin binding.The purple ribbonindicates conservedβ-turn anchor residues.Abbreviations: C-term,C-terminal; N-term,N-terminal.

that ABP1 undergoes a distinct conformational

change when auxin is bound, changing the epitope.

Circular dichroism data indicate a conformational

change upon auxin binding25. Although the

C-terminus did not have sufficient similarity to the

vicilin superfamily to model its location, a C-terminal

tryptophan or WDE sequence might occupy the

binding pocket in the absence of auxin23. The

experimental evidence presented above supports

the importance of the conserved WDE residues.

Conformational changes upon auxin binding might

release the C-terminus for signal propagation and

interaction with other proteins.

Localization versus site of action remains perplexing

The KDEL sequence of ABP1 appears to be effective

at localizing ABP1 to the endoplasmic reticulum (ER).

Paradoxically, the evidence indicates that ABP1 binds

auxin with low affinity at the pH of the ER (Ref. 28).

However, numerous visualization techniques show

that most, but not all, ABP1 is localized to the ER, not

the plasma membrane. ABP1 has been visualized at

the plasma membrane of maize cultured cells by

using immunogold labeling29. ABP1 has also been

detected throughout the Golgi and has been secreted

into culture medium29. A small population of ABP1

molecules has been detected at the plasma membrane

of maize coleoptile protoplasts using silver-enhanced

immunogold epipolarization microscopy30. The

number of ABP1 molecules at the surface was

estimated to be as low as 1000, which represents only

a small proportion of total cellular ABP1. A small

number of cell surface receptors requires less

hormone to achieve half-maximal occupancy.

Physiologically, this would allow the cell to be

sensitive to small amounts of hormone29.

Because antibodies are unlikely to enter intact

cells, the evidence that several antibodies trigger the

plasma membrane hyperpolarization response

indicates that at least some portion of the ABP1 pool

resides at the plasma membrane11,14–16. Because the

antibodies and peptides bind a protein in intact

protoplasts that affects membrane conductivity,

logically they must bind ABP1 localized to the

plasma membrane.

The C-terminal KDEL sequence was changed to

examine its role in the localization of ABP1 (Ref. 17).

KDEL was mutated to HDEL to enhance its retention

in the ER or mutated to either KDELGL or KEQL to

compromise its retention. As expected, the KDEL or

HDEL proteins localized to the ER, whereas the

KEQL and KDELGL proteins entered the Golgi

stacks. However, there was no difference in the cell

surface abundance of ABP1 in cells expressing

mutant proteins as examined by electron microscopy

or silver-enhanced immunogold epipolarization

microscopy17. Thus, even without the KDEL

sequence, quantities of ABP1 do not localize

massively to the plasma membrane.

Similar results confirming the major ER

localization were obtained by analysis of maize

coleoptile rolled leaves31. Neither immunofluorescence

nor immunogold labeling detected ABP1 at the

plasma membrane. Double-labeling experiments to

detect conformational changes that might sequester

the KDEL and allow transit to the plasma

membrane were negative. Under conditions of

auxin binding, ABP1 remained in the ER (Ref. 31).

Carbohydrate analysis further confirmed the ER

localization of ABP1. The ABP1 oligosaccharides

are high mannose types, not the more complex

carbohydrates expected if ABP1 traversed the

Golgi stacks. Less than 2% of ABP, by glycan

analysis, escaped the ER retention system31. At this

low level of escape, a special mechanism to avoid

ER retention need not be invoked. Bulk flow or

association with other proteins might be sufficient

to allow this tiny amount of ABP1 to escape to the

plasma membrane.

The mechanism of KDEL-mediated ER retention

and retrieval operates efficiently, such that an

alternative delivery mechanism to the vacuole was

proposed32. A KDEL terminus was attached to a

protein not normally localized to the ER. This

construct circumvented the Golgi to reach the

vacuole. Some such alternate mechanism might

operate for ABP1. Because overproduction of ABP1

increases the sensitivity of cells to auxin, the

presence of ABP1 at the plasma membrane might be

tightly regulated.

Never-ending story of auxin signaling

ABP1 fits the criteria for a hormone receptor. Ample

evidence indicates that ABP1 mediates auxin’s effects

on normal plant development. However, the

molecular mechanism of ABP1 remains at best a

skeletal model (Fig. 2). To reconcile the diverse effects

of auxin, two sites of ABP1 activity are proposed.

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Golgi

ER

Dockingprotein

ABP1

Ionchannel

Hyperpolarization

Vesicles to PM

Cell wall

Plasma membrane

Fig. 2. Model of ABP1localization and action.Most ABP1 resides in theendoplasmic reticulum(ER) but it is alsodetectable in the Golgiand associated with theplasma membrane (PM).ABP1 probably associateswith a transmembranedocking protein topropagate the auxinsignal to the interior of thecell, or it could interactdirectly with ion channels.A conformational changeis induced upon auxinbinding. At the plasmamembrane, auxin bindingeffects a hyperpolarizationevent, which also can bestimulated by ABP1-derived peptides. Becausethe ER does not providethe optimum pH for auxinbinding, auxin-boundABP1 in the Golgi mightdirect vesicle traffic of cellwall materials forexpansion.

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590 Review

Binding at higher or lower concentrations of auxin

might temper the response. Although ABP1 has no

hydrophobic regions, the auxin-binding signal must

be transmitted to the cell. To this end, ABP1 might

interact with a plasma membrane docking protein

(yet to be identified)3,33 or might interact directly

with the ion channel. Auxin binding induces a

conformational change in ABP1, enabling interaction

with the docking protein, or alters the ABP1–docking-

protein complex to transmit the signal. The docking

protein might be abundant at the plasma membrane;

excess docking protein would then be available to

interact with exogenously provided ABP1 or

peptides in assays, and to mediate membrane

hyperpolarization. Similarly, ER or Golgi-localized

ABP1 might interact with a transmembrane protein

to regulate the secretion of cell wall components to

mediate cell expansion.

Auxin-induced conformational changes in ABP1

might alter interactions with other membrane

proteins, perhaps heterotrimeric G-proteins. Auxin

is known to induce the transcription of several

auxin-regulated genes that are repressed by the

activation of a specific MAPK cascade34.

Furthermore, Arabidopsis cells overproducing the

plant heterotrimeric Gα protein mimic the auxin-

induced increase in cell division35. These results

suggest that the auxin signaling pathway might

involve the Gα protein to regulate cell cycle control.

However, given the varied plant responses to auxin,

there might be more than one type of auxin

receptor in the cell.

It has not been possible to cover all aspects of

auxin signaling in this article. The importance of

ABP1 in plant development is certain but more

pieces of the puzzle remain to be identified. Refining

the location and site of action of ABP1 might require

the use of non-plant in vivo systems, such as the

previously used COS cells28. The proposed docking

protein or other proteins that interact with ABP1

must be identified. Plant development, as the

ultimate result of cell division and cell elongation, is

affected by numerous hormone signals. The

mechanism of integration of these signals and

exactly where ABP1 fits into signaling cross-talk

remain to be discovered.

Review

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Acknowledgements

I am grateful to BarbaraTriplett, Sarah Lingle,Hee-Jin Kim, Amy Hermanand several anonymousreviewers for comments.Thanks to Jim Nolan forthe structure figure. I thankAlan Jones and CatherinePerrot-Rechenmann forsharing unpublishedresults.