Review Article

340

Indole-3-Acetic Acid Protein Conjugates:

Novel Players in Auxin Homeostasis
C. Seidel1, A. Walz1, 2, S. Park3, J. D. Cohen3, and J. Ludwig-Mller1
1

Institut fr Botanik, Technische Universitt Dresden, Zellescher Weg 22, 01062 Dresden, Germany
2
Vital Probes Inc., 1300 Old Plank Road, Mayfield, PA 18433, USA
3
Department of Horticultural Science, Center for Microbial and Plant Genomics, University of Minnesota, 305 Alderman Hall,
Saint Paul, MN 55108, USA
Received: October 4, 2005; Accepted: December 12, 2005

Abstract: Indole-3-acetic acid (IAA) is found in plants in both

free and conjugated forms. Within the group of conjugated IAA
there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic
group. The first gene, iap1, encoding for a protein with IAA as a
prosthetic group, was cloned from bean (Phaseolus vulgaris). It
was shown that the expression of IAP1 as a major IAA modified
protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover,
this protein underwent rapid degradation during germination.
Since further molecular analysis was difficult in bean, the iap1
gene was transformed into Arabidopsis thaliana and Medicago
truncatula. Expression of the bean iap1 gene in both plant species under the control of its native promoter targeted protein
expression to the seeds. In Arabidopsis no IAA was found to be
attached to PvIAP1. These results show that there is specificity
to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1
was predominantly associated with the microsomal fraction. In
addition, a related protein and several smaller peptides that are
conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both
advance our knowledge of IAA proteins and explore aspects of
auxin homeostasis that were not fully revealed by studies of free
IAA and lower molecular weight conjugates.
Key words: Auxin homeostasis, protein modification, Phaseolus
vulgaris, Medicago truncatula, indole-3-acetic acid, IAA protein,
Arabidopsis thaliana.

Introduction
Auxins are involved in many key processes during plant development. In vascular plants, auxins, primarily indole-3-acetic acid (IAA), regulate gene expression, cell division, cell elongation and differentiation in plant tissue. Auxins also affect
vascularization, phototropism, geotropism, fruit development,
flower development, and apical dominance (Davies, 2004).

Plant Biol. 8 (2006): 340 345

Georg Thieme Verlag KG Stuttgart New York
DOI 10.1055/s-2006-923802 Published online March 13, 2006
ISSN 1435-8603

While IAA in low concentrations stimulates growth and development, higher concentrations can be toxic to the plant (Bandurski et al., 1995). Therefore, tight control of IAA concentration is necessary for proper plant development. An understanding of such processes should allow the development of
better crop varieties and, through molecular techniques, the
improvement of existing germplasm. During seed germination, auxins control the rate of seedling growth and thus can
affect the ultimate productivity of planted fields.
Plants contain minute amounts of the phytohormone IAA as
the free acid. All plants examined so far contain most of their
IAA in conjugated form. These conjugates are thought to be involved in a variety of hormonally-related processes (Cohen,
1983): a) in the transport of IAA within the plant; b) the storage and subsequent reuse of IAA; c) protection of IAA from
enzymatic destruction; d) as components of a homeostatic
mechanism for control of IAA levels; and e) as an entry route
into the subsequent catabolism of IAA (Fig. 1 A).
Two main types of conjugated molecules have been studied:
the amide-linked IAA forms bound to one or more amino
acids and the ester-linked forms primarily bound to sugar(s)
(Fig. 1 B). These two types of conjugates appear to be found at
varying concentrations in the diverse tissues of angiosperms
(Domagalski et al., 1987). On average, 95 % of all IAA in a plant
is conjugated into these storage forms (Cohen and Bandurski,
1982; Bandurski et al., 1995). Using such mechanisms, a plant
is able to change its hormone levels and thus control its
growth and development under changing environmental conditions. In addition, some IAA conjugates are also substrates
for IAA degradation reactions (Tsurumi and Wada, 1986). Furthermore, IAA can be converted to indole-3-butyric acid (IBA)
and IBA is then also subject to conjugation via ester and amide
bonds (Ludwig-Mller, 2000). IBA may act as an auxin itself
or through its conversion to IAA (Ludwig-Mller, 2000; Bartel
et al., 2001).

Low Molecular Weight IAA Conjugates with

Amino Acids and Sugars
Despite the fact that IAA conjugates are major players in establishing auxin homeostasis, relatively few IAA conjugates have
been identified. Common IAA-ester conjugates include IAAmyo-inositol glycosides, IAA-myo-inositol, and IAA-glucose,
which have been identified in many species over the last 60

Indole-3-Acetic Acid Protein Conjugates: Novel Players in Auxin Homeostasis

Plant Biology 8 (2006)

Fig. 1 (A) Possibilities to control auxin homeostasis in plants. (B) Metabolic routes controlling auxin conjugation. The solid arrows
lead to the conjugates, the broken arrows
lead back to free IAA. The question marks
symbolize compounds and routes which have
not been proven experimentally so far. IBA =
indole-3-butyric acid.

years (Bandurski et al., 1969; Kopcewicz et al., 1974; Bandurski

and Schulze, 1977; Domagalski et al., 1987; Slovin et al., 1999).
The amide conjugates identified so far include IAA-Asp in Scots
pine (Andersson and Sandberg, 1982), IAA-Asp and IAA-Glu
in cucumber (Sonner and Purves, 1985) and soybean (Cohen,
1982). IAA-Ala has been detected in Picea abies (stin et al.,
1992) and together with IAA-Asp, IAA-Leu, and IAA-Glu in Arabidopsis thaliana (Tam et al., 2000; Kowalczyk and Sandberg,
2001).
A comparatively large amount of information is now available
on the synthesis and hydrolysis of the lower molecular weight
conjugates of IAA in higher plants. The first ester conjugate hydrolase activity was found in maize (Hall and Bandurski, 1986),
followed by whole seedling studies examining movement and
hydrolysis of IAA-inositol (Chisnell and Bandurski, 1988). More
recently, an ester conjugate hydrolase activity in maize was
identified and purified, but the corresponding gene has yet to
be cloned (Kowalczyk et al., 2003; Jakubowska and Kowalczyk,
2005).
The ILR1-like IAA amidohydrolase gene family is thought to be
involved in the regulation of free IAA concentrations. This gene
family was originally characterized in the model plant Arabidopsis thaliana (Bartel and Fink, 1995; Davies et al., 1999;

LeClere et al., 2002). The enzymes characterized to date show

distinct but overlapping substrate specificity. Another ILR1like family member, sILR1, whose gene product has substrate
specificity for IAA-Gly and IAA-Ala, has been isolated from
the related species Arabidopsis suecica (Campanella et al.,
2003 a, b). Amidohydrolase homologues have also been isolated from Brassica rapa L. (Ludwig-Mller et al., 1996; Schuller
and Ludwig-Mller, 2002). Recently, an orthologue for the Arabidopsis IAR3 auxin amidohydrolase gene (TaIAR3) has been
isolated from the monocotyledonous species wheat (Triticum
aestivum L.) and biochemically characterized (Campanella et
al., 2004). The TaIAR3 protein has, unlike its orthologue IAR3,
high specificity for the hydrolysis of indole-3-butyric acid conjugates with alanine and glycine.
IAA conjugate formation was extensively studied in maize (Michalczuk and Bandurski, 1982; Leznicki and Bandurski, 1988;
Kesy and Bandurski, 1990) and a gene encoding an IAA-glucose
synthase (iaglu) was cloned (Szerszen et al., 1994). Recently, an
orthologue from Arabidopsis has been isolated and characterized (Jackson et al., 2001, 2002). The first mechanism of auxin
amide conjugate formation was described by Staswick et al.
(2002), who showed that a family of GH3-like proteins in Arabidopsis was able to adenylate several phytohormones including IAA. Further biochemical analysis showed that various

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C. Seidel et al.

amino acids were attached to IAA by members of the GH3

family (Staswick et al., 2005). This large gene family consists of three sub-families: 1) enzyme(s) adenylating jasmonic
acid, 2) enzyme(s) adenylating IAA, and 3) proteins for which
no substrates have been found (Staswick et al., 2002, 2005).
Gain-of-function mutations in some of these genes (ydk1,
dfl1) suggest an auxin-deficient phenotype, although a knockout mutant of ydk1 did not show any apparent phenotype
(Nakazawa et al., 2001; Takase et al., 2003, 2004).

The Need to Search for Novel Types of Auxin Conjugates

Most studies done over the last fifty years showed that the
amount of total IAA (free plus conjugated) obtained by direct
hydrolysis of the tissue was much greater than the amount
readily extractable with solvents (Chen, 1987; Bialek and Cohen, 1989 a, b; Cohen et al., 1989). This discrepancy has only recently been resolved by the isolation of proteins and peptides
to which IAA was attached. In bean seeds, the majority of IAA
seems to be conjugates to large peptides and proteins. The first
studies that suggested IAA protein covalent complexes left
open the possibility that IAA attachment was a random event
or incidental to the function of either the proteins or the phytohormone. We have recently shown that a specific protein in
bean, PvIAP1, is covalently modified by IAA (Fig. 1 B). Approximately half of that protein in the tissue is modified and the expression of the single copy gene encoding this protein is developmentally regulated (Walz et al., 2002).

A Gene From Bean Encoding a Protein Modified

by Indole-3-Acetic Acid
The auxin levels in bean in relation to development were studied extensively over many years (Bialek et al., 1983). The first
attempts to isolate IAA-amino acid conjugates demonstrated
that if such compounds were present in bean seeds, they must
be present in very low amounts. Bandurski and Schulze (1977)
showed that bean seeds contained significant amounts of
amide-linked IAA. A compound with a very low mobility on
TLC was isolated and found, upon strong alkaline hydrolysis,
to yield IAA (Bialek and Cohen, 1986). The properties of this
compound were quite different from those of simple IAA-amino acid conjugates. The conjugate consisted of two moieties
of IAA attached to a 3.6 kDa peptide. In bean, the accumulation
of amide conjugates is correlated with other processes such
as storage protein accumulation during late seed maturation
(Bialek and Cohen, 1989 b, 1992).
Antibodies raised against the bean 3.6 kDa IAA peptide
(Ab3.6K; Cohen et al., 1988) immunoreacted with several other
bean seed proteins of varying molecular masses from 17
60 kDa. The major immunoreactive protein was found by
GC-MS analysis to have IAA covalently attached (Walz et al.,
2002). This protein was purified and quantitative GC-MS analysis of the purified protein showed a molar protein to IAA ratio of 2 : 1. Based on microsequencing of IAP1, the cDNA encoding a 35 kDa protein was isolated (iap1, GenBank Acc. No.
AF293023; Walz et al., 2002). The single copy gene has 65.5 %
similarity at the nucleotide level, interspersed throughout
the sequence, and 64.7 % similarity at the amino acid level to
a 35 kDa late seed maturation protein from soybean (Fig. 2).
Putative Arabidopsis homologues cluster in a separate group
(Fig. 2). The iap1 gene does not contain known signalling se-

Fig. 2 Phylogenetic tree for different iap1 homologues. Alignment

was performed with ClustalW (http://www.ebi.ac.uk/clustalw/). The
method used to calculate distances in the phylogram (a branching diagram assumed to be an estimate of a phylogeny, branch lengths are
proportional to the amount of inferred evolutionary change) was the
Neighbour Joining method.

quences nor does it appear to encode the smaller 3.6 kDa bean
IAA peptide (Cohen et al., 1988). RNA blot analysis showed that
the iap1 transcripts accumulate in significant amounts in
seeds late in their development. No iap1mRNA was found in
other bean tissues, suggesting IAP1 accumulates during seed
maturation to provide a mechanism for hormonal regulation
during seed development and germination (Walz et al., 2002).
Immunoblots using Ab3.6K detected protein accumulation
during late seed development and showed that IAP1 levels decrease dramatically in dark-germinating axes and cotyledons
of bean seedlings in the in the course of a single day after imbibition.

Tissue and Cell-Specific Localization of IAP1

An iap1-GFP reporter gene construct was designed for subcellular PvIAP1 (IAP1 from bean) protein localization. Since Phaseolus vulgaris is problematic for transformation, iap1 from
bean was heterologously expressed in two model plants, Arabidopsis and Medicago truncatula. In both plant species, iap1 expression under its native bean promoter targeted gene expression solely to the seeds (A. Walz, C. Seidel, and J. Ludwig-Mller, unpublished results). It was shown that the GFP reporter
distribution was relatively uniform throughout the embryo
of both plant species and on the cellular level PvIAP1 seemed
to accumulate predominantly in the endomembrane fraction
(C. Seidel, A. Walz, G. Rusak and J. Ludwig-Mller, unpublished
results). This finding might be of significance, even though in
this case the localization of heterologous IAP1 was analyzed,
because in Arabidopsis the proteins encoding IAA amino acid
conjugate hydrolases have N-terminal sequences making them
targets for the endoplasmic reticulum (Davies et al., 1999).
Thus, IAP1 and IAA conjugate hydrolases probably co-localize
to the same compartment and thereby facilitate hydrolysis of
IAA conjugates. Whether these proteins play a role in cleaving
IAA from naturally occurring IAP1 homologues in Arabidopsis
has yet to be shown once homologous proteins are available
for these kinds of studies. Since bean is difficult to manipulate,
we have started to isolate homologous proteins from Arabidopsis to analyze the function of these novel proteins in plant development and auxin homeostasis.

Indole-3-Acetic Acid Protein Conjugates: Novel Players in Auxin Homeostasis

Plant Biology 8 (2006)

subjected to alkaline hydrolysis. GC-MS analysis confirmed

the presence of IAA covalently bound to the protein (A. Walz,
S. Park, J. D. Cohen, and J. Ludwig-Mller, unpublished results).
These results establish the presence of IAA proteins in dicots
other than bean IAP1.
To isolate putative IAA proteins from Arabidopsis, peptide antibodies were made based on the conserved sequences between
PvIAP1 and its closest homologues in Arabidopsis and Glycine
max (A. Walz and J. Ludwig-Mller, unpublished results). The
peptide antibodies were used for immunoblotting of total protein from Arabidopsis. The antibodies strongly cross reacted
with 52 and 35 kDa proteins and were used for immunoprecipitation. Initial experiments indicated the presence of amidelinked IAA in the immunoprecipitated protein fractions (C.
Seidel, S. Park, J. D. Cohen, and J. Ludwig-Mller; unpublished
results). The proteins will be further characterized.
Fig. 3 The percentage of free IAA and IAA conjugates in Arabidopsis
seeds. Data were taken from Ljung et al. (2002).

Is it IAA Conjugation or Protein Modification by IAA?

Auxin conjugates play a major role in controlling the concentration of the free hormone. In addition, conjugates with peptides or proteins may reveal other roles for IAA besides a simple storage function. IAA conjugation may act as a novel protein modification and target proteins to specific cellular compartments. The IAA protein conjugates might be expected to
be localized within the cell and, if moved, transported by processes very different from systems acting on free IAA and the
low molecular weight conjugates.

Fig. 4 Cross-reactivity of Arabidopsis thaliana seed proteins with AB

3.6K. (A) Immunoblot of Arabidopsis seed proteins with AB 3.6K as well
as with anti-IAA-BSA antibodies. (B) Immunolocalization of cross-reacting proteins with AB 3.6K in the Arabidopsis embryo. The dark colored
parts represent positive staining with 3,3-diaminobenzidine after incubation with antirabbit IgG conjugated to peroxidase. For further details see Walz et al. (2002). c = cotyledons, r = root.

A diverse number of prosthetic groups are covalently attached

to proteins as post-translational modifications. In plants, glycoproteins, lipoproteins, phosphoproteins, hemoproteins, and
flavoproteins all represent examples of such conjugated proteins. IAP1 from bean is not glycosylated or phosphorylated
(Walz et al., 2002), but has IAA covalently attached to the molecule. Therefore, IAA-modified proteins are a distinct class of
proteins conjugated with auxin. They appear to be the major
form of auxin conjugates in bean seeds and are also found during germination. Our immunological and analytical data suggest that auxin modification of a small class of proteins may
be a feature common to many plants.

Acknowledgements

IAP1 Homologues in Other Plant Species

Previous studies have shown that Arabidopsis has both ester
and amide conjugated IAA (Tam et al., 2000), but in total the
smaller molecular weight conjugates previously identified
did not account for the bulk of the conjugate pool (Fig. 3). A
number of proteins were identified from different plant species which cross-reacted with the 3.6K antibody (Walz et al.,
2002). All cross-reacting proteins examined so far had IAA covalently attached (Cohen et al., 1995). Ab3.6K was used for immunostaining of different Arabidopsis tissues. Cross-reactivity
was found with two major seed proteins (Fig. 4 A). Immunoreactive proteins were localized to the root meristem and outer cell regions of the cotyledons and radical of Arabidopsis
seeds (Fig. 4 B; Walz et al., 2002). The Arabidopsis seed 35 kDa
immunoreactive protein also cross-reacts with anti-IAA-BSA
antibodies (Fig. 4 A). The protein was partially purified and

This work was supported by the DFG SPP 1067 (Lu500/8) and
by a grant from the National Science Foundation (IBN 0111530). We also acknowledge support from the Minnesota Agricultural Experimental Station and the Gordon and Margaret
Bailey Endowment for Environmental Horticulture.

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Plant Biology 8 (2006)

J. Ludwig-Mller
Institut fr Botanik
Technische Universitt Dresden
Zellescher Weg 22
01062 Dresden
Germany
E-mail: jutta.ludwig-mueller@mailbox.tu-dresden.de
Guest Editor: R. Reski

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