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Inputs to the Active Indole-3-Acetic
Acid Pool: De Novo Synthesis,
Conjugate Hydrolysis, and
Indole-3-Butyric Acid -Oxidation
Bonnie Bartel,* Sherry LeClere, Mo´nica Magidin, and Bethany K. Zolman
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, 77005, USA
ABSTRACT
The phytohormone auxin is important in virtually
all aspects of plant growth and development, yet our
understanding of auxin homeostasis is far from com-
plete. Plants use several mechanisms to control lev-
els of the active auxin, indole-3-acetic acid (IAA).
Plants can synthesize IAA both from tryptophan
(Trp-dependent pathways) and from a Trp precursor
but bypassing Trp (Trp-independent pathways). De-
spite progress in identifying enzymes in Trp-
dependent IAA biosynthesis, no single IAA biosyn-
thetic pathway is yet defined to the level that all of
the relevant genes, enzymes, and intermediates are
identified. In addition to de novo synthesis, vascular
plants can obtain IAA from the hydrolysis of IAA
conjugates. IAA can be conjugated to amino acids,
sugars, and peptides; endogenous conjugates that
are active in bioassays and hydrolyzed in plants are
likely to be important free IAA sources. Conjugation
is also used to permanently inactivate excess IAA,
and these conjugates may be distinct from the hy-
drolyzable conjugates. The peroxisomal -oxidation
of endogenous indole-3-butyric acid (IBA) also can
supply plants with IAA, which may account for part
of the auxin activity of exogenous IBA. Compart-
mentalization of enzymes and precursors may con-
tribute to the regulation of auxin metabolism. IAA
obtained through de novo synthesis, conjugate hy-
drolysis, or IBA -oxidation may have different
functions in plant development, and possible roles
for the IAA derived from the various pathways are
discussed.
Key words: Auxin biosynthesis; Auxin conjugate
hydrolysis; Fatty acid -oxidation; IAA; IBA; Indole;
Glucosinolates; Phytohormone homeostasis; Plant
peroxisome; Tryptophan
IAA BIOSYNTHESIS:MULTIPLE PATHWAYS
Many of the molecular components of de novo IAA
biosynthesis, an essential aspect of IAA homeostasis
(Figure 1), remain undefined despite decades of ef-
fort. Although several IAA biosynthetic pathways
are now established in plant-associated microbes
(Costacurta and Vanderleyden 1995; Patten and
Glick 1996), no plant IAA biosynthetic pathway has
been fully elucidated. The current state of our
knowledge is illustrated in Figure 2 and genes im-
plicated in IAA biosynthesis are listed in Table 1.
Plants can produce IAA from both Trp-dependent
and Trp-independent pathways (Normanly and oth-
ers 1995; Bartel 1997; Normanly and Bartel 1999;
Slovin and others 1999). Multiple routes to the same
product may allow precise regulation of IAA biosyn-
Online publication 8 November 2001
*Corresponding author; e-mail: bartel@rice.edu
J Plant Growth Regul (2001) 20:198–216
DOI: 10.1007/s003440010025
© 2001 Springer-Verlag
198
thesis, but our understanding of this control is rudi-
mentary. Two complementary approaches are con-
tributing to breakthroughs in the study of IAA bio-
synthesis: the identification and quantification of
trace amounts of indolic compounds from plants us-
ing stable isotope dilution and GC-MS, and the ad-
vance of genetic and genomic analyses in model
plants to identify the specific enzymes involved.
Trp-Independent IAA Biosynthesis
A variety of plants use both Trp-dependent and Trp-
independent IAA biosynthetic pathways (Normanly
and others 1995; Slovin and others 1999). Although
it was long assumed that plants would synthesize
IAA from Trp, early suggestions of a Trp-indepen-
dent pathway came from the demonstration that in-
dole, but not Trp, has auxin activity in an Avena
coleoptile bioassay (Winter 1966). Trp-independent
IAA biosynthesis is important not only in an-
giosperms, but also in lower land plants, including
liverworts, mosses, and ferns (Sztein and others
2000).
Feeding plants heavy isotope-labeled intermedi-
ates has been used to determine the importance of
various biosynthetic pathways. These studies are
based on the premise that, for a linear pathway, a
precursor will contain higher isotopic enrichment
than its derivative. Feeding studies with Lemna gibba
indicate that Trp availability does not limit IAA bio-
synthesis in this system (Baldi and others 1991). In
Arabidopsis seedlings, the Trp precursor [
15
N]-
anthranilate (Figure 2) labels IAA more completely
than Trp, whereas [
2
H
5
]-Trp is not efficiently con-
verted into IAA (Normanly and others 1993), sup-
porting a Trp-independent IAA biosynthetic path-
way.
Analyses of Trp biosynthetic mutants also dem-
onstrate that IAA biosynthesis is not solely Trp-
dependent. The maize orange pericarp (orp) mutant is
a Trp auxotroph with defects in two Trp synthase 
loci (Figure 2). Instead of having reduced IAA levels,
as would be expected if all IAA is derived from Trp,
the orp mutant contains more total (free plus con-
jugated) IAA than wild type (Wright and others
1991). In the mutant, D
2
O feeding enriches the IAA
pool more than the Trp pool, and [
15
N]-anthranilate
labels IAA but not Trp (Wright and others 1991).
The Arabidopsis trp3-1 and trp2-1 mutants, which
are defective in Trp synthase ␣and , respectively,
(Last and others 1991; Radwanski and others 1996),
have low soluble Trp levels (Mu¨ller and Weiler
2000b; Ouyang and others 2000) but accumulate
amide- and ester-linked IAA conjugates (Normanly
and others 1993; Ouyang and others 2000), suggest-
ing that excess IAA is inactivated through conjuga-
tion. The trp3-1 mutant also accumulates the Trp
precursor indole-3-glycerol phosphate (Figure 2),
which may be converted to IAA upon the in vitro
base hydrolysis that is used to quantify IAA-
conjugates (Mu¨ller and Weiler 2000b).
In contrast to trp2 and trp3, blocks in earlier steps
of the Trp pathway (Figure 2) do not result in IAA
accumulation. The Arabidopsis trp1-1 anthranilate
phosphoribosyltransferase mutant (Last and Fink
1988) does not accumulate excess IAA (Normanly
and others 1993). Similarly, Arabidopsis plants with
decreased indole-3-glycerol phosphate synthase lev-
els have reduced IAA accumulation (Ouyang and
others 2000). These results imply that a Trp-
independent pathway branches from indole-3-
glycerol phosphate (Figure 2). In maize, Trp syn-
thase ␣-like enzymes synthesize the indole destined
for release as a volatile (Frey and others 2000) or
used as a precursor for certain Trp-independent sec-
ondary metabolites (Frey and others 1997; Melan-
son and others 1997). It has not been reported
whether other plants use Trp synthase ␣-like en-
zymes similarly, but it is intriguing that Arabidopsis
contains a Trp synthase␣-like enzyme (GenBank ac-
cession number T01088) in addition to TSA1, the
Trp synthase ␣defective in trp3 (Radwanski and oth-
ers 1996).
None of the enzymes catalyzing Trp-independent
IAA biosynthesis have been identified. However,
maize seedling extracts efficiently convert indole to
IAA without going through a Trp intermediate (O
¨s-
tin and others 1999), and further analysis of this
Figure 1. A simplified model of IAA homeostasis. IAA
can be synthesized using at least two de novo pathways,
from Trp and from a Trp precursor, perhaps indole. In
addition, IBA -oxidation and conjugate (IAA-X) hydro-
lysis can contribute to the free IAA pool. IAA can be in-
activated by oxidation (OxIAA) or through formation of
non-hydrolyzable conjugates (IAA-Z), which may also be
oxidized (OxIAA-Z). In addition, as hydrolyzable IAA con-
jugates and IBA are likely to be derived from IAA, syn-
thesis of these compounds may contribute to IAA inacti-
vation. Formation and hydrolysis of IBA conjugates (IBA-
X) may also be important for IAA homeostasis. See text for
details.
Inputs to the Active Indole-3-Acetic Acid Pool 199
Figure 2. Proposed pathways of de novo IAA synthesis in plants. De novo IAA biosynthetic pathways initiate from Trp or
Trp precursors. Trp biosynthesis and the P450-catalyzed conversion of Trp to IAOx are chloroplastic (shown in green),
whereas many Trp-dependent IAA biosynthetic enzymes are apparently cytoplasmic (shown in blue). Suggested conver-
sions for which plant genes have not been identified are dashed. Several points of negative regulation are shown by blunt
arrows (red): anthranilate synthase is inhibited by Trp (Belser and others 1971), CYP83B1 is inhibited by tryptamine (Bak
and others 2001), TDC transcription is repressed by IAA (Gooddijn and others 1992; Pasquali and others 1992), and AAO1
levels are elevated in the rty mutant (Sekimoto and others 1998; Seo and others 1998). The IAN pathway (boxed in gray)
may be limited to families in the Capparales order that make indole glucosinolates (Bak and others 1998).
200 B. Bartel et al.
Table 1. Plant Genes Implicated in de novo IAA Biosynthesis
Gene Species Product
Putative
Localization
Loss-of-Function
(LOF) or
Overexpression
(OE) Phenotype Reference
AAO1 Arabidopsis IAA1d oxidase Cytoplasm OE in rty
LOF not reported
(Sekimoto and others
1998; Seo and others
1998
CYP79B2 Arabidopsis Cytochrome
P450
Chloroplast OE: resistant to Trp
analogs
OE: high indole
glucosinolates
OE: high IAN and
IAA-X
(Hull and others 2000)
(Mikkelsen and others
2000)
(Celenza and
Normanly, personal
communication)
CYP79B3 Arabidopsis Cytochrome
P450
Chloroplast not reported (Hull and others 2000)
CYP83B1/
SUR2
Arabidopsis Cytochrome
P450
Cytoplasm LOF: high IAA, IAA1d,
and IAA-Asp; normal
IAN; low indole
glucosinolates
(Delarue and others
1998; Barlier and
others 2000; Bak and
others 2001)
OE: high indole
glucosinolates
(Bak and others 2001)
NIT1 Arabidopsis Nitrilase Not reported LOF: IAN resistant,
normal IAA
(Normanly and others
1997)
NIT2 Arabidopsis Nitrilase Not reported OE: increased
sensitivity to IAN,
normal IAA
(Normanly and others
1997)
SUR1/
RTY/
ALF1/
HLS3
Arabidopsis Amino-
transferase-
like
Not reported LOF: high IAA and
IAA-X
(Boerjan and others
1995; Celenza and
others 1995; King
and others 1995;
Golparaj and others
1996; Lehman and
others 1996)
TDC C. roseus Trp
decarboxylase
Cytoplasm OE (canola): low
indole glucosinolates
OE (tobacco): low
Trp
(Chavadej and others
1994)
(Guillet and others
2000)
TRP2 Arabidopsis Trp
synthase 
Chloroplast LOF: high IAA-X and
IAN, normal free
IAA, low Trp
(Normanly and others
1993; Ouyang and
others 2000)
ORP Zea mays Trp
synthase 
Chloroplast LOF: high IAA-X,
normal free IAA
(Wright and others
1991)
TRP3 Arabidopsis Trp
synthase ␣
Chloroplast LOF: high IAA-X and
IAN, normal free
IAA, low Trp
(Normanly and others
1993; Ouyang and
others 2000
LOF: high IGP, IAN,
and indole
glucosinolates;
normal IAA
(Mu¨ller and Weiler
2000b)
YUCCA,
YUCCA2,
YUCCA3
Arabidopsis FMO-like Cytoplasm OE: high IAA (Zhao and others 2001)
Inputs to the Active Indole-3-Acetic Acid Pool 201
system may yield the relevant intermediates and en-
zymes.
Trp-Dependent IAA Biosynthesis:
Indole-3-Pyruvate
In contrast to the Trp-independent pathway, the
Trp-dependent pathway of IAA biosynthesis is rich
with potential intermediates (Figure 2). Enzymes
that convert Trp to indole-3-pyruvic acid (IPA),
tryptamine, and indole-3-acetaldoxime (IAOx) have
all been proposed to catalyze initial steps in Trp-
dependent IAA biosynthesis. IPA has been identified
in tomato shoots (Cooney and Nonhebel 1991), Ara-
bidopsis seedlings (Tam and Normanly 1998), and
pea root nodules (Badenoch-Jones and others
1984), and accumulates more label than IAA follow-
ing D
2
O feeding of tomato shoots (Cooney and Non-
hebel 1991), consistent with an IAA precursor role.
If IPA is an IAA precursor, as in certain IAA-
synthesizing microorganisms (Koga 1995), a Trp
aminotransferase could convert Trp to IPA and an
IPA decarboxylase could convert IPA to indole-3-
acetaldehyde (IAAld; Figure 2). However, genes for
these enzymes have not been identified in plants.
Homology inspection alone is inadequate to identify
plant versions of the microbial enzymes. For ex-
ample, Arabidopsis encodes four apparent decar-
boxylases similar to pyruvate decarboxylase, but it is
not obvious whether any are IPA decarboxylases, as
the microbial IPA decarboxylases are more similar to
microbial pyruvate decarboxylases than to any plant
protein.
Trp-Dependent IAA Biosynthesis: Renewed
Focus on Tryptamine
Tryptamine was proposed to be an IAA precursor
based on its auxin activity in Avena coleoptile elon-
gation assays (Winter 1966). Tryptamine has been
identified in tomato (Cooney and Nonhebel 1991),
and tryptamine-derived alkaloids have been identi-
fied in a variety of plants. Tryptamine accumulates
more label than IAA in D
2
O-fed tomato shoots, con-
sistent with a precursor role (Cooney and Nonhebel
1991).
Trp is converted to tryptamine by Trp decarbox-
ylase (TDC), a well-studied enzyme necessary for
the biosynthesis of pharmaceutically valuable
monoterpenoid indole alkaloids in Catharanthus ro-
seus (Facchini and others 2000). Tryptamine accu-
mulation may be subject to feedback inhibition (Fig-
ure 2), as C. roseus TDC transcription is down-
regulated by exogenous auxin (Gooddijn and others
1992; Pasquali and others 1992).
The potential importance of the tryptamine path-
way is highlighted by the identification of YUCCA,
an Arabidopsis flavin monooxygenase (FMO)-like
enzyme that apparently catalyzes the conversion of
tryptamine to N-hydroxyl-tryptamine. Plants over-
expressing YUCCA were identified by activation-
tagging (Zhao and others 2001). These plants accu-
mulate free IAA and display high auxin phenotypes,
including long hypocotyls in the light, hookless de-
velopment in the dark, epinastic cotyledons and
leaves, long petioles, auxin-independent growth in
tissue culture, and increased apical dominance
(Zhao and others 2001). YUCCA overexpression also
confers resistance to toxic Trp analogs, suggesting
that the accumulating IAA is Trp-derived (Zhao and
others 2001). Whereas TDC-overexpressing tobacco
or canola accumulate tryptamine without high
auxin phenotypes (Songstad and others 1990; Cha-
vadej and others 1994; Guillet and others 2000), the
finding that YUCCA overexpression in tobacco or
Arabidopsis leads to high auxin phenotypes (Zhao
and others 2001) suggests that YUCCA is rate-
limiting in the tryptamine pathway. This hypothesis
has not been definitively tested, as a family of
YUCCA-like enzymes is present in Arabidopsis, and
disrupting YUCCA, YUCCA2, or both does not confer
any obvious phenotypes (Zhao and others 2001).
Moreover, tryptamine has not yet been identified as
an endogenous compound in Arabidopsis. The isola-
tion of YUCCA accentuates the importance of non-
biased genetic approaches in understanding IAA
synthesis, as the tryptamine to N-hydroxyl-
tryptamine conversion catalyzed by this enzyme was
not uncovered in previous biochemical studies.
Trp-Dependent IAA Biosynthesis:
Indole-3-Acetaldoxime and
Branchpoint Control
A third potential tryptophan-derived IAA precursor,
indole-3-acetaldoxime (IAOx), also serves as an in-
dole glucosinolate precursor (Figure 2). IAOx has
been identified in Chinese cabbage (Ludwig-Mu¨ller
and Hilgenberg 1988), but is not detectable in to-
mato (Cooney and Nonhebel 1991).
Microsomal membranes from a variety of plants,
including cabbage, maize, and pea, can convert Trp
to IAOx (Ludwig-Mu¨ller and Hilgenberg 1988), and
two cytochrome P450 monooxygenases, CYP79B2
and CYP79B3, catalyze this conversion in vitro (Hull
and others 2000; Mikkelsen and others 2000).
CYP79B2 was identified in a yeast screen for Arabi-
dopsis proteins that confer resistance to 5-fluoro-
indole (Hull and others 2000). 5-Fl-indole is toxic
because it is converted to 5-Fl-Trp, which inhibits
202 B. Bartel et al.
anthranilate synthase and is incorporated into pro-
teins. CYP79B2 expression in yeast presumably con-
verts 5-Fl-Trp to 5-Fl-IAOx, which relieves the tox-
icity, and overexpression in Arabidopsis leads to re-
sistance to toxic Trp analogs (Hull and others 2000)
and increased indole glucosinolate (Mikkelsen and
others 2000), indole-3-acetonitrile (IAN), and IAA
conjugate levels (J. Celenza and J. Normanly, per-
sonal communication). Like the trp2 and trp3 mu-
tants, CYP79B2-overexpressing plants have normal
free IAA levels (J. Celenza and J. Normanly, per-
sonal communication), suggesting that the excess
IAA is inactivated through conjugation.
A third cytochrome P450, CYP83B1, converts
IAOx to its N-oxide, funneling carbon into the in-
dole-glucosinolate pathway (Figure 2; Bak and oth-
ers 2001). superroot2 (sur2), a recessive mutant de-
fective in this enzyme, was isolated based on its high
auxin phenotypes (Delarue and others 1998) and
independently in a reverse-genetic screen for cyto-
chrome P450 mutants (Winkler and others 1998).
The mutant accumulates free IAA (Delarue and oth-
ers 1998; Barlier and others 2000), indole-3-
acetaldehyde (IAAld), and IAA-Asp and IAA-Glu
conjugates; but has normal IAN, IAA-Leu, and IAA-
Ala levels (Barlier and others 2000). It is likely that
IAOx accumulates in the sur2 mutant and is con-
verted to IAA, perhaps through IAAld (see below).
The sur2 mutant phenotype can be rescued by
growth at low pH or on low concentrations of IAA
(Barlier and others 2000). Exogenous IAA might in-
hibit de novo IAA biosynthesis, thus reducing the IAA
accumulation caused by the sur2 block. It will be
interesting to determine whether yucca also can be
rescued by low pH or exogenous IAA, as the chlo-
roplast targeting signals on CYP79B2/3 and probable
cytoplasmic localization of YUCCA suggest that yucca
and sur2 might accumulate IAOx in different com-
partments (Figure 2; Table 1).
TDC overexpression in Brassica napus leads to
tryptamine accumulation and dramatically reduces
indole glucosinolate content (Chavadej and others
1994). The observation that tryptamine inhibits
CYP83B1/SUR2 in vitro (Bak and others 2001) could
explain this decrease. Regulation of branchpoint en-
zymes, such as CYP83B1, may be necessary to en-
sure adequate IAA production, as Trp-derived sec-
ondary metabolites, such as indole glucosinolates
and indole alkaloids, compete with IAA for precur-
sors.
Trp-Dependent IAA Biosynthesis: Pathways
Converge at Indole-3-Acetaldehyde
Indole-3-acetaldehyde (IAAld) is a possible conver-
gence point for the proposed Trp-dependent IAA
biosynthetic pathways discussed above (Figure 2).
The Trp →IPA →IAAld →IAA pathway has been
thoroughly characterized in microorganisms (Patten
and Glick 1996) and is considered a significant path-
way in plants (Nonhebel and others 1993). IAAld
could also be derived from IAOx (Rajagopal and
Larsen 1972; Rajagopal and others 1991); a soluble
protein activity from Chinese cabbage converts
IAOx to IAA, apparently through an IAAld interme-
diate (Helmlinger and others 1987). Both CYP79B2
overexpressors and cyp83b1/sur2 mutants are ex-
pected to accumulate IAOx, although this has not
been directly demonstrated. sur2 accumulates IAAld
(Barlier and others 2000), which is probably derived
from IAOx. YUCCA overexpression probably in-
creases conversion of tryptamine to N-hydroxyl-
tryptamine (Zhao and others 2001), which could be
dehydrogenated to IAOx or dehydrogenated and
hydrolyzed to IAAld in a third pathway of IAAld
production. However, enzymes that catalyze IAAld
formation from N-hydroxyl-tryptamine or IAOx
have not been identified.
The conversion of IAAld into IAA is catalyzed by
IAAld oxidases, which have been characterized from
several plants (Bower and others 1978; Sekimoto
and others 1997; Seo and others 1998). An Arabi-
dopsis aldehyde oxidase isozyme (AAO1) specific for
IAAld accumulates in the rooty (rty) mutant (Seo and
others 1998). The rty mutant (King and others
1995), also isolated as sur1 (Boerjan and others
1995), hookless3 (Lehman and others 1996), and alf1
(Celenza and others1995), has high auxin pheno-
types. This recessive mutant, defective in a tyrosine
aminotransferase-like enzyme (Golparaj and others
1996), accumulates free IAA and IAA conjugates
(Boerjan and others 1995; King and others 1995;
Lehman and others 1996). By analogy to CYP83B1/
SUR2, RTY might normally act to divert an IAA pre-
cursor to a secondary metabolite (Celenza 2001). It
has not been reported if other indolic metabolites
accumulate in rooty or whether the excess IAA is
produced from Trp or independently of Trp. The ob-
servation that AAO1 transcription and enzymatic ac-
tivity are elevated in the rty mutant (Sekimoto and
others 1998; Seo and others 1998) indicates that
RTY acts genetically as a negative regulator of AAO1
expression (Figure 2). It will be interesting to deter-
mine the level at which this regulation occurs, and
whether AAO1 disruption results in IAA deficiency
or rescue of the rty high auxin phenotypes.
Trp-Dependent IAA Biosynthesis: A Limited
Role for Indole-3-Acetonitrile
The importance and ubiquity of IAN in IAA biosyn-
thesis remains uncertain. IAOx is converted to IAN
Inputs to the Active Indole-3-Acetic Acid Pool 203
in Chinese cabbage and maize tissues (Helmlinger
and others 1985), and IAN can be hydrolyzed to IAA
by nitrilases encoded by the Arabidopsis NIT genes
(Bartling and others 1992; Bartel and Fink 1994;
Bartling and others 1994). Extracts from the Cru-
ciferae, Graminae, and Musaceae families can hy-
drolyze IAN to IAA, suggesting the existence of a
nitrilase pathway in these families (Thimann and
Mahadevan 1964). Arabidopsis NIT1, NIT2, and NIT3
enzymes have apparent K
m
values for IAN that are
an order of magnitude greater than those for their
preferred substrate, 3-phenyl-propionate (Vorwerk
and others 2001), so IAN may not be the most rel-
evant substrate. However, NIT1 and NIT2 can hy-
drolyze IAN in vivo (Schmidt and others 1996; Nor-
manly and others 1997), and an Arabidopsis enzy-
matic complex with nitrilase immunoreactivity
converts Trp to IAA in vitro (Mu¨ller and Weiler
2000a), making it possible that substrate channeling
in vivo compensates for the high K
m
.
Rather than hydrolyzing IAN made directly from
IAOx, nitrilases may act on IAN formed following
hydrolysis of indole glucosinolates by myrosinases
(Figure 2). Plants with excess indole glucosinolates,
including CYP79B2 overexpressors (Mikkelsen and
others 2000), the trp3 mutant (Mu¨ller and Weiler
2000b), and the bus mutant (Reintanz and others
2001), also accumulate IAN (Celenza and Nor-
manly, personal communication; Normanly and
others 1993; Mu¨ller and Weiler 2000b; Reintanz and
others 2001). The trp3 mutant has higher nitrilase
immunoreactivity than wild type (Mu¨ller and
Weiler 2000b), suggesting that increased nitrilase
mediated hydrolysis of indole glucosinolate-derived
IAN might supply the extra IAA in the mutant. The
NIT2 gene is induced by bacterial pathogen infiltra-
tion of leaves (Bartel and Fink 1994) and by Plasmo-
diophora infection of roots (Grsic-Rausch and others
2000), situations in which myrosinases might hy-
drolyze glucosinolates. The observations that IAN
levels are normal in the sur2 mutant (Barlier and
others 2000), and that the IAN-resistant nit1 mutant
(Normanly and others 1997) fails to rescue sur2 de-
fects (Bak and others 2001), also suggest that nit-
rilases act downstream of glucosinolates. Determi-
nation of the importance of nitrilases in Arabidopsis
IAA biosynthesis awaits identification of an enzyme
that converts IAOx to IAN and isolation of a triple
nit1 nit2 nit3 mutant.
Regulation of De Novo IAA Synthesis
Trp-dependent and Trp-independent pathways are
developmentally regulated. Carrot cells switch from
Trp-dependent to Trp-independent IAA biosynthesis
when callus cultures are induced to form embryos
(Michalczuk and others 1992). In zygotic carrot em-
bryogenesis, an 80-fold surge in free IAA concentra-
tion is derived from Trp, but basal IAA biosynthesis
is Trp-independent (Ribnicky and others 2001).
During Arabidopsis leaf senescence, IAN levels de-
crease and IAA levels increase, which is correlated
with a strong induction of NIT2 expression, implying
that the increased IAA might be IAN-derived
(Quirino and others 1999). Scots pine seedlings be-
gin synthesizing IAA from Trp approximately 3 days
after sowing, and induce a Trp-independent path-
way after 6 days (Ljung and others 2001). Develop-
mental differences can be recapitulated in vitro; ex-
tracts from maize coleoptile tips (Koshiba and Mat-
suyama 1993; Koshiba and others 1995) or
endosperm (Ilic’and others 1999; Glawischnig and
others 2000) synthesize IAA from Trp, whereas
seedling extracts use a Trp-independent pathway
(O
¨stin and others 1999). Tobacco protoplasts from
vegetative shoot apices use both Trp-dependent and
Trp-independent IAA biosynthetic pathways (Sitbon
and others 2000), suggesting that both sources may
be important later in development.
An emerging theme in IAA biosynthesis is that
plants use Trp-independent pathways for IAA main-
tenance, but switch to Trp-dependent pathways
when high IAA levels are required (Ribnicky and
others 2001; Sztein and others 2001). Intact Arabi-
dopsis seedlings convert more [
15
N]-anthranilate
into IAA than into Trp, and do not efficiently con-
vert [
2
H
5
]-Trp into IAA (Normanly and others
1993), indicating that Trp-independent biosynthesis
is important during normal growth. In contrast, Ara-
bidopsis shoot or root explants efficiently convert
[
2
H
5
]-Trp to IAA (Mu¨ller and others 1998b; Mu¨ller
and Weiler 2000b), suggesting that a Trp-dependent
pathway is wound-induced. Similarly, Trp-
independent IAA biosynthesis predominates in
6-day-old bean seedlings, but a Trp-dependent path-
way is induced by wounding (Sztein and others
2001).
IAA CONJUGATES:STORAGE AND INACTIVATION
In addition to de novo synthesis, hydrolysis of IAA
conjugates is an important source of free IAA in
higher plants (Figure 1). Whereas most IAA is con-
jugated in vascular plants (Slovin and others 1999;
Sztein and others 1999), conjugation and hydrolysis
are apparently less important in nonvascular plants
such as liverworts, which rely mostly on IAA bio-
synthesis and degradation to modulate IAA levels
(Sztein and others 1995; Sztein and others 1999;
Sztein and others 2000). IAA can be conjugated ei-
204 B. Bartel et al.
ther to sugars via ester linkages or to amino acids
and peptides via amide linkages, and these conju-
gates may act in IAA storage or transport, protection
of IAA against peroxidative degradation, and com-
partmentalization or detoxification of excess IAA
(Cohen and Bandurski 1982). Despite the complex-
ity of conjugate metabolism, which differs not only
between species but also between developmental
stages of the same species, patterns of conjugate
functions are emerging. Conjugates that are biologi-
cally active, present endogenously, and hydrolyzed
in planta are likely to be important IAA stores,
whereas biologically inactive conjugates are prob-
ably precursors for IAA degradation.
Several approaches are elucidating conjugate
roles in auxin metabolism. IAA conjugates present
in unstressed plants and those formed in response to
high exogenous auxin are being identified and
quantified (Slovin and others 1999), the effects of
conjugates in bioassays are being analyzed (Feung
and others 1977; Hangarter and others 1980; Han-
garter and Good 1981; Bialek and others 1983), and
mutant screens are identifying loci important in
conjugate perception and metabolism (Bartel and
Fink 1995; Campanella and others 1996; Barratt and
others 1999; Davies and others 1999; Lasswell and
others 2000).
IAA Conjugates Identified in Plants
Conjugate synthesis pathways in higher plants ap-
pear complex and diverged. Many identified conju-
gates have only been found in a single plant species,
which may result from incomplete analysis or bio-
logical differences. Because IAA-ester and IAA-
amide conjugates are hydrolyzed under different
conditions (Bialek and Cohen 1986; Baldi and oth-
ers 1989), total ester- and amide-linked conjugates
can be quantified. In general, monocots appear to
accumulate ester conjugates, whereas dicots accu-
mulate mostly amide conjugates (Cohen and
Bandurski 1982; Slovin and others 1999).
In maize, soybean, and bean seeds, the identified
and quantified IAA conjugates account for most of
the stored IAA. Maize kernels contain primarily es-
ter-linked conjugates, including IAA-myo-inositol,
IAA-myo-inositol glycosides, IAA-glucose, and a
large cellulosic glucan conjugate (Cohen and
Bandurski 1982). In soybean seeds, IAA-Asp and
IAA-Glu are the predominant IAA conjugates (Co-
hen 1982; Epstein and others 1986). Bean seeds ap-
parently lack amino acid conjugates, and the IAA is
instead conjugated to several polypeptides ranging
in size from 3–60 kDa (Bialek and Cohen 1986;
Walz and others 2001). One of these IAA-modified
bean proteins, IAP1, is encoded by a homolog of a
soybean late seed maturation protein (Walz and
others 2001), suggesting a new role for seed storage
proteins in phytohormone action.
In other species, quantification of identified con-
jugates does not account for all of the IAA released
following base hydrolysis, so additional conjugates
may remain to be identified. Oats contain an IAA-
glycoprotein ester conjugate (Percival and Bandur-
ski 1976), and rice contains IAA-myo-inositol (Hall
1980). Arabidopsis conjugates include IAA-Ala, IAA-
Leu (Barlier and others 2000), IAA-Asp, IAA-Glu,
IAA-glucose (Tam and others 2000), and an IAA-
peptide (Walz and others 2001).
IAA conjugates that accumulate following expo-
sure to exogenous IAA often differ from those ap-
parently used for IAA storage (Figure 1). In response
to high auxin levels from IAA application or iaaM
transgene expression (Sitbon and others 1993), a
distinct pathway is activated to detoxify the excess
IAA. Arabidopsis inactivates low levels of IAA by oxi-
dation (O
¨stin and others 1998), and forms conju-
gates to Asp and Glu (O
¨stin and others 1998) or Asp,
Glu, and glucose (Barratt and others 1999) in re-
sponse to high exogenous IAA. Similarly, IAA ap-
plied to the epicarp of orange fruit is primarily inac-
tivated by oxidation and by conjugation to Asp
(Chamarro and others 2001). This high-auxin con-
jugation pathway is evolutionarily conserved, as a
variety of vascular plants challenged with IAA ap-
parently accumulate IAA-Asp, IAA-Glu, or IAA-
glucose (Sztein and others 1995). IAA-Asp and IAA-
Glu can be further oxidized to oxIAA-conjugates
(O
¨stin and others 1992; O
¨stin and others 1998;
Chamarro and others 2001), which permanently in-
activates the hormone. Additional catabolites, such
as N-linked products of the indole ring, have also
been identified (O
¨stin and others 1995). Asp and
Glu conjugates of the synthetic auxin 2,4-
dichlorophenoxyacetic acid (2,4-D) are apparently
sequestered in the vacuole (Davidonis and others
1982), supporting the hypothesis that these conju-
gates are catabolites.
Activity of IAA Conjugates
Certain endogenous IAA conjugates elicit auxin re-
sponses in bioassays, and conjugate activity often
correlates with hydrolysis. For example, IAA-Ala is
present in Arabidopsis (Barlier and others 2000) and
Picea abies (O
¨stin and others 1992) and has auxin
activity in numerous bioassays (Hangarter and oth-
ers 1980; Hangarter and Good 1981; Bialek and
others 1983; Magnus and others 1992b; Davies
and others 1999). Similarly, IAA-Leu is an endog-
Inputs to the Active Indole-3-Acetic Acid Pool 205
enous Arabidopsis conjugate (Barlier and others
2000) and has activity in some bioassays (Feung and
others 1977; Hangarter and others 1980; Bartel and
Fink 1995). Bioactive conjugates often can be hy-
drolyzed in plants. IAA-Ala is hydrolyzed by bean
stem sections (Bialek and others 1983), Arabidopsis
extracts (LeClere and Bartel, unpublished), and the
Arabidopsis IAR3 amidohydrolase (Davies and others
1999). IAA-Leu is hydrolyzed by the Arabidopsis
ILR1 amidohydrolase (Bartel and Fink 1995), and
IAA-Ala and IAA-Leu are hydrolyzed in Chinese
cabbage extracts (Ludwig-Mu¨ller and others 1996).
Hydrolysis rates do not always fully account for
conjugate bioactivity, however. For example, IAA-
Ala has unique effects on tomato tissue cultures;
pretreatment of cultures with IAA-Ala can inhibit
IAA-induced shoot growth and root initiation, sug-
gesting a possible competition between IAA and
some conjugates for a common binding site(s) (Mag-
nus and others 1992a).
Other conjugates found in plants do not consis-
tently elicit auxin effects in bioassays. Although
IAA-Asp promotes Avena coleoptile (Feung and oth-
ers 1977) and soybean hypocotyl (Cohen and Baldi
1983) elongation, it has only low auxin activity in
other systems, including bean stems (Bialek and
others 1983) and Arabidopsis roots (Campanella and
others 1996). IAA-Glu has auxin activity and is hy-
drolyzed in bean stem sections (Bialek and others
1983). However, neither IAA-Asp nor IAA-Glu are
hydrolyzed by intact Lemna gibba plants (Slovin
1997), extracts from uninfected Chinese cabbage
(Ludwig-Mu¨ller and others 1996), or Arabidopsis
seedlings (O
¨stin and others 1998).
Uptake and transport differences may contribute
to the activities of IAA conjugates in bioassays. In
Lemna gibba, IAA-Glu is taken up faster than IAA-
Ala, whereas IAA-myo-inositol is taken up more
slowly than either amide-linked conjugate (Slovin
1997). Conjugate uptake differences may reflect dif-
ferences in influx carrier binding affinities, conju-
gate compartmentalization, or metabolism inside
the cell. The Arabidopsis aux1 auxin influx carrier
mutant (Bennett and others 1996) is resistant to
IAA-Ala and IAA-Leu, suggesting that at least some
conjugates enter cells similarly to free IAA (our un-
published data). However, it is not known how con-
jugates exit cells. IAA-Ala and IAA-Gly are appar-
ently not subject to polar auxin transport in pea
stem sections, but rather disperse through diffusion
(Hangarter and others 1980), suggesting that conju-
gates are not substrates of the auxin efflux carrier. In
contrast, IAA-myo-inositol is transported 400 times
faster than IAA from maize endosperm to shoots
(Nowacki and Bandurski 1980). One possibility is
that vascular transport contributes to the high rate
of ester conjugate transport (Ludwig-Mu¨ller and
others 1996). It is intriguing to speculate that sugar-
linked conjugates may be directed to the vascular
system for rapid movement through the plant,
whereas amide-linked conjugates provide more lo-
calized responses. Analysis of different auxin trans-
port mutants on various conjugates may allow fur-
ther dissection of conjugate roles in IAA trafficking.
Developmental Regulation of Conjugation
and Conjugate Hydrolysis
IAA conjugates stored in seeds can provide free IAA
to seedlings. IAA-ester hydrolysis supplies free IAA
to germinating maize seedlings (Epstein and others
1980), and amide conjugates are rapidly hydrolyzed
after bean seed imbibition (Bialek and Cohen 1992).
Conjugate hydrolysis in Scots pine peaks just prior
to radicle emergence, and the disappearance of es-
ter-linked conjugates correlates with an increase in
free IAA (Ljung and others 2001). Interestingly,
amide-linked IAA is low in these seeds, and IAA-Asp
conjugation and IAA catabolism are initiated con-
currently with de novo synthesis, which occurs after
seed-stored ester conjugates are hydrolyzed (Ljung
and others 2001).
The catabolic conjugation system is probably
present during normal growth because IAA-Asp,
IAA-Glu, and IAA-glucose are present at low levels
in Arabidopsis seedlings (Tam and others 2000). In
response to elevated IAA levels, storage conjugation
pathways may be down-regulated as catabolic path-
ways are up-regulated. For example, the sur2 mu-
tant, which accumulates free IAA (see above), has a
reduced ability to make IAA-Leu (Barlier and others
2000), a putative Arabidopsis IAA storage form (see
above). However, sur2 plants do accumulate IAA-
Asp (Barlier and others 2000), an intermediate in
permanent IAA inactivation (Normanly 1997;
Slovin and others 1999). It appears that plants use
different conjugates to detoxify excess IAA and to
store IAA, suggesting that the conjugated moiety
may dictate the fate of the attached IAA for storage,
transport, or degradation (Cohen and Bandurski
1982).
GENETIC ANALYSIS OF IAA
CONJUGATE FUNCTIONS
The isolation and characterization of IAA-conjugate
resistant mutants that remain sensitive to IAA pro-
vide an additional tool to examine conjugate func-
tions. A number of genes are implicated in IAA con-
jugate responses (Table 2), and several Arabidopsis
206 B. Bartel et al.
IAA-amino acid conjugate resistant mutants have
been identified: ilr1 (Bartel and Fink 1995) and ilr2
(Magidin and Bartel, unpublished) are IAA-Leu and
IAA-Phe resistant; iar3 (Davies and others 1999) and
iar4 (LeClere and Bartel, unpublished) are IAA-Ala
resistant; icr1 and icr2 (IAA conjugate resistant) are
resistant to IAA-Ala, IAA-Phe, and IAA-Gly (Cam-
panella and others 1996); and iar1 is resistant to
several IAA conjugates, including IAA-Ala, IAA-
Leu, and IAA-Phe (Lasswell and others 2000).
If conjugates are IAA precursors, then conjugate-
resistant mutants may be defective in conjugate hy-
drolysis or uptake. If conjugates have additional
roles, however, it may be possible to genetically
separate conjugate functions from those of IAA.
ILR1 and IAR3 are amidohydrolases that cleave
IAA-Leu and IAA-Phe (Bartel and Fink 1995) or
IAA-Ala (Davies and others 1999), respectively.
IAR1 (Lasswell and others 2000) is a multi-pass
transmembrane protein with weak similarity to the
ZIP family of metal transporters (Guerinot 2000).
The biochemical activity of IAR1 is unknown, but it
may transport a co-factor to facilitate amidohydro-
lase activity (Lasswell and others 2000). To date, the
analysis of IAA conjugate-resistant mutants suggests
that IAA conjugates with auxin activity act via their
hydrolysis to free IAA. Although the genes defective
in icr1, icr2, ilr2, and iar4 have not been reported, it
will be interesting to learn whether these mutants
are also defective in some aspect of conjugate hy-
drolysis or if they reveal hydrolysis-independent
conjugate functions.
INDOLE-3-BUTYRICACID:ANOTHER IAA
STORAGE FORM
Indole-3-butyric acid (IBA) was long regarded as a
synthetic auxin, but recent evidence demonstrates
that IBA is present in numerous plant species. IBA is
used widely in horticulture because of its efficacy in
inducing lateral and adventitious roots on cuttings.
Most IBA research has focused on identifying opti-
mal conditions for secondary root development for
the vegetative propagation of commercially impor-
tant trees and ornamental plants.
Examination of the molecular mechanisms of IBA
action indicates that IBA -oxidation provides a
source of free IAA in addition to de novo biosynthesis
and conjugate hydrolysis (Figure 1). Some evidence
suggests that IBA acts as an auxin on its own, inde-
pendently of IAA. However, biochemical studies in
numerous plants and genetic studies of Arabidopsis
IBA-response mutants indicate that IBA acts primar-
Table 2. Plant Genes Implicated in IAA Conjugate Metabolism
Gene Species Product
Putative
Localization Mutant Phenotype Reference
ILR1 Arabidopsis IAA-amino acid
amidohydrolase
ER lumen IAA-Leu resistant (Bartel and Fink 1995)
IAR3 Arabidopsis IAA-amino acid
amidohydrolase
ER lumen IAA-Ala resistant (Davies and others 1999)
ILL1 Arabidopsis IAA-amino acid
amidohydrolase
ER lumen Not reported (Bartel and Fink 1995)
ILL2 Arabidopsis IAA-amino acid
amidohydrolase
ER lumen Not reported (Bartel and Fink 1995)
IAR1 Arabidopsis Transporter? Membrane IAA-amino acid
conjugate resistant
(Lasswell and others 2000)
ILR2 Arabidopsis Not reported Not reported IAA-Leu resistant (Magidin and Bartel,
unpublished)
IAR4 Arabidopsis Not reported Not reported IAA-Ala resistant (LeClere and Bartel,
unpublished)
ICR1 Arabidopsis Not reported Not reported IAA-Phe resistant (Campanella and others 1996)
ICR2 Arabidopsis Not reported Not reported IAA-Phe resistant (Campanella and others 1996)
iaglu Zea mays IAA glucosyl-
transferase
Not reported Not reported (Szerszen and others 1994)
UGT84B1 Arabidopsis IAA glucosyl-
transferase
Not reported Not reported (Jackson and others 2001)
IAP1 Phaseolus
vulgaris
Seed protein
modified by
IAA
Not reported Not reported (Walz and others 2001)
Inputs to the Active Indole-3-Acetic Acid Pool 207
ily via its conversion to IAA, which occurs in a
mechanism similar to peroxisomal fatty acid -oxi-
dation.
IBA is an Endogenous Auxin
IBA is found in a variety of plants and tissues, in-
cluding Arabidopsis seedlings (Ludwig-Mu¨ller and
Epstein 1993; Ludwig-Mu¨ller and others 1993); car-
rot roots (Epstein and others 1991); tobacco leaves
(Sutter and Cohen 1992); maize kernels, leaves, and
roots (Epstein and others 1989; Ludwig-Mu¨ller and
Epstein 1991); and pea roots, epicotyls, and shoots
(Schneider and others 1985; Nordstro¨m and others
1991). A few studies have quantified IAA and IBA
levels in plant tissues. For example, tobacco leaves
contain 9 ng/g free IBA compared to 26 ng/g free
IAA (Sutter and Cohen 1992) and there is slightly
less total IBA than IAA in Arabidopsis (Ludwig-
Mu¨ller and others 1993). However, these compari-
sons are complicated by variations in auxin concen-
trations under different growth conditions and be-
tween species (Ludwig-Mu¨ller and Epstein 1993).
For instance, potato peelings contain elevated IBA
levels at the start of sprouting (Blommaert 1954),
and tumor-prone tobacco genotypes have higher
IBA levels than other lines (Bayer 1969). In addi-
tion, auxin concentration varies during develop-
ment. For example, free IAA peaks in 3-day-old Ara-
bidopsis seedlings, whereas free IBA peaks between 5
and 9 days (Ludwig-Mu¨ller and Epstein 1993; Lud-
wig-Mu¨ller and others 1993).
IBA has auxin effects in many bioassays and ini-
tiates rooting significantly better than IAA in nu-
merous plant species (Hartmann and others 1990),
including adventitious root formation in pea (Nord-
stro¨m and others 1991), mung bean (Wiesman and
others 1988, 1989), and apple cuttings (Alvarez and
others 1989; van der Krieken and others 1992; van
der Krieken and others 1993). In addition, exog-
enous IBA stimulates stem elongation similarly to
IAA in intact pea plants (Yang and Davies 1999).
IBA also has auxin effects on Arabidopsis seedlings:
IBA inhibits root elongation (Zolman and others
2000) and induces lateral (Zolman and others 2000)
and adventitious (King and Stimart 1998) root for-
mation. Whereas IBA and IAA responses are quali-
tatively similar, effective IBA concentrations are
generally higher than those of IAA.
The increased ability of IBA versus IAA to initiate
lateral and adventitious roots may result from dif-
ferences in receptor binding, compartmentalization,
stability, tissue sensitivity, uptake, transport, or con-
jugation between the two auxins (Epstein and Lud-
wig-Mu¨ller 1993; de Klerk and others 1999; Lud-
wig-Mu¨ller 2000). IBA is more stable than IAA, both
in vivo and in solution (Robbins and others 1988;
Nissen and Sutter 1990; Nordstro¨m and others
1991). In addition, plants form inactive oxidation
products following exposure to exogenous IAA
(Normanly 1997; Slovin and others 1999); after this
irreversible oxidation, IAA levels can drop below the
optimal concentration for lateral root initiation at
the time when auxin is required. IBA may be better
at initiating lateral roots because it is not readily
oxidized (Epstein and Ludwig-Mu¨ller 1993; de Klerk
and others 1997; de Klerk and others 1999).
IBA is taken up and transported more slowly than
IAA in a variety of systems, perhaps leaving more
hormone at the plant base where it can affect root
initiation (Epstein and Ludwig-Mu¨ller 1993; Lud-
wig-Mu¨ller 2000). The aux1 mutant, defective in an
auxin influx carrier (Bennett and others 1996), is
resistant to the inhibitory effects of IBA (Zolman and
others 2000), suggesting that IBA enters cells similar
to IAA and IAA-conjugates (see above). In contrast,
the eir1/agr1/pin2 auxin efflux carrier mutant (Chen
and others 1998; Luschnig and others 1998; Mu¨ller
and others 1998a; Utsuno and others 1998) re-
sponds differently to IBA and IAA, suggesting that
IBA is not an EIR1 substrate (Poupart and Waddell
2000; Zolman and others 2000).
Like IAA (see above), much of the IBA in plants is
conjugated to other moieties through amide- and
ester-linkages (Epstein and Ludwig-Mu¨ller 1993;
Ludwig-Mu¨ller 2000). Although the IBA and IAA
conjugate levels are similar in tobacco leaves (Sutter
and Cohen 1992), differences in metabolism may
contribute to the increased efficacy of IBA in lateral
root induction (Wiesman and others 1988; Epstein
and Ludwig-Mu¨ller 1993; van der Krieken and oth-
ers 1997; Ludwig-Mu¨ller 2000). Unlike IAA, which
is predominantly amide linked in dicots, IBA is
largely ester linked (Ludwig-Mu¨ller and others
1993), and IBA conjugates may be more easily hy-
drolyzed (Epstein and Ludwig-Mu¨ller 1993) or dif-
ferentially transported than IAA conjugates (see
above). In addition, it has been reported that certain
IBA conjugates, including IBA-Asp, are themselves
active in secondary root initiation (Wiesman and
others 1989).
IBA is Converted to IAA
One explanation for the auxin activity of IBA is that
IBA is a “slow-release”form of IAA (van der Krieken
and others 1997), similar to certain IAA conjugates
(see above). IBA may supply plants with a continu-
ous IAA source, providing auxin when it is required
for root initiation. Even-chain length derivatives of
208 B. Bartel et al.
the synthetic auxin 2,4-D, including 2,4-
dichlorophenoxybutyric acid (2,4-DB), have auxin
activity in wheat cylinder tests, pea curvature assays,
and tomato leaf epinasty tests (Wain and Wightman
1954). Similarly, even chain-length derivatives of
IAA, including IBA, are active in auxin bioassays
(Fawcett and others 1960). Wheat and pea extracts
shorten these derivatives two carbons at a time
(Fawcett and others 1960), suggesting that IBA and
2,4-DB are converted to IAA and 2,4-D similarly to
fatty acid -oxidation, which shortens fatty acids in
two carbon increments.
Radiolabeled IBA is converted to IAA by a variety
of plants, including apple (van der Krieken and oth-
ers 1992), olive (Epstein and Lavee 1984), grape
(Epstein and Lavee 1984), pear (Baraldi and others
1993), pea (Nordstro¨m and others 1991), bean
(Wiesman and others 1988), and maize (Ludwig-
Mu¨ller and Epstein 1991). In pea shoots, IBA treat-
ment causes a transient tenfold increase in IAA lev-
els during the first day of exposure, but IAA levels
return to normal by day 4 (Nordstro¨m and others
1991). After apple shoots are incubated with [
3
H]-
IBA for 2 days, 4% of the label is in free IBA and 1%
is in free IAA, which is a higher proportion of la-
beled free IAA than after treatment with [
3
H]-IAA
(van der Krieken and others 1992; van der Krieken
and others 1993), consistent with a “slow-release”
function for IBA. However, when shoots are treated
with IBA or IAA concentrations that produce similar
free IAA levels, IBA induces more secondary roots,
suggesting that IBA also may act on its own or syn-
ergistically with IAA (van der Krieken and others
1992; van der Krieken and others 1993).
IBA to IAA Conversion is Peroxisomal
Our understanding of IBA action is being clarified by
the isolation and characterization of Arabidopsis mu-
tants with IBA-resistant root elongation that re-
spond normally to IAA (Poupart and Waddell 2000;
Zolman and others 2000; Zolman and others 2001a;
Zolman and others 2001b). As discussed below, de-
fects in -oxidation can lead to IBA resistance, sup-
porting the hypothesis that IBA is converted to IAA
in a mechanism that parallels fatty acid -oxidation
(Figure 3). In plants, -oxidation is exclusively per-
oxisomal (Gerhardt 1992; Kindl 1993). Peroxisomes
are small organelles that contain no DNA; proteins
acting in the peroxisomal matrix are imported from
the cytoplasm post-translationally. At least 20 pro-
teins are required for peroxisomal import, biogen-
esis, and function (Olsen 1998; Subramani 1998;
Tabak and others 1999). It is likely that most of these
proteins will be required for IBA to IAA conversion.
Long-chain fatty acids stored in seeds undergo
peroxisomal -oxidation to provide energy during
Arabidopsis germination. Mutants defective in fatty
acid utilization therefore do not develop after ger-
mination unless provided with sucrose (Hayashi and
others 1998). Analysis of sucrose-dependence and
auxin-related phenotypes, including lateral root ini-
tiation and responses to synthetic auxins and auxin
transport inhibitors, allows classification of IBA-
response mutants into four groups (Zolman and oth-
Figure 3. The conversion of IBA to IAA. IBA is likely to
be converted to IAA-CoA in peroxisomes in a process that
parallels fatty acid -oxidation to acetyl-CoA. The IBA
analog 2,4-DB is probably converted to the synthetic
auxin 2,4-D by the same enzymes. Mutations in genes
encoding the ABC-transporter-like protein PXA1 (Zolman
and others 2001b), the medium chain acyl-CoA oxidase
ACX3 (Eastmond and others 2000), the multifunctional
protein AIM1 (Richmond and Bleecker 1999), and the
thiolase PED1 (Hayashi and others 1998) each confer re-
duced sensitivity to IBA and 2,4-DB (Zolman and others
2000; Zolman and others 2001b). Moreover, mutations in
PEX5 and PEX14, which are necessary for the import of
most matrix enzymes, including AIM1, also confer IBA
and/or 2,4-DB resistance (Hayashi and others 2000; Zol-
man and others 2000). The acyl-CoA synthase that esteri-
fies IBA to IBA-CoA, the hydrolase that releases the free
acid, and the transporter that effluxes IAA from the per-
oxisome remain to be identified. The acyl-CoA hydrolase
is shown in the peroxisome, but alternatively could be
cytoplasmic.
Inputs to the Active Indole-3-Acetic Acid Pool 209
ers 2000). Some mutants are IBA resistant in both
root elongation and lateral root initiation, and are
sucrose-dependent during early seedling develop-
ment, indicating possible defects in enzymes re-
quired for the peroxisomal -oxidation of IBA and
long-chain fatty acids. A second subset of mutants
are resistant to IBA in root elongation and have
slight peroxisomal defects, but induce lateral roots
normally in response to IBA, perhaps reflecting de-
fects in tissue-specific -oxidation isozymes. Other
mutants are IBA resistant in root elongation and
lateral root initiation, but lack obvious peroxisomal
defects; these may be defective in isozymes specific
to short-chain substrates and IBA. Finally, addi-
tional IBA-response mutants, including rib1 (resis-
tant to IBA; Poupart and Waddell 2000), respond
normally to IAA but have altered responses to syn-
thetic auxins and auxin transport inhibitors, and
thus may function in an aspect of IBA action, such as
transport, that is independent of its conversion to
IAA.
Additional Arabidopsis peroxisomal -oxidation
mutants have been isolated using resistance to the
IBA analog 2,4-DB. 2,4-DB is converted to 2,4-D
similarly to IBA -oxidation (Wain and Wightman
1954; Hayashi and others 1998). 2,4-DB resistant
mutants that also have defects in growth without
sucrose include ped1, ped2, ped3 (Hayashi and others
1998), acx3 (Eastmond and others 2000), aim1
(Richmond and Bleecker 1999), and dbr5 (Lange
and Graham 2000). acx3, aim1, and ped1 mutants are
also IBA resistant (Zolman and others 2000; Zolman
and Bartel, unpublished data), suggesting that 2,4-
DB resistant mutants will be IBA resistant as well,
and that the defects in these mutants will affect the
conversion of IBA to IAA in addition to the conver-
sion of 2,4-DB to 2,4-D.
The identification of the genes defective in several
IBA-response mutants has confirmed the impor-
tance of peroxisomal -oxidation in IBA action (Fig-
ure 3). One mutant is defective in PEX5 (Zolman
and others 2000), a receptor that binds and trans-
ports proteins from the cytoplasm into the peroxi-
somal matrix (Olsen 1998; Subramani 1998). Simi-
larly, the ped2 (peroxisome defective) mutant is de-
fective in PEX14, a peroxisomal membrane protein
essential for the import of peroxisomal matrix pro-
teins (Hayashi and others 2000). It is likely that pex5
and ped2 have defects in importing peroxisomal ma-
trix proteins required for -oxidation, which slow
-oxidation and cause IBA-resistant, sucrose-
dependent phenotypes. A third IBA response mu-
tant is defective in PXA1 (Zolman and others 2001b).
PXA1 is approximately 30% identical to human and
yeast ATP-binding cassette transporters implicated
in importing or activating long-chain fatty acids for
-oxidation (Dubois-Dalcq and others 1999; Hol-
land and Blight 1999). Because pxa1 is resistant to
IBA and 2,4-DB and displays sucrose-dependent
seedling development, it is likely that PXA1 imports
IBA, 2,4-DB, and fatty acids into peroxisomes.
In contrast to peroxisome biogenesis defects,
other IBA-response mutants have defects in -oxi-
dation enzymes (Figure 3). One IBA-response mu-
tant is an acx3 allele (Zolman and Bartel, unpub-
lished). acx3 is defective in a gene encoding an acyl-
CoA oxidase catalyzing the second step of
-oxidation (Eastmond and others 2000). The weak
sucrose-dependence of this mutant may reflect the
specificity of ACX3 for medium-chain fatty acids
(Eastmond and others 2000). The aim1 (abnormal
inflorescence meristem) mutant contains a mutation
in a multifunctional protein acting in fatty acid
-oxidation (Richmond and Bleecker 1999), and
ped1 is defective in a thiolase that acts in the final
step of -oxidation (Hayashi and others 1998). Be-
cause Arabidopsis IBA-response mutants are defec-
tive in fatty acid -oxidation enzymes and peroxi-
some biogenesis proteins, it is likely that IBA acts as
an IAA precursor, and that IBA is converted to IAA
in a peroxisomal process that uses at least a subset of
fatty acid -oxidation enzymes (Figure 3). The iden-
tification of genes defective in IBA-response mu-
tants with apparently normal IBA to IAA conversion
(Poupart and Waddell 2000; Zolman and others
2000) may elucidate IAA-independent roles for IBA.
COMPARTMENTALIZATION AND INPUTS TO
THE IAA POOL
It is becoming clear that the subcellular localizations
of the intermediates and enzymes controlling IAA
homeostasis are potential control points. The en-
zymes in the Trp biosynthetic pathway are appar-
ently chloroplastic. In contrast, most of the down-
stream proteins implicated in Trp-dependent IAA
biosynthesis, including Trp decarboxylase, YUCCA,
AAO1, and CYP83B1, appear cytoplasmic (Figure 2;
Table 1), and a partially purified Arabidopsis IAA-
synthase that converts Trp to IAA is soluble (Mu¨ller
and Weiler 2000a). Only CYP79B2 and CYP79B3,
the P450s that convert Trp to IAOx, possess appar-
ent chloroplast-targeting signals (Hull and others
2000). It is possible that the IAOx made by these
chloroplastic P450s is channeled to glucosinolate
production, whereas the IAOx made via the YUCCA
pathway is destined for IAA biosynthesis (Celenza
2001). Direct comparisons of the IAA and Trp me-
tabolites that accumulate in yucca and sur2 mutants
210 B. Bartel et al.
might reveal whether this subcellular distribution
contributes to IAA homeostasis. It also will be inter-
esting to learn where the Trp-independent IAA bio-
synthetic enzymes are localized.
Whereas Trp-dependent IAA biosynthesis is likely
to be largely cytoplasmic, IBA to IAA conversion is
almost certainly peroxisomal (Zolman and others
2000), and sequence analysis suggests that the IAA-
amino acid conjugate hydrolases reside in the ER
lumen (Bartel and Fink 1995; Davies and others
1999). It is intriguing that ABP1, an essential auxin
binding protein (Chen and others 2001), is also pre-
dominantly localized in the ER lumen (Jones 1994),
suggesting a role for auxin in this compartment. It
will be informative to identify the membrane in
which IAR1 resides, as the iar1 mutant is resistant to
the known substrates of the putative ER-resident
hydrolases (Lasswell and others 2000). Further-
more, it is important to learn where the enzymes
that catalyze IAA-amino acid conjugate formation
are localized, as conjugates may need to move into
the ER for hydrolysis. Although dramatic progress in
identifying intercellular IAA transporters has been
made in recent years (Estelle 1998; Palme and Ga¨l-
weiler 1999), the apparent compartmentalization of
auxin metabolism implies that intracellular trans-
porters that move IAA out of the ER and the per-
oxisome (Figure 3) remain to be identified.
FUTURE DIRECTIONS
Recent advances in genetic technologies and ana-
lytical chemistry have advanced our understanding
of auxin homeostasis, and are likely to contribute to
future breakthroughs. Activation tagging (Weigel
and others 2000) and related technologies (Wilson
and others 1996; LeClere and Bartel 2001) may cir-
cumvent the redundancy (Zhao and others 2001)
that has plagued analysis of auxin metabolism to
date (Normanly and Bartel 1999). In addition, sys-
tematic reverse genetic screens for loss-of-function
mutations in genes of interest are now routine (Kry-
san and others 1999), and classical genetic analysis
has been accelerated by the Arabidopsis genome se-
quence completion (The Arabidopsis Genome Initia-
tive 2000). Furthermore, screens in heterologous
hosts based on enzyme activity (Corey and others
1993) or resistance to toxic compounds (Hull and
others 2000) expanded to additional steps in auxin
metabolism may complement traditional biochemi-
cal approaches.
As the various inputs to the IAA pool are eluci-
dated, it will also be important to identify the en-
zymes that inactivate IAA (Figure 1) and determine
how they are regulated. The high auxin phenotype
in the yucca mutant is suppressed by expression of
iaaL (Zhao and others 2001), a microbial IAA con-
jugating enzyme (Glass and Kosuge 1986), suggest-
ing that IAA-inactivating activities are not sufficient
in yucca. In contrast, endogenous conjugation path-
ways apparently inactivate the IAA that accumu-
lates in trp2 and trp3 mutants (Normanly and others
1993) and in CYP79B2 overexpressors (Celenza and
Normanly, personal communication). Although
genes encoding enzymes responsible for IAA-
glucose synthesis have been cloned from maize (Sz-
erszen and others 1994) and Arabidopsis (Jackson
and others 2001), plant enzymes responsible for
IAA- and IBA-amino acid conjugate synthesis re-
main to be identified. In addition, as IAA can be
converted to IBA in plant extracts (Ludwig-Mu¨ller
2000), IBA synthase might contribute to IAA inac-
tivation (Figure 1). A maize IBA synthase that is
regulated by a variety of biotic and abiotic stresses
(Ludwig-Mu¨ller 2000) has been partially purified
(Ludwig-Mu¨ller and Hilgenberg 1995). It will be in-
teresting to determine whether IBA synthesis im-
pacts the free IAA pool.
A future challenge will be to determine whether
de novo synthesis, conjugate hydrolysis, and IBA
-oxidation (Figure 1) are redundant IAA sources or
instead supply specific auxin needs during develop-
ment. For example, free IAA accumulates in Arabi-
dopsis seedlings grown at high temperature (Gray
and others 1998), resulting in high auxin pheno-
types (Gray and others 1998; Rogg and others
2001), but the source of this IAA has not been iden-
tified. Mutants defective in various facets of IAA ho-
meostasis may aid in assigning functions to indi-
vidual IAA sources. Several peroxisome defective
IBA-response mutants have reduced lateral root ini-
tiation, not only following exposure to IBA (Zolman
and others 2000; Zolman and others 2001a), but
also in the absence of exogenous auxin (Zolman and
others 2001b). This defect implies that the IAA
formed from endogenous IBA -oxidation during
seedling development is important for lateral root
initiation. In support of this hypothesis, IBA appar-
ently is not converted to IAA in a subset of pear
plants with defects in lateral root formation (Baraldi
and others 1993). In contrast, the ilr1 and iar3 ami-
dohydrolase mutants have normal numbers of lat-
eral roots (Magidin and Bartel, unpublished); the
IAA released from conjugates may be less important
for this process. Plants may use different IAA sources
for different auxin requirements during develop-
ment, and it will be interesting to learn which func-
tions are specific to particular precursors and which
can be supplied through any of several pathways.
Inputs to the Active Indole-3-Acetic Acid Pool 211
ACKNOWLEDGMENTS
We are grateful to John Celenza, Jerry Cohen, and
Jennifer Normanly for sharing unpublished results,
and Raquel Adham, Melanie Monroe-Augustus,
Jennifer Normanly, Rebekah Rampey, Luise Rogg,
and Andrew Woodward for critical comments on
the manuscript. Work in the authors’lab is sup-
ported by grants from the National Institutes of
Health (GM54749), the National Science Founda-
tion (IBN-9982611, DBI-0077769), the United
States Department of Agriculture (2001-35304-
09925), and the Robert A. Welch Foundation (C-
1309).
REFERENCES
Alvarez R, Nissen SJ, Sutter EG. 1989. Relationship between in-
dole-3-acetic acid levels in apple (Malus pumila Mill) rootstocks
cultured in vitro and adventitious root formation in the pres-
ence of indole-3-butyric acid. Plant Physiol 89:439–443.
Badenoch-Jones J, Summons RE, Rolfe BG, Letham DS. 1984.
Phytohormones, Rhizobium mutants, and nodulation in le-
gumes. IV. Auxin metabolites in pea root nodules. J Plant
Growth Regul 3:23–39.
Bak S, Nielsen HL, Halkier BA. 1998. The presence of CYP79
homologues in glucosinolate-producing plants shows evolu-
tionary conservation of the enzymes in the conversion of
amino acid to aldoxime in the biosynthesis of cyanogenic glu-
cosides and glucosinolates. Plant Mol Biol 38:725–734.
Bak S, Tax FE, Feldmann KA, Galbraith DW, Feyereisen R. 2001.
CYP83B1, a cytochrome P450 at the metabolic branch point in
auxin and indole glucosinolate biosynthesis in Arabidopsis.
Plant Cell 13:101–111.
Baldi BG, Maher BR, Cohen JD. 1989. Hydrolysis of indole-3-
acetic acid esters exposed to mild alkaline conditions. Plant
Physiol 91:9–12.
Baldi BG, Maher BR, Slovin JP, Cohen JD. 1991. Stable isotope
labeling, in vivo, of D- and L-tryptophan pools in Lemna gibba
and the low incorporation of label into indole-3-acetic acid.
Plant Physiol 95:1203–1208.
Baraldi R, Bertazza G, Predieri S, Bregoli AM, Cohen JD. 1993.
Uptake and metabolism of indole-3-butyric acid during the in
vitro rooting phase in pear cultivars (Pyrus communis). Acta
Hort 329:289–291.
Barlier I, Kowalczyk M, Marchant A, Ljung K, Bhalerao R, Ben-
nett M, Sandberg G, Bellini C. 2000. The SUR2 gene of Arabi-
dopsis thaliana encodes the cytochrome P450 CYP83B1, a
modulator of auxin homeostasis. Proc Natl Acad Sci USA
97:14819–14824.
Barratt NM, Dong W, Gage DA, Magnus V, Town CD. 1999. Me-
tabolism of exogenous auxin by Arabidopsis thaliana: identifi-
cation of the conjugate N
␣
-(indol-3-ylacetyl)-glutamine and
initiation of a mutant screen. Physiol Plant 105:207–217.
Bartel B. 1997. Auxin biosynthesis. Ann Rev Plant Physiol Plant
Mol Biol 48:51–66.
Bartel B, Fink GR. 1994. Differential regulation of an auxin-
producing nitrilase gene family in Arabidopsis thaliana. Proc
Natl Acad Sci USA 91:6649–6653.
Bartel B, Fink GR. 1995. ILR1, an amidohydrolase that releases
active indole-3-acetic acid from conjugates. Science 268:1745–
1748.
Bartling D, Seedorf M, Mitho¨fer A, Weiler EW. 1992. Cloning and
expression of an Arabidopsis nitrilase which can convert indole-
3-acetonitrile to the plant hormone, indole-3-acetic acid. Eur J
Biochem 205:417–424.
Bartling D, Seedorf M, Schmidt RC, Weiler EW. 1994. Molecular
characterization of two cloned nitrilases from Arabidopsis
thaliana: key enzymes in the biosynthesis of the plant hormone
indole-3-acetic acid. Proc Natl Acad Sci USA 91:6021–6025.
Bayer MH. 1969. Gas chromatographic analysis of acidic indole
auxins in Nicotiana. Plant Physiol 44:267–271.
Belser WL, Murphy JB, Delmer DP, Mills SE. 1971. End product
control of tryptophan biosynthesis in extracts and intact cells of
the higher plant Nicotiana tabacum var. Wisconsin 38. Biochim
Biophys Acta 237:1–10.
Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner
PA, Walker AR, Schultz B, Feldmann KA. 1996. Arabidopsis
AUX1 gene: a permease-like regulator of root gravitropism.
Science 273:948–950.
Bialek K, Cohen JD. 1986. Isolation and partial characterization
of the major amide-linked conjugate of indole-3-acetic acid
from Phaseolus vulgaris L. Plant Physiol 80:99–104.
Bialek K, Cohen JD. 1992. Amide-linked indoleacetic acid con-
jugates may control levels of indoleacetic acid in germinating
seedlings of Phaseolus vulgaris. Plant Physiol 100:2002–2007.
Bialek K, Meudt WJ, Cohen JD. 1983. Indole-3-acetic acid (IAA)
and IAA conjugates applied to bean stem sections. Plant Phys-
iol 73:130–134.
Blommaert K. 1954. Growth and inhibiting substances in relation
to the rest period of the potato tuber. Nature 174:970–972.
Boerjan W, Cervera M, Delarue M, Beeckman I, Dewitte W,
Bellini C, Caboche M, Onckelen H, Van Montagu M, Inze´D.
1995. Superroot, a recessive mutation in Arabidopsis, confers
auxin overproduction. Plant Cell 7:1405–1419.
Bower PJ, Brown HM, Purves WK. 1978. Cucumber seedling
indoleacetaldehyde oxidase. Plant Physiol 61:107–110.
Campanella JJ, Ludwig-Mueller J, Town CD. 1996. Isolation and
characterization of mutants of Arabidopsis thaliana with in-
creased resistance to growth inhibition by indoleacetic acid-
amino acid conjugates. Plant Physiol 112:735–745.
Celenza JL. 2001. Metabolism of tyrosine and tryptophan - new
genes for old pathways. Curr Opin Plant Biol 4:234–240.
Celenza JL, Grisafi PL, Fink GR. 1995. A pathway for lateral root
formation in Arabidopsis thaliana. Genes Dev 9:2131–2142.
Chamarro J, O
¨stin A, Sandberg G. 2001. Metabolism of indole-
3-acetic acid by orange (Citrus sinensis) flavedo tissue during
fruit development. Phytochemistry 57:179–187.
Chavadej S, Brisson N, McNeil JN, De Luca V. 1994. Redirection
of tryptophan leads to production of low indole glucosinolate
canola. Proc Natl Acad Sci USA 91:2166–2170.
Chen J-G, Ullah H, Young JC, Sussman MR, Jones AM. 2001.
ABP1 is required for organized cell elongation and division in
Arabidopsis embryogenesis. Genes Dev 15:902–911.
Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH.
1998. The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a
component of the polar-auxin-transport efflux carrier. Proc
Natl Acad Sci USA 95:15112–15117.
Cohen JD. 1982. Identification and quantitative analysis of in-
dole-3-acetyl-L-aspartate from seeds of Glycine max L. Plant
Physiol 70:749–753.
Cohen JD, Baldi BG. 1983. Studies of endogenous indole-3-
acetyl-L-aspartate during germination of soybeans. Proc Plant
Growth Reg Soc Amer 10:117–122.
212 B. Bartel et al.
Cohen JD, Bandurski RS. 1982. Chemistry and physiology of the
bound auxins. Ann Rev Plant Physiol 33:403–430.
Cooney TP, Nonhebel HM. 1991. Biosynthesis of indole-3-acetic
acid in tomato shoots: measurement, mass-spectral identifica-
tion and incorporation of
2
H from
2
H
2
O into indole-3-acetic
acid, D- and L-tryptophan, indole-3-pyruvate and tryptamine.
Planta 184:368–376.
Corey EJ, Matsuda SPT, Bartel B. 1993. Isolation of an Arabidopsis
thaliana gene encoding cycloartenol synthase by functional ex-
pression in a yeast mutant lacking lanosterol synthase by the
use of a chromatographic screen. Proc Natl Acad Sci USA
90:11628–11632.
Costacurta A, Vanderleyden J. 1995. Synthesis of phytohormones
by plant-associated bacteria. Crit Rev Microbiol 21:1–18.
Davidonis GH, Hamilton RH, Mumma RO. 1982. Evidence for
compartmentalization of conjugates of 2,4-dichlorophenoxy-
acetic acid in soybean callus tissue. Plant Physiol 70:939–942.
Davies RT, Goetz DH, Lasswell J, Anderson MN, Bartel B. 1999.
IAR3 encodes an auxin conjugate hydrolase from Arabidopsis.
Plant Cell 11:365–376.
de Klerk G-J, Brugge JT, Marinova S. 1997. Effectiveness of in-
doleacetic acid, indolebutyric acid and napthaleneacetic acid
during adventitious root formation in vitro in Malus ‘Jork 9’.
Plant Cell, Tissue, Organ Culture 49:39–44.
de Klerk G-J, van der Krieken WM, de Jong JC. 1999. The for-
mation of adventitious roots: new concepts, new possibilities.
In Vitro Cell Dev. Biol.-Plant 35:189–199.
Delarue M, Prinsen E, Van Onckelen H, Caboche M, Bellini C.
1998. Sur2 mutations of Arabidopsis thaliana define a new locus
involved in the control of auxin homeostasis. Plant J 14:603–
611.
Dubois-Dalcq M, Feigenbaum V, Aubourg P. 1999. The neurobi-
ology of X-linked adrenoleukodystrophy, a demyelinating per-
oxisomal disorder. Trends Neurosci 22:4–12.
Eastmond PJ, Hooks MA, Williams D, Lange P, Bechtold N, Sar-
robert C, Nussaume L, Graham IA. 2000. Promoter trapping of
a novel medium-chain acyl-CoA oxidase, which is induced
transcriptionally during Arabidopsis seed germination. J Biol
Chem 275:34375–34381.
Epstein E, Baldi BG, Cohen JD. 1986. Identification of indole-3-
acetylglutamate from seeds of Glycine max L. Plant Physiol
80:256–258.
Epstein E, Chen K-H, Cohen JD. 1989. Identification of indole-
3-butyric acid as an endogenous constituent of maize kernels
and leaves. Plant Growth Regul 8:215–223.
Epstein E, Cohen JD, Bandurski RS. 1980. Concentration and
metabolic turnover of indoles in germinating kernels of Zea
mays L. Plant Physiol 65:415–421.
Epstein E, Lavee S. 1984. Conversion of indole-3-butyric acid to
indole-3-acetic acid by cuttings of grapevine (Vitus vinifera) and
olive (Olea europa). Plant Cell Physiol 25:697–703.
Epstein E, Ludwig-Mu¨ller J. 1993. Indole-3-butyric acid in plants:
occurrence, synthesis, metabolism, and transport. Physiol Plant
88:382–389.
Epstein E, Nissen SJ, Sutter EG. 1991. Indole-3-acetic acid and
indole-3-butyric acid in tissues of carrot inoculated with Agro-
bacterium rhizogenes. J Plant Growth Regul 10:97–100.
Estelle M. 1998. Polar auxin transport: new support for an old
model. Plant Cell 10:1775–1778.
Facchini PJ, Huber-Allanach KL, Tari LW. 2000. Plant aromatic
L-amino acid decarboxylases: evolution, biochemistry, regula-
tion, and metabolic engineering applications. Phytochemistry
54:121–138.
Fawcett CH, Wain RL, Wightman F. 1960. The metabolism of
3-indolylalkanecarboxylic acids, and their amides, nitriles and
methyl esters in plant tissues. Proc Royal Soc London, Series B
152:231–254.
Feung C-S, Hamilton RH, Mumma RO. 1977. Metabolism of in-
dole-3-acetic acid. IV. Biological properties of amino acid con-
jugates. Plant Physiol 59:91–93.
Frey M, Chomet P, Glawischnig E, Stettner C, Gru¨n S, Winklmair
A, Eisenreich W, Bacher A, Meeley RB, Briggs SP, Simcox K,
Gierl A. 1997. Analysis of a chemical plant defense mechanism
in grasses. Science 277:696–699.
Frey M, Stettner C, Pare´PW, Schmelz EA, Tumlinson JH, Gierl A.
2000. An herbivore elicitor activates the gene for indole emis-
sion in maize. Proc Natl Acad Sci USA 97:14801–14806.
Gerhardt B. 1992. Fatty acid degradation in plants. Prog Lipid Res
31:417–446.
Glass NL, Kosuge T. 1986. Cloning of the gene for indoleacetic
acid-lysine synthetase from Pseudomonas syringae subsp, savas-
tanoi. J Bacteriol 166:598–603.
Glawischnig E, Tomas A, Eisenreich W, Spiteller P, Bacher A,
Gierl A. 2000. Auxin biosynthesis in maize kernels. Plant Phys-
iol 123:1109–1119.
Golparaj M, Tseng T-S, Olszewski N. 1996. The Rooty gene of
Arabidopsis encodes a protein with highest similarity to ami-
notransferases. Plant Physiol 111 (suppl.):114
Goddijn OJM, de Kam RJ, Zanetti A, Schilperoort RA, Hoge HC.
1992. Auxin rapidly down-regulates transcription of the tryp-
tophan decarboxylase gene from Catharanthus roseus. Plant Mol
Biol 18:1113–1120.
Gray WM, O
¨stin A, Sandberg G, Romano CP, Estelle M. 1998.
High temperature promotes auxin-mediated hypocotyl elonga-
tion in Arabidopsis. Proc Natl Acad Sci USA 95:7197–7202.
Grsic-Rausch S, Kobelt P, Siemens JM, Bischoff M, Ludwig-
Mu¨ller J. 2000. Expression and localization of nitrilase during
symptom development of the clubroot disease in Arabidopsis.
Plant Physiol 122:369–378.
Guerinot ML. 2000. The ZIP family of metal transporters. Biochim
Biophys Acta 1465:190–198.
Guillet G, Poupart J, Basurco J, De Luca V. 2000. Expression of
tryptophan decarboxylase and tyrosine decarboxylase genes in
tobacco results in altered biochemical and physiological phe-
notypes. Plant Physiol 122:933–943.
Hall PJ. 1980. Indole-3-acetyl-myo-inositol in kernels of Oryza
sativa. Phytochemistry 19:2121–2123.
Hangarter RP, Good NE. 1981. Evidence that IAA conjugates are
slow-release sources of free IAA in plant tissues. Plant Physiol
68:1424–1427.
Hangarter RP, Peterson MD, Good NE. 1980. Biological activities
of indoleacetylamino acids and their use as auxins in tissue
culture. Plant Physiol 65:761–767.
Hartmann HT, Kester DE, Davies FT. 1990. Plant propagation:
principles and practices. Englewood Cliffs, N.J.: Prentice-Hall. p
Hayashi M, Nito K, Toriyama-Kato K, Kondo M, Yamaya T, Nish-
imura M. 2000. AtPex14p maintains peroxisomal functions by
determining protein targeting to three kinds of plant peroxi-
somes. EMBO J 19:5701–5710.
Hayashi M, Toriyama K, Kondo M, Nishimura M. 1998. 2,4-
Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis
have defects in glyoxysomal fatty acid -oxidation. Plant Cell
10:183–195.
Inputs to the Active Indole-3-Acetic Acid Pool 213
Helmlinger J, Rausch T, Hilgenberg W. 1985. Metabolism of
14
C-
indole-3-acetaldoxime by hypocotyls of Chinese cabbage. Phy-
tochemistry 24:2497–2502.
Helmlinger J, Rausch T, Hilgenberg W. 1987. A soluble protein
factor from Chinese cabbage converts indole-3-acetaldoxime to
IAA. Phytochemistry 26:615–618.
Holland IB, Blight MA. 1999. ABC-ATPases, adaptable energy
generators fuelling transmembrane movement of a variety of
molecules in organisms from bacteria to humans. J Mol Biol
293:381–399.
Hull AK, Vij R, Celenza JL. 2000. Arabidopsis cytochrome P450s
that catalyze the first step of tryptophan-dependent indole-3-
acetic acid biosynthesis. Proc Natl Acad Sci USA 97:2379–2384.
Ilic’N, O
¨stin A, Cohen JD. 1999. Differential inhibition of indole-
3-acetic acid and tryptophan biosynthesis by indole analogues.
I. Tryptophan-dependent IAA biosynthesis. Plant Growth
Regul 27:57–62.
Jackson RG, Lim E-K, Li Y, Kowalczyk M, Sandberg G, Hoggett J,
Ashford DA, Bowles DJ. 2001. Identification and biochemical
characterization of an Arabidopsis indole-3-acetic acid glucosyl-
transferase. J Biol Chem 276:4350–4356.
Jones AM. 1994. Auxin-binding proteins. Ann Rev Plant Physiol
Plant Mol Biol 45:393–420.
Kindl H. 1993. Fatty acid degradation in plant peroxisomes: func-
tion and biosynthesis of the enzymes involved. Biochimie
75:225–230.
King JJ, Stimart DP. 1998. Genetic analysis of variation for auxin-
induced adventitious root formation among eighteen ecotypes
of Arabidopsis thaliana L. Heynh. J. Heredity 89:481–487.
King JJ, Stimart DP, Fisher RH, Bleecker AB. 1995. A mutation
altering auxin homeostasis and plant morphology in Arabidop-
sis. Plant Cell 7:2023–2037.
Koga J. 1995. Structure and function of indolepyruvate decar-
boxylase, a key enzyme in indole-3-acetic acid biosynthesis.
Biochim Biophys Acta 1249:1–13.
Koshiba T, Kamiya Y, Iino M. 1995. Biosynthesis of indole-3-
acetic acid from L-tryptophan in coleoptile tips of maize (Zea
mays L.). Plant Cell Physiol 36:1503–1510.
Koshiba T, Matsuyama H. 1993. An in vitro system of indole-3-
acetic acid formation from tryptophan in maize (Zea mays) co-
leoptile extracts. Plant Physiol 102:1319–1324.
Krysan PJ, Young JC, Sussman MR. 1999. T-DNA as an inser-
tional mutagen in Arabidopsis. Plant Cell 11:2283–2290.
Lange PR, Graham I. 2000. Arabidopsis thaliana mutants disrupted
in lipid mobilization. Biochem Soc Trans 28:762–765.
Lasswell J, Rogg LE, Nelson DC, Rongey C, Bartel B. 2000. Clon-
ing and characterization of IARI, a gene required for auxin
conjugate sensitivity in Arabidopsis. Plant Cell 12:2395–2408.
Last RL, Bissinger PH, Mahoney DJ, Radwanski ER, Fink GR.
1991. Tryptophan mutants in Arabidopsis: the consequences of
duplicated tryptophan synthase genes. Plant Cell 3:345–358.
Last RL, Fink GR. 1988. Tryptophan-requiring mutants of the
plant Arabidopsis thaliana. Science 240:305–310.
LeClere S, Bartel B. 2001. A library of Arabidopsis 35S-cDNA lines
for identifying novel mutants. Plant Mol Biol 46:695–703.
Lehman A, Black R, Ecker JR. 1996. HOOKLESS1, an ethylene
response gene, is required for differential cell elongation in the
Arabidopsis hypocotyl. Cell 85:183–194.
Ljung K, O
¨stin A, Lioussanne L, Sandberg G. 2001. Developmen-
tal regulation of indole-3-acetic acid turnover in Scots Pine
seedlings. Plant Physiol 125:464–475.
Ludwig-Mu¨ller J. 2000. Indole-3-butyric acid in plant growth and
development. Plant Growth Regul 32:219–230.
Ludwig-Mu¨ller J, Epstein E. 1991. Occurrence and in vivo biosyn-
thesis of indole-3-butyric acid in corn (Zea mays L.). Plant Phys-
iol 97:765–770.
Ludwig-Mu¨ller J, Epstein E. 1993. Analysis of indole-3-butyric
acid in Arabidopsis thaliana. Acta Hort 329:109–111.
Ludwig-Mu¨ller J, Epstein E, Hilgenberg W. 1996. Auxin-
conjugate hydrolysis in Chinese cabbage: characterization of an
amidohydrolase and its role during infection with clubroot dis-
ease. Physiol Plant 97:627–634.
Ludwig-Mu¨ller J, Hilgenberg W. 1988. A plasma membrane-
bound enzyme oxidizes l-tryptophan to indole-3-
acetaldoxime. Physiol Plant 74:240–250.
Ludwig-Mu¨ller J, Hilgenberg W. 1995. Characterization and par-
tial purification of indole-3-butyric acid synthetase from maize
(Zea mays). Physiol Plant 94:651–660.
Ludwig-Mu¨ller J, Sass S, Sutter EG, Wodner M, Epstein E. 1993.
Indole-3-butyric acid in Arabidopsis thaliana. I. Identification
and quantification. Plant Growth Regul 13:179–187.
Luschnig C, Gaxiola RA, Grisafi P, Fink GR. 1998. EIR1, a root-
specific protein involved in auxin transport, is required for
gravitropism in Arabidopsis thaliana. Genes Dev 12:2175–2187.
Magnus V, Hangarter RP, Good NE. 1992a. Interaction of free
indole-3-acetic acid and its amino acid conjugates in tomato
hypocotyl cultures. J Plant Growth Regul 11:67–75.
Magnus V, Nigovic B, Hangarter RP, Good NE. 1992b. N-(Indol-
3-ylacetyl)amino acids as sources of auxin in plant tissue cul-
ture. J Plant Growth Regul 11:19–28.
Melanson D, Chilton M-D, Masters-Moore D, Chilton WS. 1997.
A deletion in an indole synthase gene is responsible for the
DIMBOA-deficient phenotype of bxbx maize. Proc Natl Acad
Sci USA 94:13345–13350.
Michalczuk L, Ribnicky DM, Cooke TJ, Cohen JD. 1992. Regula-
tion of indole-3-acetic acid biosynthetic pathways in carrot cell
cultures. Plant Physiol 100:1346–1353.
Mikkelsen MD, Hansen CH, Wittstock U, Halkier BA. 2000. Cy-
tochrome P450 CYP79B2 from Arabidopsis catalyzes the con-
version of tryptophan to indole-3-acetaldoxime, a precursor of
indole glucosinolates and indole-3-acetic acid. J Biol Chem
275:33712–33717.
Mu¨ller A, Guan C, Ga¨lweiler L, Ta¨nzler P, Huijser P, Marchant A,
Parry G, Bennett M, Wisman E, Palme K. 1998a. AtPNI2 de-
fines a locus of Arabidopsis for root gravitropism control. EMBO
J 17:6903–6911.
Mu¨ller A, Hillebrand H, Weiler EW. 1998b. Indole-3-acetic acid is
synthesized from L-tryptophan in roots of Arabidopsis thaliana.
Planta 206:362–369.
Mu¨ller A, Weiler EW. 2000a. IAA-synthase, an enzyme complex
from Arabidopsis thaliana catalyzing the formation of indole-3-
acetic acid from (S)-tryptophan. Biol Chem 381:679–686.
Mu¨ller A, Weiler EW. 2000b. Indolic constituent and indole-3-
acetic acid biosynthesis in the wild-type and a tryptophan aux-
otroph mutant of Arabidopsis thaliana. Planta 211:855–863.
Nissen SJ, Sutter EG. 1990. Stability of IAA and IBA in nutrient
medium to several tissue culture procedures. Hort Science
25:800–802.
Nonhebel HM, Cooney TP, Simpson R. 1993. The route, control
and compartmentation of auxin synthesis. Aust J Plant Physiol
20:527–539.
Nordstro¨m A-C, Jacobs FA, Eliasson L. 1991. Effect of exogenous
indole-3-acetic acid and indole-3-butyric acid on internal lev-
214 B. Bartel et al.
els of the respective auxins and their conjugation with aspartic
acid during adventitious root formation in pea cuttings. Plant
Physiol 96:856–861.
Normanly J. 1997. Auxin metabolism. Physiol Plant 100:431–
442.
Normanly J, Bartel B. 1999. Redundancy as a way of life - IAA
metabolism. Curr Op in Plant Biol 2:207–213.
Normanly J, Cohen JD, Fink GR. 1993. Arabidopsis thaliana aux-
otrophs reveal a tryptophan-independent biosynthetic path-
way for indole-3-acetic acid. Proc Natl Acad Sci USA 90:10355–
10359.
Normanly J, Grisafi P, Fink GR, Bartel B. 1997. Arabidopsis mu-
tants resistant to the auxin effects of indole-3-acetonitrile are
defective in the nitrilase encoded by the NIT1 gene. Plant Cell
9:1781–1790.
Normanly J, Slovin JP, Cohen JD. 1995. Rethinking auxin bio-
synthesis and metabolism. Plant Physiol 107:323–329.
Nowacki J, Bandurski RS. 1980. Myo-inositol esters of indole-3-
acetic acid as seed auxin precursors of Zea mays L. Plant Physiol
65:422–427.
Olsen LJ. 1998. The surprising complexity of peroxisome biogen-
esis. Plant Mol Biol 38:163–189.
O
¨stin A, Catala´C, Chamarro J, Sandberg G. 1995. Identification
of glucopyranosyl--1,4-glucopyranosyl -1-Noxindole-3-
acetyl-N-aspartic acid, a new IAA catabolite, by liquid chroma-
tography/tandem mass spectrometry. J Mass Spectrometry
30:1007–1017.
O
¨stin A, Ilı´c N, Cohen JD. 1999. An in vitro system from maize
seedlings for tryptophan-independent indole-3-acetic acid bio-
synthesis. Plant Physiol 119:173–178.
O
¨stin A, Kowalyczyk M, Bhalerao R, Sandberg G. 1998. Metabo-
lism of indole-3-acetic acid in Arabidopsis. Plant Physiol
118:285–296.
O
¨stin A, Moritz T, Sandberg G. 1992. Liquid chromatography/
mass spectrometry of conjugates and oxidative metabolites of
indole-3-acetic acid. Biol Mass Spectrometry 21:292–298.
Ouyang J, Shao X, Li J. 2000. Indole-3-glycerol phosphate, a
branchpoint of indole-3-acetic acid biosynthesis from the tryp-
tophan biosynthetic pathway in Arabidopsis thaliana. Plant J 24
:327–333.
Palme K, Ga¨ lweiler L. 1999. PIN-pointing the molecular basis of
auxin transport. Curr Opin Plant Biol 2:375–381.
Pasquali G, Goddijn OJM, de Waal A, Verpoorte R, Schilperoort
RA, Hoge JHC, Memelink J. 1992. Coordinated regulation of
two indole alkaloid biosynthetic genes from Cantharanthus ro-
seus by auxin and elicitors. Plant Mol Biol 18:1121–1131.
Patten CL, Glick BR. 1996. Bacterial biosynthesis of indole-3-
acetic acid. Can J Microbiol 42:207–220.
Percival FW, Bandurski RS. 1976. Esters of indole-3-acetic acid
from Avena seeds. Plant Physiol 58:60–67.
Poupart J, Waddell CS. 2000. The rib1 mutant is resistant to in-
dole-3-butyric acid, an endogenous auxin in Arabidopsis. Plant
Physiol 124:1739–1751.
Quirino B, Normanly J, Amasino RM. 1999. Diverse range of
gene activity during Arabidopsis thaliana leaf senescence in-
cludes pathogen-independent induction of defense-related
genes. Plant Mol Biol 40:267–278.
Radwanski ER, Barczak AJ, Last RL. 1996. Characterization of
tryptophan synthase alpha subunit mutants of Arabidopsis
thaliana. Mol Gen Genet 253:353–361.
Rajagopal R, Larsen P. 1972. Metabolism of indole-3-
acetaldoxime in plants. Planta 103:45–54.
Rajagopal R, Moller I, Olsen CE. 1991. Synthesis, biological ac-
tivity and metabolism of N-hydroxytryptophan. Phytochemis-
try 30:1405–1408.
Reintanz B, Lehnen M, Reichelt M, Gershenzon J, Kowalczyk M,
Sandberg G, Godde M, Uhl R, Palme K. 2001. bus, a bushy
Arabidopsis CYP79F1 knockout mutant with abolished synthe-
sis of short-chain aliphatic glucosinolates. Plant Cell 13:351–
367.
Ribnicky DM, Cohen JD, Hu W-S, Cooke TJ. 2001. An auxin
surge following fertilization in carrots: a mechanism for regu-
lating plant totipotency. Planta in press
Richmond TA, Bleecker AB. 1999. A defect in -oxidation causes
abnormal inflorescence development in Arabidopsis. Plant Cell
11:1911–1923.
Robbins JA, Campidonica MJ, Burger DW. 1988. Chemical and
biological stability of indole-3-butryic acid (IBA) after long-
term storage at selected temperatures and light regimes. J En-
viron Hort 6:33–38.
Rogg LE, Lasswell J, Bartel B. 2001. A gain-of-function mutation
in IAA28 suppresses lateral root development. Plant Cell
13:465–480.
Schmidt R-C, Mu¨ ller A, Hain R, Bartling D, Weiler EW. 1996.
Transgenic tobacco plants expressing the Arabidopsis thaliana
nitrilase II enzyme. Plant J 9:683–691.
Schneider EA, Kazakoff CW, Wightman F. 1985. Gas chromatog-
raphy-mass spectrometry evidence for several endogenous
auxins in pea seedling organs. Planta 165:232–241.
Sekimoto H, Seo M, Dohmae N, Takio K, Kamiya Y, Koshiba T.
1997. Cloning and molecular characterization of plant alde-
hyde oxidase. J Biol Chem 272:15280–15285.
Sekimoto H, Seo M, Kawakami N, Komano T, Desloire S, Lioten-
berg S, Marion-Poll A, Caboche M, Kamiya Y, Koshiba T. 1998.
Molecular cloning and characterization of aldehyde oxidases in
Arabidopsis thaliana. Plant Cell Physiol 39:433–442.
Seo M, Akaba S, Oritani T, Delarue M, Bellini C, Caboche M,
Koshiba T. 1998. Higher activity of an aldehyde oxidase in the
auxin-overproducing superroot1 mutant of Arabidopsis thaliana.
Plant Physiol 116:687–693.
Sitbon F, A
˚stot C, Edlund A, Crozier A, Sandberg G. 2000. The
relative importance of tryptophan-dependent and tryptophan-
independent biosynthesis of indole-3-acetic acid in tobacco
during vegetative growth. Planta 211:715–721.
Sitbon F, O
¨stin A, Sundberg B, Olsson O, Sandberg G. 1993.
Conjugation of indole-3-acetic acid (IAA) in wild-type and
IAA-overproducing transgenic tobacco plants, and identifica-
tion of the main conjugates by frit-fast atom bombardment
liquid chromatography-mass spectrometry. Plant Physiol
101:313–320.
Slovin JP. 1997. Phytotoxic conjugates of indole-3-acetic acid:
potential agents for biochemical selection of mutants in con-
jugate hydrolysis. Plant Growth Regul 21:215–221.
Slovin JP, Bandurski RS, Cohen JD. 1999. Auxin. In: Hooykaas
PJJ, Hall MA, Libbenga KR, editors. Biochemistry and molecu-
lar biology of plant hormones. Elsevier: Amsterdam. p 115–
140.
Songstad DD, De Luca V, Brisson N, Kurz WGW, Nessier CL.
1990. High levels of tryptamine accumulation in transgenic
tobacco expressing tryptophan decarboxylase. Plant Physiol
94:1410–1413.
Subramani S. 1998. Components involved in peroxisome import,
biogenesis, proliferation, turnover, and movement. Physiol
Rev 78:171–188.
Inputs to the Active Indole-3-Acetic Acid Pool 215
Sutter EG, Cohen JD. 1992. Measurement of indolebutyric acid in
plant tissues by isotope dilution gas chromatography-mass
spectrometry analysis. Plant Physiol 99:1719–1722.
Szerszen JB, Szczyglowski K, Bandurski RS. 1994. iaglu, a gene
from Zea mays involved in conjugation of the growth hormone
indole-3-acetic acid. Science 265:1699–1701.
Sztein AE, Cohen JD, Cooke TJ. 2000. Evolutionary patterns in
the auxin metabolism of green plants. Int J Plant Sci 161:849–
859.
Sztein AE, Cohen JD, Cooke TJ. 2001. Indole-3-acetic acid bio-
synthesis in isolated axes from germinating bean seeds: the
effect of wounding on the biosynthetic pathway. Plant Growth
Regul in press
Sztein AE, Cohen JD, Garcı´a de la Fuente I, Cooke TJ. 1999.
Auxin metabolism in mosses and liverworts. Am J Bot
86:1544–1555.
Sztein AE, Cohen JD, Slovin JP, Cooke TJ. 1995. Auxin metabo-
lism in representative land plants. Am J Bot 82:1514–1521.
Tabak HF, Braakman I, Distel B. 1999. Peroxisomes: simple in
function but complex in maintenance. Trends Cell Biol 9:447–
453.
Tam YY, Epstein E, Normanly J. 2000. Characterization of auxin
conjugates in Arabidopsis. Low steady-state levels of indole-3-
acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-
acetyl-glucose. Plant Physiol 123:589–595.
Tam YY, Normanly J. 1998. Determination of indole-3-pyruvic
acid levels in Arabidopsis thaliana by gas chromatography-
selected ion monitoring-mass spectrometry. J Chromatogr A
800:101–108.
The Arabidopsis Genome Initiative. Analysis of the genome se-
quence of the flowering plant Arabidopsis thaliana. Nature
408:796–815.
Thimann KV, Mahadevan S. 1964. Nitrilase. I. Occurrence,
preparation, and general properties of the enzyme. Arch Bio-
chem Biophys 105:133–141.
Utsuno K, Shikanai T, Yamada Y, Hashimoto T. 1998. AGR, an
agravitropic locus of Arabidopsis thaliana, encodes a novel mem-
brane-protein family member. Plant Cell Physiol 39:1111–
1118.
van der Krieken WM, Breteler H, Visser MHM. 1992. The effect of
the conversion of indolebutyric acid into indoleacetic acid on
root formation on microcuttings of Malus. Plant Cell Physiol
33:709–713.
van der Krieken WM, Breteler H, Visser MHM, Mavridou D.
1993. The role of the conversion of IBA into IAA on root re-
generation in apple: introduction of a test system. Plant Cell
Reports 12:203–206.
van der Krieken WM, Kodde J, Visser MHM, Tsardakas D, Blaak-
meer A, de Groot K, Leegstra L. 1997. Increased induction of
adventitious rooting by slow release auxins and elicitors. In:
Altman A, Waisel Y, Altman A, Waisel Y, editors. Biology of
root formation and development. New York: Plenum Press. p
95–104.
Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Mu¨ller A, Weiler
EW, Pitrowski M. 2001. Enzymatic characterization of the re-
combinant Arabidopsis thaliana nitrilase subfamily encoded by
the NIT1/NIT2/NIT3-gene cluster. Planta 212:508–516.
Wain RL, Wightman F. 1954. The growth-regulating activity of
certain -substituted alkyl carboxylic acids in relation to their
-oxidation within the plant. Proc Royal Soc London, Series B
142:525–536.
Walz A, Park S, Momonoki YS, Slovin JP, Ludwig-Mu¨ller J, Co-
hen JD. 2001. A gene encoding a protein modified by the phy-
tohormone indoleacetic acid. Plant Biology 2001. Providence,
RI: American Society of Plant Biologists. p
Weigel D, Ahn JH, Bla´zquez MA, Borevitz JO, Christensen SK,
Fankhauser C, Ferra´ndiz C, Kardailsky I, Malancharuvil EJ,
Neff MM, Nguyen JT, Sato S, Wang Z-Y, Xia Y, Dixon RA,
Harrison MJ, Lamb CJ, Yanofsky MF, Chory J. 2000. Activa-
tion tagging in Arabidopsis. Plant Physiol 122:1003–1013.
Wiesman Z, Riov J, Epstein E. 1988. Comparison of movement
and metabolism of indole-3-acetic acid and indole-3-butyric
acid in mung bean cuttings. Physiol Plant 74:556–560.
Wiesman Z, Riov J, Epstein E. 1989. Characterization and rooting
ability of indole-3-butyric acid conjugates formed during root-
ing of mung bean cuttings. Plant Physiol 91:1080–1084.
Wilson K, Long D, Swinburne J, Coupland G. 1996. A Dissociation
insertion causes a semidominant mutation that increases ex-
pression of TINY, an Arabidopsis gene related to APETELA2.
Plant Cell 8:659–671.
Winkler RG, Frank MR, Gallbraith DW, Feyereisen R, Feldmann
KA. 1998. Systematic reverse genetics of transfer-DNA-tagged
lines of Arabidopsis: isolation of mutations in the cytochrome
P450 gene superfamily. Plant Physiol 118:743–750.
Winter A. 1966. A hypothetical route for the biogenesis of IAA.
Planta 71:229–239.
Wright AD, Sampson MB, Neuffer MG, Michalczuk L, Slovin JP,
Cohen JD. 1991. Indole-3-acetic acid biosynthesis in the mu-
tant maize orange pericarp, a tryptophan auxotroph. Science
254:998–1000.
Yang T, Davies PJ. 1999. Promotion of stem elongation by indole-
3-butyric acid in intact plants of Pisum sativum L. Plant Growth
Regul 27:157–160.
Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD,
Weigel D, Chory J. 2001. A role for flavin monooxygenase-like
enzymes in auxin biosynthesis. Science 291:306–309.
Zolman BK, Monroe-Augustus M, Thompson B, Hawes JW,
Krukenberg KA, Matsuda SPT, Bartel B. 2001a. chy1, an Ara-
bidopsis mutant with impaired -oxidation, is defective in a
peroxisomal -hydroxyisobutyryl-CoA hydrolase. J Biol Chem
276:31037–31046.
Zolman BK, Silva ID, Bartel B. 2001b. The Arabidopsis pxa1 mu-
tant is defective in an ABC transporter-like protein required for
fatty acid -oxidation in peroxisomes. Plant Physiol in press
Zolman BK, Yoder A, Bartel B. 2000. Genetic analysis of indole-
3-butyric acid responses in Arabidopsis thaliana reveals four
mutant classes. Genetics 156:1323–1337.
216 B. Bartel et al.
