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207
Redundancy as a way of life — IAA metabolism
Jennifer Normanly* and Bonnie Bartel†
Plants have evolved elaborate systems for regulating cellular
levels of indole-3-acetic acid (IAA). The redundancy of this
network has complicated the elucidation of IAA metabolism,
but molecular genetic studies and precise analytical
methods have begun to expose the circuitry. It is now clear
that plants synthesize, inactivate and catabolize IAA by
multiple pathways, and multiple genes can encode a
particular enzyme within a pathway. A number of these
genes are now cloned, which greatly facilitates the future
dissection of IAA metabolism.
Addresses
*Department of Biochemistry and Molecular Biology, University of
Massachusetts, Amherst, MA 01003, USA;
e-mail: [email protected]
†Department of Biochemistry and Cell Biology, Rice University,
Houston, TX 77005, USA; e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:207–213
may be overestimated. Rapparini et al. [4••] simultaneously pulse-labeled duckweed fronds with 15N-anthranilate
and 2H5-Trp and monitored incorporation of label into
IAA and Trp. Within the first hour, IAA incorporated 15N
from anthranilate faster than did Trp, indicating that initial synthesis of IAA was Trp-independent. By two hours
post-labeling, Trp was equally labeled with 15N and 2H5,
yet the IAA pool was more enriched with 2H5 than 15N.
Thus, exogenous 2H5-Trp is more accessible to IAA synthesis than denovo synthesized N-Trp. Previous work has
suggested that plants have at least two physically separate
Trp pools, one of which may be vacuolar [5]. Future studies will need to account for and identify the cellular
locations of multiple precursor pools. Further characterization of Trp-dependent IAA synthesis will be facilitated
by recently identified indole analogs that inhibit the conversion of 14C-indole to 14C-IAA but not the conversion of
14C-indole to 14C-Trp [6].
http://biomednet.com/elecref/1369526600200207
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
AO
aldehyde oxidase
IAA
indole-3-acetic acid
IAAld
indole-3-acetaldehyde
IAM
indole-3-acetamide
IAN
indole-3-acetonitrile
IPA
indole-3-pyruvic acid
JA
jasmonic acid
Introduction
The coordinated control of indole-3-acetic acid (IAA)
biosynthesis, inactivation, catabolism and transport is a very
dynamic process that responds to developmental and environmental signals. Biochemical approaches have
demonstrated that plants can synthesize IAA from tryptophan (Trp), and several Trp-dependent pathways have been
proposed (Figure 1; reviewed in [1]). Stable isotope labeling
of intact plants has revealed that IAA synthesis can also
occur from a Trp precursor in Trp-independent pathways
(Figure 1; reviewed in [1–3]). Trp-dependent pathways predominate during early embryogenesis and seed
germination, whereas Trp-independent pathways predominate during later embryogenesis and vegetative growth
[2,3]. IAA is inactivated either reversibly through conjugation, or irreversibly by oxidizing IAA or IAA-conjugates
(reviewed in [2,3]). The components of these pathways
have been recalcitrant to biochemical approaches and are
only now being identified. Here we focus on recent progress
in the characterization of IAA metabolism and transport.
Tryptophan as an IAA precursor
In vivo labeling experiments with maize, Arabidopsis, carrot and duckweed indicate that Trp can be metabolized to
IAA ([1–3]); however, its contribution to IAA synthesis
Although several pathways have been proposed for Trpdependent IAA synthesis (Figure 1), both Trp-dependent
and Trp-independent pathways remain poorly defined in
terms of the enzymes, their intermediates and cellular
locations. Recently, an in vitro assay for Trp-independent
conversion of 14C-indole to 14C-IAA has been developed
using extracts from maize seedlings [7••]. The pH (8 to
8.5) optimum and requirement for a reducing environment
suggest that the activity is plastid-localized.
Establishing a role for nitrilases in
IAA biosynthesis
The indole-3-acetonitrile (IAN) pathway (Figure 1) is
apparently limited to three plant families [8] and most of the
characterization of this pathway has been done in the
Brassicaceae ([9] and references therein). Crucifers can convert IAN to IAA, but the redundancy of nitrilase genes
confounds genetic analysis [10•] and the potential for nonenzymatic conversion of IAN to IAA complicates
biochemical analysis [11]. The cumulative data suggest that
if nitrilase converts IAN to IAA in vivo, then the rate-limiting step is substrate access. Since the subcellular localization
of nitrilase and IAN are not well defined, how and when this
pathway is utilized are major unanswered questions. There
is some evidence that IAN utilization could be developmentally controlled [12•,13] and pathogen-induced [9,13].
Micromolar concentrations of exogenous IAN inhibit
Arabidopsis seedling growth in a manner similar to that of
excess IAA [10•]. Arabidopsis encodes four differentially
expressed nitrilases [13–15] and each converts IAN to IAA
in vitro. NIT1 and NIT2 have Km values for IAN in the
mM range [13,14,16] and IAN is present in seedlings at
~3–6 nmol/g fresh weight. In vivo labeling studies demonstrated exogenous 13C-IAN conversion to 13C-IAA in
208
Physiology and metabolism
Figure 1
de novo IAA biosynthesis
Trp biosynthesis
IAA inactivation and conjugate hydrolysis
O
OH
OH
O
O
OH
O
O
N
H
Chorismate
Anthranilate
Trp-ind
e
penden
5-Phosphoribosylanthranilate
HO
t IAA sy
IAA-glucose
synthase
(iaglu)
nthesis
Indole-3-methylglucosinolate
1-(o-carboxyphenylamino)-1deoxyribulose 5-P
IPA
decarboxylase
(ipdC∗)
+ Serine
N
H
se
ra
Trpnsfe
a
r
t
ino
Indole
am
COOH
O
N
H
)
*
aH
O
N
H
Indole-3-acetaldehyde
(IAAld)
Indole-3-pyruvic acid
(IPA)
O
Trp monoxygenase (iaaM*)
N
H
M
IA
?
IAA-amino acid
hydrolases
(ILR1, IAR3)
H
N
dr
hy
O
OxIAA
IAA
ia
e(
s
ola
COOH
N
H
O
COOH
R
N
H
NH2
IAA-amino acid conjugates
N
H
N
H
Trp
Indole-3-acetamide
(IAM)
Oxidized
IAA-amino acid conjugates
NH2
IAAinositol
hydrolase
IAA-glucose
hydrolase
H
-1)
Indole-3-acetonitrile
AO
(At
e
(IAN)
as
xid
ld o
A
A
I
COOH
OH
OH
COOH
N
H
Indole-3-glycerol
phosphate
HO
IAA-myo-inositol
Nitrilase
(NIT1, 2)
N
Indole-3-acetaldoxime
N
H
OH
IAA-glucose OH
Current Opinion in Plant Biology
Simplified diagram of IAA metabolism. IAA can be synthesized via
several Trp-dependent pathways (open arrows) that are named after
unique intermediates, or by a Trp-independent pathway (dashed arrow)
using indole as a precursor. Genes are in italics, microbial genes are
marked with an asterisk. Further catabolism of oxidized IAA and
IAA–Asp is reviewed in [3]. IAA–myo-inositol can be further
metabolized to other conjugates, many of which can also be
hydrolyzed [31•]. It is not known whether IAA–amino acid conjugates
are synthesized directly or use IAA–glucose as an intermediate.
wild-type Arabidopsis and also in transgenic tobacco and
Arabidopsis overexpressing NIT2 [10•,17]. In response to
exogenous IAN, wild-type Arabidopsis accumulates nitrilase protein, and NIT1, NIT2 and NIT4 gene expression is
induced 1.4-, 21- and 4.8-fold, respectively [18].
Overexpressing nitrilase genes in Arabidopsis or tobacco
also increases nitrilase protein and activity [17,18], yet only
NIT2 overexpressing lines show increased sensitivity to
exogenous IAN [10•,17]. An Arabidopsis nit1 mutant is
resistant to IAN, but metabolizes IAN normally and is
morphologically normal in the absence of exogenous IAN
[10•]. The existence of three wild-type nitrilases (NIT2–4)
may compensate for the nit1 defect, or tissue-specific differences in IAN metabolism are masked in whole
seedlings. Label from 2H5-Trp is incorporated into both
IAN and IAA when added to 5 week-old Arabidopsis root
explants [19]. NIT1 is expressed in five-week-old
Arabidopsis roots [13,19], but it is important to consider that
exogenous Trp can be preferentially utilized for IAA synthesis [4••], and that Trp-dependent IAA biosynthetic
pathways can be activated by wounding [20,21].
siliques [13], but is dramatically induced by pathogenic
bacteria [13], exogenous IAN [18], and at the onset of
senescence [12•]. In senescing Arabidopsis leaves, free IAA
levels increase two-fold, whereas IAN and conjugated IAA
levels drop two-fold [12•]. Infection of Arabidopsis or
Chinese cabbage with Plasmodiophora brassicae, the causal
agent of clubroot disease, increases nitrilase activity [9]
and perturbs IAA metabolism [22]. Arabidopsis mutants
with diminished indole-3-methyl-glucosinolate levels (a
putative IAN precursor, Figure 1) appear unaffected in
IAA synthesis and turnover under normal conditions, but
upon P. brassicae infection have lower IAA and IAN levels
and less severe clubroot symptoms than wild-type [23].
There is correlative evidence for IAN pathway utilization
in response to specific developmental and environmental
cues. NIT2 is normally expressed at levels four- to eightfold lower than NIT1 and is restricted to root tips and
Indole pyruvic acid pathway of
IAA biosynthesis
Lastly, two-hybrid screens have identified a nitrilase-associated protein in Arabidopsis (Genbank accession number
Z96936), and a nitrilase-like protein in tobacco that interacts
with an ethylene-response element binding protein [24]. The
functions of these proteins remain to be determined but they
are intriguing in light of the need for some event that renders
endogenous IAN available to nitrilase for IAA synthesis.
Indole-3-pyruvic acid (IPA) ([25] and references therein)
and aldehyde oxidase (AO) activities that convert
Redundancy as a way of life — IAA metabolism Normanly and Bartel
209
Table 1
Plant genes potentially involved in IAA metabolism and transport.
Gene
Species
(Map position)
Gene product
Expression pattern
Mutant phenotype
NIT1
Arabidopsis
(Chr 3, middle)
Nitrilase
Expressed strongly at base of
hypocotyl [13], where exogenous
IAN induces lateral roots [10•]
IAN insensitive hypocotyl and roots [10•]
NIT2
Arabidopsis
(Chr 3, middle)
Nitrilase
AtAO-1
Arabidopsis
(Chr 5, middle)
Aldehyde oxidase [26•,30]
Expressed in roots, induced in IAA
overproducing mutant (sur1)
[26•,29••]
?
ILR1
Arabidopsis
(Chr 3, top)
IAA–amino acid
hydrolase
?
IAA–Leu insensitive root growth [32]
IAR3
Arabidopsis
(Chr 1, middle)
IAA–amino acid
hydrolase
Developmentally expressed in
roots, induced in leaves in response
to JA and wounding [35•]
IAA–Ala insensitive root growth [33••]
iaglu
Maize
IAA–glucose conjugating
enzyme [50]
?
Transgenic tomato that overexpress maize
iaglu are unable to form roots [62]
RTY
(= ALF1
= SUR1
= HLS3)
Arabidopsis
(Chr 2, middle)
Aminotransferase [28]
?
Overproduces IAA and IAA conjugates,
adventitious rooting, epinastic cotyledons
(reviewed in [1]); increased AtAO-1
expression [26•] and AO activity [29••]
SUR2
Arabidopsis
(Chr 4, bottom)
IAA inactivation?
?
Overproduces IAA, adventitious rooting,
epinastic cotyledons [49••]
AUX1
Arabidopsis
(Chr 2, bottom)
Putative root auxin influx
carrier, similar to amino
acid permeases [56]
Root tip and root epidermal cells
Agravitropic, auxin- and ethyleneresistant root growth, enhanced root
elongation
PIN1
Arabidopsis
(Chr 1, bottom)
Putative auxin efflux
carrier [57••]
Vascular tissue (basal end of xylem
cells)
Reduced polar auxin transport, altered
vascular, floral and embryonic
development
EIR1
(= AGR1
= PIN2
= WAV6)
Arabidopsis
(Chr 5, bottom)
Putative root auxin
efflux carrier
[58••,59••,60•,61••]
Basal end of cortical and epidermal
cells in root elongation zone of
light-grown plants [61••]; root and
cotyledon and/or hypocotyl of
dark-grown plants [59••]
Agravitropic root, ethylene and auxin
transport inhibitor-resistant root growth,
enhanced root elongation
Induced leaves in response to
Overexpression increases IAN sensitivity
[10•,17]
pathogens [13] and senescence [12•]
indole-3-acetaldehyde (IAAld) to IAA (Figure 1; [26•] and
references therein) have been identified in several plants.
As the final step of abscisic acid biosynthesis is also catalyzed by an AO [27], it has been difficult to assign in vivo
functions to individual isozymes. The Arabidopsis rty
mutant, which is allelic to sur1, hls3, ivr, and alf1 (Table 1;
reviewed in [1]), overproduces IAA and is defective in a
putative aminotransferase gene [28]. Roots and hypocotyls
of sur1 contain increased AO activity with an apparent
preference for IAAld [29••] and accumulate mRNA from
AtAO-1 [26•], one of four differentially expressed AO genes
[26•,30]. Although the molecular details of RTY action
remain unclear, these observations suggest a role for AtAO1 in IAA biosynthesis. With the AO genes in hand, it is now
possible to screen for insertion mutants. A mutation in
AtAO-1 alone may not affect IAA levels if other IAA
biosynthetic pathways can compensate; however, the
labeling patterns of IAA precursors in such a mutant may
be informative.
Increasing free IAA by conjugate hydrolysis
Most of the IAA in plants is conjugated to a variety of
amino acids, peptides, sugars, and myo-inositol [45]. The
physiological significance of the individual conjugate
moieties is yet to be determined, but one theory is that
they direct the IAA either to catabolism or to storage for
subsequent hydrolosis. Biochemical approaches are being
used to isolate IAA-glucose and IAA-inositol hydrolase
genes [31•], and genetic approaches have uncovered IAAamino acid conjugate hydrolases. A screen for
insensitivity to IAA-Leu identified the Arabidopsis ILR1
gene [32], which is homologous to the gene defective in
iar3, an IAA-Ala insensitive mutant [33••]. Arabidopsis has
a family of at least six ILR1/IAR3-like enzymes [33••].
Whereas past screens for conjugate hydrolysis mutants
have been based on the toxicity of excess free IAA, IAAconjugate derivatives with increased phytotoxicity might
facilitate future screens [34]. Among the conjugates tested, the IAR3 enzyme is specific for IAA-Ala [33••],
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Physiology and metabolism
whereas ILR1 prefers IAA-Phe and IAA-Leu to IAA-Ala
[32]. Comparison of ILR1 and IAR3 to rice homologs suggests that this diversity in specificity arose before the
divergence of monocots and dicots. Interestingly, an
Arabidopsis cDNA (JR3), that is 96% identical to IAR3,
was isolated as a jasmonic acid (JA)-induced gene [35•].
JR3 is also rapidly and transiently induced by wounding,
both at the wound site and systemically [35•]. This
wound- and JA-induced expression is blocked in the JAinsensitive mutant coi1 and is mediated by a
phosphorylation-dependent signal transduction pathway
[35•,36••]. As auxin inhibits the expression of some JAinduced genes, JR3 might be involved in feedback
inhibition of the JA response by hydrolyzing conjugates
and increasing the free IAA concentration [36••]. It will
be interesting to determine whether the other conjugate
hydrolase genes are similarly regulated and whether the
iar3 or iar3 ilr1 mutants show altered responses to JA,
wounding, or pathogens.
Like many IAA metabolic enzymes, IAA-conjugate
hydrolases are not exclusive to plants. The Enterobacter
agglomerans iaaspH gene encodes a hydrolase specific for
IAA-Asp [37•]. This enzyme is 20% identical to the
Arabidopsis ILR1 and IAR3 enzymes. IAA-Asp is of particular importance in plants, as it is found in a variety of
species and can be an intermediate in IAA catabolism (see
below). Thus, overexpressing iaaspH in transgenic plants
might short-circuit IAA inactivation.
Differential regulation of redundant IAA
biosynthetic pathways
bean leaves, but once the leaves dry the decrease in viability is an order of magnitude more severe in the ipdC–
strain. Reporter gene activity indicates that the iaaH promoter is induced dramatically during pathogenic
infection, whereas ipdC expression is lower than that of
iaaH during pathogenic or non-pathogenic propagation.
The regulation of these pathways no doubt determines
overall IAA levels, and this may be the key to their differential utilization.
In addition to developmental regulation, IAA metabolism can be altered by temperature and light. Hypocotyls
of light-grown Arabidopsis seedlings grown at 29°C elongate four- to five-fold more than those of control
seedlings at 20°C. This elongation is blocked in IAA
transport mutants, in auxin response mutants and by the
auxin transport inhibitor NPA [43••]. Endogenous free
IAA and IAA conjugate levels rise under these conditions [43••], implying that increased IAA synthesis and
decreased IAA inactivation mediate the elongation. In
wild-type duckweed, IAA turnover decreases in continuous light compared to darkness, yet IAA levels remain
constant, suggesting that biosynthesis also slows in continuous light. The MTR1 mutant, which contains a
feedback insensitive anthranilate synthase, maintains a
high rate of turnover under all light conditions, but accumulates IAA in continuous light [44•]. Perhaps high
levels of IAA precursors in this strain prevent the normal
light-induced slowing of IAA biosynthesis. As IAA metabolic genes are cloned (Table 1), it will be interesting to
examine transcriptional regulation by such factors as
light and temperature.
Plant developmental processes requiring transient, high
levels of free IAA may rely on a Trp-dependent pathway,
whereas the Trp-independent pathway probably maintains
low levels of IAA during vegetative growth. For example,
IAA levels are low in unfertilized carrot ovules, increase 80fold by the late globular and early heart stages and then
decline to pre-fertilization levels by the torpedo stage (DM
Ribnicky, JD Cohen, W-S Hu, TJ Cooke, personal communication). Conjugated IAA levels also rise, so both synthesis
and inactivation rates are changing. Trp-dependent pathways are active in carrot prior to the onset of somatic
embryogenesis and then subsequently decline [38].
It has long been recognized that higher plants maintain
most IAA as conjugates [45]. Lower land plants (e.g. several liverworts, mosses, a fern, and a lycophyte) contain
significant levels of amide- and ester-linked IAA [46•] and
conjugate exogenous IAA [47], indicating that the ability
to regulate IAA levels via conjugation was an early development of land plants. The liverworts conjugate
exogenous IAA more slowly than the other taxa [46•], suggesting that these plants may rely on alternative means of
IAA inactivation, such as direct oxidation.
Microbes may also differentially regulate multiple IAA
biosynthetic pathways. Microbial IAA production is evident during symbiotic nodule formation, in
rhizosphere-inhabiting bacteria and in many pathogenic
interactions (reviewed in [39–41]). Erwinia herbicola
requires the indole-3-acetamide (IAM) pathway for pathogenicity and the IPA pathway for epiphytic fitness [42•].
Disrupting the IPA decarboxylase gene (ipdC, Figure 1)
from E. herbicola does not affect gall size or formation on
susceptible plants, whereas disrupting the IAM hydrolase
gene (iaaH, Figure 1) decreases gall size. Strains disrupted in either IAA biosynthetic pathway are equally viable
when propagated as non-pathogenic epiphytes on wet
IAA conjugates serve not only as reservoirs of inactive IAA,
but can also function as intermediates in IAA catabolism
(reviewed in [2]). For example, 14-day-old sterile
Arabidopsis seedlings fed low levels of IAA (in darkness)
convert labeled IAA to oxindole-3-acetic acid (OxIAA),
OxIAA-hexose, IAA-Glu and IAA-Asp. IAA-Asp is oxidized on the indole ring, rendering the IAA irreversibly
inactivated, but IAA-Glu is not further metabolized [48••].
Interestingly, when exogenous IAA levels are low IAA oxidation predominates whereas conjugation is more
prevalent at higher levels. This is consistent with the
observation that IAA-Asp formation is induced by high
concentrations of exogenous IAA (reviewed in [2,45]).
IAA inactivation and catabolism
Redundancy as a way of life — IAA metabolism Normanly and Bartel
Like the rty mutant, the Arabidopsis superroot2 (sur2)
mutant displays phenotypes suggestive of auxin overproduction, including epinastic cotyledons and adventitious
hypocotyl rooting. sur2 seedlings have elevated free IAA
and slightly reduced conjugate levels, suggesting a defect
in inactivation [49••], whereas the rty mutant has higher
levels of both free and conjugated IAA, suggesting a defect
in synthesis (reviewed in [1]). Only one gene directly
involved in IAA inactivation has been cloned; the maize
iaglu gene that encodes IAA-glucose synthase [50].
Cloning the SUR2 gene should reveal whether it encodes
another IAA inactivating enzyme.
IAA transport versus metabolism
Auxin transport assays indicate that free IAA is the preferred substrate for the polar auxin transport machinery
[51]. In the absence of details about the localization (or
identity) of the proteins responsible for the synthesis,
inactivation, activation and transport of IAA it has not
been possible to determine the relative contributions of
metabolism and transport to the level of IAA in any given
cell. While inhibition of auxin transport from the shoot to
the root inhibits lateral root formation in Arabidopsis [52•],
it is difficult to imagine that polar transport could be the
sole source of IAA in complex root systems and large trees.
Sundberg and Uggla [53•] replaced the apices of Pinus
sylvestris saplings with 13C6-IAA and measured the ratio of
labeled, exogenous IAA to unlabeled endogenous IAA in
serial stem sections. If the 13C6-IAA were the sole source
of IAA distal to the apex then the ratio of labeled to unlabeled IAA should remain the same throughout the stem.
However, the labeled IAA was diluted, indicating IAA
synthesis or IAA conjugate hydrolysis in regions other
than the apex.
Molecular genetic approaches have recently yielded several genes that may encode the efflux and influx carriers
postulated to transport IAA (reviewed in [54•,55•]). AUX1,
a putative auxin influx carrier, is similar to amino acid permeases and the AUX1 gene is expressed in root tip and root
epidermal cells (Table 1; [56]). PIN1 [57••] localizes to the
basal end of stem xylem cells while EIR1 [58••], which was
also isolated as AGR1 [59••,60•] and PIN2 [61••], localizes
to the basal end of root epidermal cells in the elongation
zone (Table 1). In addition to the localization data, genetic
and biochemical evidence suggest that PIN1 and
EIR1/AGR1/PIN2 may serve as auxin efflux carriers
(reviewed in [54•,55•]). These proteins are encoded by
gene families, so homologs that localize to other tissues
that require auxin transport (e.g. embryos, root cap cells)
should be forthcoming. It will be interesting to compare
the expression patterns of the auxin carriers with those of
enzymes involved in IAA metabolism (Table 1), all of
which potentially modulate free IAA levels.
Conclusions and perspectives
A thorough understanding of IAA metabolism will require
identification and analysis of the intermediates, enzymes,
211
and genes involved in IAA biosynthesis, inactivation, catabolism and transport, as well as of mutants defective in each
pathway. Although the enzymes and intermediates in IAA
biosynthesis have not yet been definitively established, substantial progress is being made on the biochemical
characterization of these pathways. Candidate IAA biosynthetic genes have been identified as members of gene
families (Table 1) and a variety of approaches are available
to assess their substrate specificities and participation in IAA
synthesis. The expanding collection of mutants specifically
defective in IAA metabolic pathways are valuable analytical
tools. The diverse substrate specificities of IAA-conjugate
hydrolases [33••] implies that Arabidopsis contains several
IAA-amino acid conjugates, making identification of the
endogenous conjugates in this species particularly important. Further characterization of the maize iaglu [50] and the
Arabidopsis SUR2 [49••] genes should complement our
largely biochemical understanding of IAA inactivation. The
identification of gene families that are likely to encode
auxin efflux and influx carriers [54•,55•,56,57••–59••,60•,61••] represents an enormous leap forward in the
characterization of IAA transport and ultimately the manner
by which IAA transport ties into IAA metabolism. Now that
many of the genes involved in auxin metabolism have been
isolated (Table 1), analysis of the substrate specificities of
the encoded enzymes, their loss of function phenotypes,
and their expression during development and in response to
environmental challenges will reveal the roles of particular
pathways in regulating free IAA levels.
Acknowledgements
We thank Seiichi Matsuda, John Celenza, and members of the Bartel lab for
critical comments on the manuscript. Work in the authors’ laboratories is
supported by the DOE (DE-FG02-95ER20204, J Normanly), the NSF
(MCB9870798, J Normanly), the NIH (R29 GM54749, B Bartel), and the
Robert A Welch Foundation (C-1309, B Bartel).
References and recommended reading
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• of special interest
•• of outstanding interest
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free IAA levels increase two-fold in senescing leaves, whereas IAN and conjugated IAA levels drop two-fold.
13. Bartel B, Fink GR: Differential regulation of an auxin-producing
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14. Bartling D, Seedorf M, Schmidt RC, Weiler EW: Molecular
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•
Liotenberg S, Marion-Poll A, Caboche M, Kamiya Y, Koshiba T:
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Arabidopsis thaliana. Plant Cell Physiol 1998, 39:433-442.
Arabidopsis encodes at least four aldehyde oxidases. The mRNA for one of
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increased AO activity and higher IAA levels in this mutant thereby implicating aldehyde oxidase in IAA synthesis.
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Aldehyde oxidase activity with a preference for indole acetaldehyde is
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biochemical experiments have revealed multiple inputs and outputs to the
IAA pool in a single tissue.
32. Bartel B, Fink GR: ILR1, an amidohydrolase that releases active
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33. Davies RT, Goetz DH, Lasswell J, Anderson MN, Bartel B: IAR3
•• encodes an auxin conjugate hydrolase from Arabidopsis. Plant
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The IAA-Ala insensitive iar3 mutant was isolated, and the defective gene
shown to encode an IAA conjugate hydrolase with specificity for IAA-Ala.
The IAR3 gene is similar to the previously isolated ILR1 gene [32] and at
least four additional ILR1-like (ILL) genes in Arabidopsis.
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35. Titarenko E, Rojo E, León J, Sánchez-Serrano JJ: Jasmonic acid
•
dependent and -independent signaling pathways control woundinduced gene activation in Arabidopsis thaliana. Plant Physiol
1997, 115:817-826.
The authors use differential display to isolate cDNAs induced by wounding
or JA. One of these genes, JR3, is 96% identical to IAR3, which encodes
an IAA conjugate hydrolase [33••]. JR3 induction by wounding is blocked
in the JA-insensitive coi1 mutant.
36. Rojo E, Titarenko E, León J, Berger S, Vancanneyt G,
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jasmonic acid-dependent and -independent wound signal
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A pharmacological approach is used to determine that wound signal transduction in Arabidopsis involves reversible phosphorylation. JR3 [35•] is
unique among the JA-induced genes studied in that it is induced by cycloheximide and its induction by JA is not reversed by auxin. The authors suggest that JR3 might dampen the JA response by increasing IAA levels.
37.
•
Chou J-C, Mulbry WW, Cohen JD: The gene for indole-3-acetyl-L
aspartic acid hydrolase from Enterobacter agglomerans:
molecular cloning, nucleotide sequence, and expression in
Escherichia coli. Mol Gen Genet 1998, 259:172-178.
The bacterial iaaspH gene, encoding an IAA-Asp hydrolase similar to
Arabidopsis IAA-amino acid hydrolases, was cloned. The authors suggest
that overexpression of iaaspH in plants might disrupt IAA inactivation.
Redundancy as a way of life — IAA metabolism Normanly and Bartel
38. Michalczuk L, Ribnicky DM, Cooke TJ, Cohen JD: Regulation of
indole-3-acetic acid biosynthetic pathways in carrot cell cultures.
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•
Differential involvement of indole-3-acetic acid biosynthetic
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The construction of mutant E. herbicola strains with defects in the IAM pathway, the IPA pathway, or both sheds light upon the differential utilization of
these two pathways.
43. Gray WM, Östin A, Sandberg G, Romano CP, Estelle M: High
•• temperature promotes auxin-mediated hypocotyl elongation in
Arabidopsis. Proc Natl Acad Sci USA 1998, 95:7197-7202.
High temperature is found to promote hypocotyl elongation in the light, and
is accompanied by an increase in free and conjugated IAA levels. The expansion is specifically blocked in IAA transport and response mutants.
44. Tam YY, Slovin JP, Cohen JD: Continuous light alters indole-3
•
acetic acid metabolism in Lemna gibba. Phytochemistry 1998,
49:17-21.
The authors measured IAA turnover in wild-type and mutant duckweed fronds
under various light conditions. For wild-type, IAA levels remain constant in either
continuous light or darkness due to adjustments in the rates of biosynthesis and
turnover. A feedback-insensitive anthranilate synthase mutant accumulated IAA,
presumably because IAA biosynthesis rates were less well modulated.
45. Cohen JD, Bandurski RS: Chemistry and physiology of the bound
auxins. Annu Rev Plant Physiol 1982, 33:403-430.
46. Sztein AE, Cohen JD, García de la Fuente I, Cooke TJ: Auxin
•
metabolism in mosses and liverworts. Amer J Bot 1999, in press.
IAA metabolism was characterized in five liverworts, four mosses, and two
tracheophytes. The liverworts and mosses have low levels of both free and
conjugated IAA, whereas the tracheophhytes have low free IAA but high levels of conjugates. Conjugation rates are faster in the mosses and tracheophytes than in the liverworts.
47.
Sztein AE, Cohen JD, Slovin JP, Cooke TJ: Auxin metabolism in
representative land plants. Amer J Botany 1995, 82:1514-1521.
48. Östin A, Kowalyczk M, Bhalerao RP, Sandberg G: Metabolism of
•• indole-3-acetic acid in Arabidopsis. Plant Physiol 1998, 118:285-296.
The authors fed Arabidopsis seedlings low levels of labeled IAA and followed its metabolism using MS. Oxidation was most prevalent at low IAA
concentrations and formation of conjugates with Asp and Glu was observed
at higher IAA concentrations. The authors also show that the IAA-Asp
becomes oxidized, indicating that this conjugate can be an intermediate in
the irreversible inactivation of IAA in Arabidopsis, as it is in other plants.
49. Delarue M, Prinsen E, Van Onckelen H, Caboche M, Bellini C: Sur2
•• mutations of Arabidopsis thaliana define a new locus involved in
the control of auxin homeostasis. Plant Journal 1998, 14:603-611.
The superroot2 mutant contains elevated levels of free IAA and displays phenotypes that reflect this increase. The decrease of conjugated IAA in this
mutant suggests that it is defective in IAA inactivation.
50. Szerszen JD, Szczyglowski K, Bandurski RS: iaglu, a gene from Zea
mays involved in conjugation of growth hormone indole-3-acetic
acid. Science 1994, 265:1699-1701.
51. Lomax TL, Muday GK, Rubery PH: Auxin transport. In Plant
Hormones. Edited by Davies PJ . Dordrecht, The Netherlands: Kluwer
Academic Publishers; 1995:509-530.
213
52. Reed RC, Brady SR, Muday GK: Inhibition of auxin movement from
•
the shoot to the root inhibits lateral root development in
Arabidopsis. Plant Physiol 1998, 118:1369-1378.
Local applications of an auxin transport inhibitor at the root-shoot junction
prevented formation of lateral roots without affecting root elongation, and
this inhibition was reversed after application of IAA. These results suggest
that IAA from the shoot is required for lateral root induction.
53. Sundberg B, Uggla C: Origin and dynamics of indoleacteic acid
•
under polar transport in Pinus sylvestris. Physiologia Plantarum
1998, 104:22-29.
The apical source of IAA in pine saplings was replaced with 13C6-IAA. The
decreasing ratio of labeled to unlabeled IAA in serial stem sections indicates
that apical IAA is not the only source of free IAA in these trees.
54. Jones A: Auxin transport: down and out and up again. Science
•
1998, 282:2201-2202.
Review article summarizing the recent cloning of genes likely to be central to
polar auxin transport.
55. Estelle M: Polar auxin transport: new support for an old model.
•
Plant Cell 1998, 10:1775-1778.
Review article summarizing the recent cloning of genes likely to be central to
polar auxin transport.
56. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,
Walker AR, Schultz B, Feldmann KA: Arabidopsis AUX1 gene: a
permease-like regulator of root gravitropism. Science 1996,
273:948-950.
57.
••
Gälweiler L, Guan C, Müller A, Wiseman E, Mendgen K, Yephremov A,
Palme K: Regulation of polar auxin transport by AtPIN1 in
Arabidopsis vascular tissue. Science 1998, 282:2226-2230.
The pin1 mutant, which has a pin-formed inflorescence and defects in embryo
symmetry, was previously shown to be defective in polar auxin transport. Here
the PIN1 gene was isolated by transposon tagging and shown to be
expressed in all organs of the plant. The PIN1 protein was immunolocalized
to the basal end of elongated xylem cells in stems, consistent with its proposed role as an auxin efflux carrier.
58. Luschnig C, Gaxiola RA, Grisafi P, Fink GR: EIR1, a root-specific
•• protein involved in auxin transport, is required for gravitropism in
Arabidopsis thaliana. Genes Dev 1998, 12:2175-2187.
The eir1 mutant, which was previously isolated based on ethylene insensitivity in the root, has agravitropic roots and is allelic with agr1, pin2 and
wav6–52. The EIR1 gene was cloned by transposon tagging [58••], positional cloning [59••,60•], and homology to PIN1 [61••], and is expressed in
roots [58••,59••,60•,61••]. Double mutant analysis indicates that eir1 roots
have reduced sensitivity to endogenous auxin [58••]. EIR1 expressed in
yeast increases resistance to fluorinated indolic compounds [58,59], consistent with its proposed role as a auxin efflux carrier.
59. Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH: The
•• Arabidopsis thaliana AGRAVITROPIC1 gene encodes a
component of the polar-auxin-transport efflux carrier. Proc Natl
Acad Sci USA 1998, 95:15112-15117.
See [58••]. AGR1 expressed in yeast promotes increased IAA efflux, consistent with its proposed role as an auxin efflux carrier.
60. Utsuno K, Shikanai T, Yamada Y, Hashimoto T: AGR, an Agravitropic
•
locus of Arabidopsis thaliana, encodes a novel membrane-protein
family member. Plant Cell Physiol 1998, 39:1111-1118.
See [58••]. Vertically grown agr1 roots grow into media containing IAA or
NAA, but not media containing 2,4-D. Since 2,4-D diffuses out of cells
whereas IAA and NAA are transported via an efflux carrier, this result suggests a specific defect in auxin efflux.
61. Müller A, Guan C, Gälweiler L, Tänzler P, Huijser P, Marchant A,
•• Parry G, Bennett M, Wisman E, Palme K: AtPIN2 defines a locus of
Arabidopsis for root gravitropism control. EMBO J 1998,
17:6903-6911.
See [58••]. The PIN2 protein was immunolocalized to the basal end of epidermis cells in the root elongation zone, consistent with its propsed role as
an auxin efflux carrier.
62. Iyer M, Cohen JD, Slovin JP: Molecular manipulation of IAA
metabolism in tomato (Abstract). Plant Physiol 1997, 114(suppl.):158.
Redundancy as a way of life — IAA metabolism
Jennifer Normanly* and Bonnie Bartel†
Plants have evolved elaborate systems for regulating cellular
levels of indole-3-acetic acid (IAA). The redundancy of this
network has complicated the elucidation of IAA metabolism,
but molecular genetic studies and precise analytical
methods have begun to expose the circuitry. It is now clear
that plants synthesize, inactivate and catabolize IAA by
multiple pathways, and multiple genes can encode a
particular enzyme within a pathway. A number of these
genes are now cloned, which greatly facilitates the future
dissection of IAA metabolism.
Addresses
*Department of Biochemistry and Molecular Biology, University of
Massachusetts, Amherst, MA 01003, USA;
e-mail: [email protected]
†Department of Biochemistry and Cell Biology, Rice University,
Houston, TX 77005, USA; e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:207–213
may be overestimated. Rapparini et al. [4••] simultaneously pulse-labeled duckweed fronds with 15N-anthranilate
and 2H5-Trp and monitored incorporation of label into
IAA and Trp. Within the first hour, IAA incorporated 15N
from anthranilate faster than did Trp, indicating that initial synthesis of IAA was Trp-independent. By two hours
post-labeling, Trp was equally labeled with 15N and 2H5,
yet the IAA pool was more enriched with 2H5 than 15N.
Thus, exogenous 2H5-Trp is more accessible to IAA synthesis than denovo synthesized N-Trp. Previous work has
suggested that plants have at least two physically separate
Trp pools, one of which may be vacuolar [5]. Future studies will need to account for and identify the cellular
locations of multiple precursor pools. Further characterization of Trp-dependent IAA synthesis will be facilitated
by recently identified indole analogs that inhibit the conversion of 14C-indole to 14C-IAA but not the conversion of
14C-indole to 14C-Trp [6].
http://biomednet.com/elecref/1369526600200207
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
AO
aldehyde oxidase
IAA
indole-3-acetic acid
IAAld
indole-3-acetaldehyde
IAM
indole-3-acetamide
IAN
indole-3-acetonitrile
IPA
indole-3-pyruvic acid
JA
jasmonic acid
Introduction
The coordinated control of indole-3-acetic acid (IAA)
biosynthesis, inactivation, catabolism and transport is a very
dynamic process that responds to developmental and environmental signals. Biochemical approaches have
demonstrated that plants can synthesize IAA from tryptophan (Trp), and several Trp-dependent pathways have been
proposed (Figure 1; reviewed in [1]). Stable isotope labeling
of intact plants has revealed that IAA synthesis can also
occur from a Trp precursor in Trp-independent pathways
(Figure 1; reviewed in [1–3]). Trp-dependent pathways predominate during early embryogenesis and seed
germination, whereas Trp-independent pathways predominate during later embryogenesis and vegetative growth
[2,3]. IAA is inactivated either reversibly through conjugation, or irreversibly by oxidizing IAA or IAA-conjugates
(reviewed in [2,3]). The components of these pathways
have been recalcitrant to biochemical approaches and are
only now being identified. Here we focus on recent progress
in the characterization of IAA metabolism and transport.
Tryptophan as an IAA precursor
In vivo labeling experiments with maize, Arabidopsis, carrot and duckweed indicate that Trp can be metabolized to
IAA ([1–3]); however, its contribution to IAA synthesis
Although several pathways have been proposed for Trpdependent IAA synthesis (Figure 1), both Trp-dependent
and Trp-independent pathways remain poorly defined in
terms of the enzymes, their intermediates and cellular
locations. Recently, an in vitro assay for Trp-independent
conversion of 14C-indole to 14C-IAA has been developed
using extracts from maize seedlings [7••]. The pH (8 to
8.5) optimum and requirement for a reducing environment
suggest that the activity is plastid-localized.
Establishing a role for nitrilases in
IAA biosynthesis
The indole-3-acetonitrile (IAN) pathway (Figure 1) is
apparently limited to three plant families [8] and most of the
characterization of this pathway has been done in the
Brassicaceae ([9] and references therein). Crucifers can convert IAN to IAA, but the redundancy of nitrilase genes
confounds genetic analysis [10•] and the potential for nonenzymatic conversion of IAN to IAA complicates
biochemical analysis [11]. The cumulative data suggest that
if nitrilase converts IAN to IAA in vivo, then the rate-limiting step is substrate access. Since the subcellular localization
of nitrilase and IAN are not well defined, how and when this
pathway is utilized are major unanswered questions. There
is some evidence that IAN utilization could be developmentally controlled [12•,13] and pathogen-induced [9,13].
Micromolar concentrations of exogenous IAN inhibit
Arabidopsis seedling growth in a manner similar to that of
excess IAA [10•]. Arabidopsis encodes four differentially
expressed nitrilases [13–15] and each converts IAN to IAA
in vitro. NIT1 and NIT2 have Km values for IAN in the
mM range [13,14,16] and IAN is present in seedlings at
~3–6 nmol/g fresh weight. In vivo labeling studies demonstrated exogenous 13C-IAN conversion to 13C-IAA in
208
Physiology and metabolism
Figure 1
de novo IAA biosynthesis
Trp biosynthesis
IAA inactivation and conjugate hydrolysis
O
OH
OH
O
O
OH
O
O
N
H
Chorismate
Anthranilate
Trp-ind
e
penden
5-Phosphoribosylanthranilate
HO
t IAA sy
IAA-glucose
synthase
(iaglu)
nthesis
Indole-3-methylglucosinolate
1-(o-carboxyphenylamino)-1deoxyribulose 5-P
IPA
decarboxylase
(ipdC∗)
+ Serine
N
H
se
ra
Trpnsfe
a
r
t
ino
Indole
am
COOH
O
N
H
)
*
aH
O
N
H
Indole-3-acetaldehyde
(IAAld)
Indole-3-pyruvic acid
(IPA)
O
Trp monoxygenase (iaaM*)
N
H
M
IA
?
IAA-amino acid
hydrolases
(ILR1, IAR3)
H
N
dr
hy
O
OxIAA
IAA
ia
e(
s
ola
COOH
N
H
O
COOH
R
N
H
NH2
IAA-amino acid conjugates
N
H
N
H
Trp
Indole-3-acetamide
(IAM)
Oxidized
IAA-amino acid conjugates
NH2
IAAinositol
hydrolase
IAA-glucose
hydrolase
H
-1)
Indole-3-acetonitrile
AO
(At
e
(IAN)
as
xid
ld o
A
A
I
COOH
OH
OH
COOH
N
H
Indole-3-glycerol
phosphate
HO
IAA-myo-inositol
Nitrilase
(NIT1, 2)
N
Indole-3-acetaldoxime
N
H
OH
IAA-glucose OH
Current Opinion in Plant Biology
Simplified diagram of IAA metabolism. IAA can be synthesized via
several Trp-dependent pathways (open arrows) that are named after
unique intermediates, or by a Trp-independent pathway (dashed arrow)
using indole as a precursor. Genes are in italics, microbial genes are
marked with an asterisk. Further catabolism of oxidized IAA and
IAA–Asp is reviewed in [3]. IAA–myo-inositol can be further
metabolized to other conjugates, many of which can also be
hydrolyzed [31•]. It is not known whether IAA–amino acid conjugates
are synthesized directly or use IAA–glucose as an intermediate.
wild-type Arabidopsis and also in transgenic tobacco and
Arabidopsis overexpressing NIT2 [10•,17]. In response to
exogenous IAN, wild-type Arabidopsis accumulates nitrilase protein, and NIT1, NIT2 and NIT4 gene expression is
induced 1.4-, 21- and 4.8-fold, respectively [18].
Overexpressing nitrilase genes in Arabidopsis or tobacco
also increases nitrilase protein and activity [17,18], yet only
NIT2 overexpressing lines show increased sensitivity to
exogenous IAN [10•,17]. An Arabidopsis nit1 mutant is
resistant to IAN, but metabolizes IAN normally and is
morphologically normal in the absence of exogenous IAN
[10•]. The existence of three wild-type nitrilases (NIT2–4)
may compensate for the nit1 defect, or tissue-specific differences in IAN metabolism are masked in whole
seedlings. Label from 2H5-Trp is incorporated into both
IAN and IAA when added to 5 week-old Arabidopsis root
explants [19]. NIT1 is expressed in five-week-old
Arabidopsis roots [13,19], but it is important to consider that
exogenous Trp can be preferentially utilized for IAA synthesis [4••], and that Trp-dependent IAA biosynthetic
pathways can be activated by wounding [20,21].
siliques [13], but is dramatically induced by pathogenic
bacteria [13], exogenous IAN [18], and at the onset of
senescence [12•]. In senescing Arabidopsis leaves, free IAA
levels increase two-fold, whereas IAN and conjugated IAA
levels drop two-fold [12•]. Infection of Arabidopsis or
Chinese cabbage with Plasmodiophora brassicae, the causal
agent of clubroot disease, increases nitrilase activity [9]
and perturbs IAA metabolism [22]. Arabidopsis mutants
with diminished indole-3-methyl-glucosinolate levels (a
putative IAN precursor, Figure 1) appear unaffected in
IAA synthesis and turnover under normal conditions, but
upon P. brassicae infection have lower IAA and IAN levels
and less severe clubroot symptoms than wild-type [23].
There is correlative evidence for IAN pathway utilization
in response to specific developmental and environmental
cues. NIT2 is normally expressed at levels four- to eightfold lower than NIT1 and is restricted to root tips and
Indole pyruvic acid pathway of
IAA biosynthesis
Lastly, two-hybrid screens have identified a nitrilase-associated protein in Arabidopsis (Genbank accession number
Z96936), and a nitrilase-like protein in tobacco that interacts
with an ethylene-response element binding protein [24]. The
functions of these proteins remain to be determined but they
are intriguing in light of the need for some event that renders
endogenous IAN available to nitrilase for IAA synthesis.
Indole-3-pyruvic acid (IPA) ([25] and references therein)
and aldehyde oxidase (AO) activities that convert
Redundancy as a way of life — IAA metabolism Normanly and Bartel
209
Table 1
Plant genes potentially involved in IAA metabolism and transport.
Gene
Species
(Map position)
Gene product
Expression pattern
Mutant phenotype
NIT1
Arabidopsis
(Chr 3, middle)
Nitrilase
Expressed strongly at base of
hypocotyl [13], where exogenous
IAN induces lateral roots [10•]
IAN insensitive hypocotyl and roots [10•]
NIT2
Arabidopsis
(Chr 3, middle)
Nitrilase
AtAO-1
Arabidopsis
(Chr 5, middle)
Aldehyde oxidase [26•,30]
Expressed in roots, induced in IAA
overproducing mutant (sur1)
[26•,29••]
?
ILR1
Arabidopsis
(Chr 3, top)
IAA–amino acid
hydrolase
?
IAA–Leu insensitive root growth [32]
IAR3
Arabidopsis
(Chr 1, middle)
IAA–amino acid
hydrolase
Developmentally expressed in
roots, induced in leaves in response
to JA and wounding [35•]
IAA–Ala insensitive root growth [33••]
iaglu
Maize
IAA–glucose conjugating
enzyme [50]
?
Transgenic tomato that overexpress maize
iaglu are unable to form roots [62]
RTY
(= ALF1
= SUR1
= HLS3)
Arabidopsis
(Chr 2, middle)
Aminotransferase [28]
?
Overproduces IAA and IAA conjugates,
adventitious rooting, epinastic cotyledons
(reviewed in [1]); increased AtAO-1
expression [26•] and AO activity [29••]
SUR2
Arabidopsis
(Chr 4, bottom)
IAA inactivation?
?
Overproduces IAA, adventitious rooting,
epinastic cotyledons [49••]
AUX1
Arabidopsis
(Chr 2, bottom)
Putative root auxin influx
carrier, similar to amino
acid permeases [56]
Root tip and root epidermal cells
Agravitropic, auxin- and ethyleneresistant root growth, enhanced root
elongation
PIN1
Arabidopsis
(Chr 1, bottom)
Putative auxin efflux
carrier [57••]
Vascular tissue (basal end of xylem
cells)
Reduced polar auxin transport, altered
vascular, floral and embryonic
development
EIR1
(= AGR1
= PIN2
= WAV6)
Arabidopsis
(Chr 5, bottom)
Putative root auxin
efflux carrier
[58••,59••,60•,61••]
Basal end of cortical and epidermal
cells in root elongation zone of
light-grown plants [61••]; root and
cotyledon and/or hypocotyl of
dark-grown plants [59••]
Agravitropic root, ethylene and auxin
transport inhibitor-resistant root growth,
enhanced root elongation
Induced leaves in response to
Overexpression increases IAN sensitivity
[10•,17]
pathogens [13] and senescence [12•]
indole-3-acetaldehyde (IAAld) to IAA (Figure 1; [26•] and
references therein) have been identified in several plants.
As the final step of abscisic acid biosynthesis is also catalyzed by an AO [27], it has been difficult to assign in vivo
functions to individual isozymes. The Arabidopsis rty
mutant, which is allelic to sur1, hls3, ivr, and alf1 (Table 1;
reviewed in [1]), overproduces IAA and is defective in a
putative aminotransferase gene [28]. Roots and hypocotyls
of sur1 contain increased AO activity with an apparent
preference for IAAld [29••] and accumulate mRNA from
AtAO-1 [26•], one of four differentially expressed AO genes
[26•,30]. Although the molecular details of RTY action
remain unclear, these observations suggest a role for AtAO1 in IAA biosynthesis. With the AO genes in hand, it is now
possible to screen for insertion mutants. A mutation in
AtAO-1 alone may not affect IAA levels if other IAA
biosynthetic pathways can compensate; however, the
labeling patterns of IAA precursors in such a mutant may
be informative.
Increasing free IAA by conjugate hydrolysis
Most of the IAA in plants is conjugated to a variety of
amino acids, peptides, sugars, and myo-inositol [45]. The
physiological significance of the individual conjugate
moieties is yet to be determined, but one theory is that
they direct the IAA either to catabolism or to storage for
subsequent hydrolosis. Biochemical approaches are being
used to isolate IAA-glucose and IAA-inositol hydrolase
genes [31•], and genetic approaches have uncovered IAAamino acid conjugate hydrolases. A screen for
insensitivity to IAA-Leu identified the Arabidopsis ILR1
gene [32], which is homologous to the gene defective in
iar3, an IAA-Ala insensitive mutant [33••]. Arabidopsis has
a family of at least six ILR1/IAR3-like enzymes [33••].
Whereas past screens for conjugate hydrolysis mutants
have been based on the toxicity of excess free IAA, IAAconjugate derivatives with increased phytotoxicity might
facilitate future screens [34]. Among the conjugates tested, the IAR3 enzyme is specific for IAA-Ala [33••],
210
Physiology and metabolism
whereas ILR1 prefers IAA-Phe and IAA-Leu to IAA-Ala
[32]. Comparison of ILR1 and IAR3 to rice homologs suggests that this diversity in specificity arose before the
divergence of monocots and dicots. Interestingly, an
Arabidopsis cDNA (JR3), that is 96% identical to IAR3,
was isolated as a jasmonic acid (JA)-induced gene [35•].
JR3 is also rapidly and transiently induced by wounding,
both at the wound site and systemically [35•]. This
wound- and JA-induced expression is blocked in the JAinsensitive mutant coi1 and is mediated by a
phosphorylation-dependent signal transduction pathway
[35•,36••]. As auxin inhibits the expression of some JAinduced genes, JR3 might be involved in feedback
inhibition of the JA response by hydrolyzing conjugates
and increasing the free IAA concentration [36••]. It will
be interesting to determine whether the other conjugate
hydrolase genes are similarly regulated and whether the
iar3 or iar3 ilr1 mutants show altered responses to JA,
wounding, or pathogens.
Like many IAA metabolic enzymes, IAA-conjugate
hydrolases are not exclusive to plants. The Enterobacter
agglomerans iaaspH gene encodes a hydrolase specific for
IAA-Asp [37•]. This enzyme is 20% identical to the
Arabidopsis ILR1 and IAR3 enzymes. IAA-Asp is of particular importance in plants, as it is found in a variety of
species and can be an intermediate in IAA catabolism (see
below). Thus, overexpressing iaaspH in transgenic plants
might short-circuit IAA inactivation.
Differential regulation of redundant IAA
biosynthetic pathways
bean leaves, but once the leaves dry the decrease in viability is an order of magnitude more severe in the ipdC–
strain. Reporter gene activity indicates that the iaaH promoter is induced dramatically during pathogenic
infection, whereas ipdC expression is lower than that of
iaaH during pathogenic or non-pathogenic propagation.
The regulation of these pathways no doubt determines
overall IAA levels, and this may be the key to their differential utilization.
In addition to developmental regulation, IAA metabolism can be altered by temperature and light. Hypocotyls
of light-grown Arabidopsis seedlings grown at 29°C elongate four- to five-fold more than those of control
seedlings at 20°C. This elongation is blocked in IAA
transport mutants, in auxin response mutants and by the
auxin transport inhibitor NPA [43••]. Endogenous free
IAA and IAA conjugate levels rise under these conditions [43••], implying that increased IAA synthesis and
decreased IAA inactivation mediate the elongation. In
wild-type duckweed, IAA turnover decreases in continuous light compared to darkness, yet IAA levels remain
constant, suggesting that biosynthesis also slows in continuous light. The MTR1 mutant, which contains a
feedback insensitive anthranilate synthase, maintains a
high rate of turnover under all light conditions, but accumulates IAA in continuous light [44•]. Perhaps high
levels of IAA precursors in this strain prevent the normal
light-induced slowing of IAA biosynthesis. As IAA metabolic genes are cloned (Table 1), it will be interesting to
examine transcriptional regulation by such factors as
light and temperature.
Plant developmental processes requiring transient, high
levels of free IAA may rely on a Trp-dependent pathway,
whereas the Trp-independent pathway probably maintains
low levels of IAA during vegetative growth. For example,
IAA levels are low in unfertilized carrot ovules, increase 80fold by the late globular and early heart stages and then
decline to pre-fertilization levels by the torpedo stage (DM
Ribnicky, JD Cohen, W-S Hu, TJ Cooke, personal communication). Conjugated IAA levels also rise, so both synthesis
and inactivation rates are changing. Trp-dependent pathways are active in carrot prior to the onset of somatic
embryogenesis and then subsequently decline [38].
It has long been recognized that higher plants maintain
most IAA as conjugates [45]. Lower land plants (e.g. several liverworts, mosses, a fern, and a lycophyte) contain
significant levels of amide- and ester-linked IAA [46•] and
conjugate exogenous IAA [47], indicating that the ability
to regulate IAA levels via conjugation was an early development of land plants. The liverworts conjugate
exogenous IAA more slowly than the other taxa [46•], suggesting that these plants may rely on alternative means of
IAA inactivation, such as direct oxidation.
Microbes may also differentially regulate multiple IAA
biosynthetic pathways. Microbial IAA production is evident during symbiotic nodule formation, in
rhizosphere-inhabiting bacteria and in many pathogenic
interactions (reviewed in [39–41]). Erwinia herbicola
requires the indole-3-acetamide (IAM) pathway for pathogenicity and the IPA pathway for epiphytic fitness [42•].
Disrupting the IPA decarboxylase gene (ipdC, Figure 1)
from E. herbicola does not affect gall size or formation on
susceptible plants, whereas disrupting the IAM hydrolase
gene (iaaH, Figure 1) decreases gall size. Strains disrupted in either IAA biosynthetic pathway are equally viable
when propagated as non-pathogenic epiphytes on wet
IAA conjugates serve not only as reservoirs of inactive IAA,
but can also function as intermediates in IAA catabolism
(reviewed in [2]). For example, 14-day-old sterile
Arabidopsis seedlings fed low levels of IAA (in darkness)
convert labeled IAA to oxindole-3-acetic acid (OxIAA),
OxIAA-hexose, IAA-Glu and IAA-Asp. IAA-Asp is oxidized on the indole ring, rendering the IAA irreversibly
inactivated, but IAA-Glu is not further metabolized [48••].
Interestingly, when exogenous IAA levels are low IAA oxidation predominates whereas conjugation is more
prevalent at higher levels. This is consistent with the
observation that IAA-Asp formation is induced by high
concentrations of exogenous IAA (reviewed in [2,45]).
IAA inactivation and catabolism
Redundancy as a way of life — IAA metabolism Normanly and Bartel
Like the rty mutant, the Arabidopsis superroot2 (sur2)
mutant displays phenotypes suggestive of auxin overproduction, including epinastic cotyledons and adventitious
hypocotyl rooting. sur2 seedlings have elevated free IAA
and slightly reduced conjugate levels, suggesting a defect
in inactivation [49••], whereas the rty mutant has higher
levels of both free and conjugated IAA, suggesting a defect
in synthesis (reviewed in [1]). Only one gene directly
involved in IAA inactivation has been cloned; the maize
iaglu gene that encodes IAA-glucose synthase [50].
Cloning the SUR2 gene should reveal whether it encodes
another IAA inactivating enzyme.
IAA transport versus metabolism
Auxin transport assays indicate that free IAA is the preferred substrate for the polar auxin transport machinery
[51]. In the absence of details about the localization (or
identity) of the proteins responsible for the synthesis,
inactivation, activation and transport of IAA it has not
been possible to determine the relative contributions of
metabolism and transport to the level of IAA in any given
cell. While inhibition of auxin transport from the shoot to
the root inhibits lateral root formation in Arabidopsis [52•],
it is difficult to imagine that polar transport could be the
sole source of IAA in complex root systems and large trees.
Sundberg and Uggla [53•] replaced the apices of Pinus
sylvestris saplings with 13C6-IAA and measured the ratio of
labeled, exogenous IAA to unlabeled endogenous IAA in
serial stem sections. If the 13C6-IAA were the sole source
of IAA distal to the apex then the ratio of labeled to unlabeled IAA should remain the same throughout the stem.
However, the labeled IAA was diluted, indicating IAA
synthesis or IAA conjugate hydrolysis in regions other
than the apex.
Molecular genetic approaches have recently yielded several genes that may encode the efflux and influx carriers
postulated to transport IAA (reviewed in [54•,55•]). AUX1,
a putative auxin influx carrier, is similar to amino acid permeases and the AUX1 gene is expressed in root tip and root
epidermal cells (Table 1; [56]). PIN1 [57••] localizes to the
basal end of stem xylem cells while EIR1 [58••], which was
also isolated as AGR1 [59••,60•] and PIN2 [61••], localizes
to the basal end of root epidermal cells in the elongation
zone (Table 1). In addition to the localization data, genetic
and biochemical evidence suggest that PIN1 and
EIR1/AGR1/PIN2 may serve as auxin efflux carriers
(reviewed in [54•,55•]). These proteins are encoded by
gene families, so homologs that localize to other tissues
that require auxin transport (e.g. embryos, root cap cells)
should be forthcoming. It will be interesting to compare
the expression patterns of the auxin carriers with those of
enzymes involved in IAA metabolism (Table 1), all of
which potentially modulate free IAA levels.
Conclusions and perspectives
A thorough understanding of IAA metabolism will require
identification and analysis of the intermediates, enzymes,
211
and genes involved in IAA biosynthesis, inactivation, catabolism and transport, as well as of mutants defective in each
pathway. Although the enzymes and intermediates in IAA
biosynthesis have not yet been definitively established, substantial progress is being made on the biochemical
characterization of these pathways. Candidate IAA biosynthetic genes have been identified as members of gene
families (Table 1) and a variety of approaches are available
to assess their substrate specificities and participation in IAA
synthesis. The expanding collection of mutants specifically
defective in IAA metabolic pathways are valuable analytical
tools. The diverse substrate specificities of IAA-conjugate
hydrolases [33••] implies that Arabidopsis contains several
IAA-amino acid conjugates, making identification of the
endogenous conjugates in this species particularly important. Further characterization of the maize iaglu [50] and the
Arabidopsis SUR2 [49••] genes should complement our
largely biochemical understanding of IAA inactivation. The
identification of gene families that are likely to encode
auxin efflux and influx carriers [54•,55•,56,57••–59••,60•,61••] represents an enormous leap forward in the
characterization of IAA transport and ultimately the manner
by which IAA transport ties into IAA metabolism. Now that
many of the genes involved in auxin metabolism have been
isolated (Table 1), analysis of the substrate specificities of
the encoded enzymes, their loss of function phenotypes,
and their expression during development and in response to
environmental challenges will reveal the roles of particular
pathways in regulating free IAA levels.
Acknowledgements
We thank Seiichi Matsuda, John Celenza, and members of the Bartel lab for
critical comments on the manuscript. Work in the authors’ laboratories is
supported by the DOE (DE-FG02-95ER20204, J Normanly), the NSF
(MCB9870798, J Normanly), the NIH (R29 GM54749, B Bartel), and the
Robert A Welch Foundation (C-1309, B Bartel).
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polar auxin transport.
55. Estelle M: Polar auxin transport: new support for an old model.
•
Plant Cell 1998, 10:1775-1778.
Review article summarizing the recent cloning of genes likely to be central to
polar auxin transport.
56. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,
Walker AR, Schultz B, Feldmann KA: Arabidopsis AUX1 gene: a
permease-like regulator of root gravitropism. Science 1996,
273:948-950.
57.
••
Gälweiler L, Guan C, Müller A, Wiseman E, Mendgen K, Yephremov A,
Palme K: Regulation of polar auxin transport by AtPIN1 in
Arabidopsis vascular tissue. Science 1998, 282:2226-2230.
The pin1 mutant, which has a pin-formed inflorescence and defects in embryo
symmetry, was previously shown to be defective in polar auxin transport. Here
the PIN1 gene was isolated by transposon tagging and shown to be
expressed in all organs of the plant. The PIN1 protein was immunolocalized
to the basal end of elongated xylem cells in stems, consistent with its proposed role as an auxin efflux carrier.
58. Luschnig C, Gaxiola RA, Grisafi P, Fink GR: EIR1, a root-specific
•• protein involved in auxin transport, is required for gravitropism in
Arabidopsis thaliana. Genes Dev 1998, 12:2175-2187.
The eir1 mutant, which was previously isolated based on ethylene insensitivity in the root, has agravitropic roots and is allelic with agr1, pin2 and
wav6–52. The EIR1 gene was cloned by transposon tagging [58••], positional cloning [59••,60•], and homology to PIN1 [61••], and is expressed in
roots [58••,59••,60•,61••]. Double mutant analysis indicates that eir1 roots
have reduced sensitivity to endogenous auxin [58••]. EIR1 expressed in
yeast increases resistance to fluorinated indolic compounds [58,59], consistent with its proposed role as a auxin efflux carrier.
59. Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH: The
•• Arabidopsis thaliana AGRAVITROPIC1 gene encodes a
component of the polar-auxin-transport efflux carrier. Proc Natl
Acad Sci USA 1998, 95:15112-15117.
See [58••]. AGR1 expressed in yeast promotes increased IAA efflux, consistent with its proposed role as an auxin efflux carrier.
60. Utsuno K, Shikanai T, Yamada Y, Hashimoto T: AGR, an Agravitropic
•
locus of Arabidopsis thaliana, encodes a novel membrane-protein
family member. Plant Cell Physiol 1998, 39:1111-1118.
See [58••]. Vertically grown agr1 roots grow into media containing IAA or
NAA, but not media containing 2,4-D. Since 2,4-D diffuses out of cells
whereas IAA and NAA are transported via an efflux carrier, this result suggests a specific defect in auxin efflux.
61. Müller A, Guan C, Gälweiler L, Tänzler P, Huijser P, Marchant A,
•• Parry G, Bennett M, Wisman E, Palme K: AtPIN2 defines a locus of
Arabidopsis for root gravitropism control. EMBO J 1998,
17:6903-6911.
See [58••]. The PIN2 protein was immunolocalized to the basal end of epidermis cells in the root elongation zone, consistent with its propsed role as
an auxin efflux carrier.
62. Iyer M, Cohen JD, Slovin JP: Molecular manipulation of IAA
metabolism in tomato (Abstract). Plant Physiol 1997, 114(suppl.):158.