Auxin in action: Signalling, transport and the control of plant growth and development

Abstract

Hormones have been at the centre of plant physiology research for more than a century. Research into plant hormones (phytohormones) has at times been considered as a rather vague subject, but the systematic application of genetic and molecular techniques has led to key insights that have revitalized the field. In this review, we will focus on the plant hormone auxin and its action. We will highlight recent mutagenesis and molecular studies, which have delineated the pathways of auxin transport, perception and signal transduction, and which together define the roles of auxin in controlling growth and patterning.

Full text

The word ‘
αυξανω
´
’ in Greek, or ‘to grow’ in English,
gives us ‘auxin’: the name of a small class of molecules
with a powerful ability to induce growth responses in
plants. In plants, growth is defined as an irreversible
increase in size, and is achieved by the enlargement of
individual cells driven by the uptake of water. Auxin
refers to an important group of phytohormones that
has been implicated in most of the quantitative growth
changes that occur during a plant’s life cycle. Exactly
how these changes are brought about is now becoming
clear after more than a century of research. We will
give an overview of auxin research, and summarize the
remarkable advances that have been made over the past
few years. This recent progress will be the foundation
on which our understanding of the molecular mecha-
nisms of auxin signal transduction is built, and is begin-
ning to explain how auxin can not only have a direct
influence on cell growth, but also control numerous
and diverse aspects of plant development.
Auxin and its effects on plants
Since Julius von Sachs first discussed the concept
of a phytohormone in 1887
(REF. 1), several chemi-
cal growth regulators have been identified in plants.
Phytohormones are chemicals that have specific effects
on plant growth, and are active at low concentrations.
Plants use a wide variety of hormones, including ster-
oids and peptides, as well as the five classical classes
of phytohormones (auxins, abscisic acid, cytokinins,
ethylene and gibberellins), which are all relatively
small molecules. The extent and significance of phy-
tohormone transport is not well understood for all of
these classes, but is particularly significant to the action
of auxin and to the story of its discovery. The effect of
auxin was first documented when Charles and Francis
Darwin published The Power of Movement in Plants.
They noted that after the perception of light in one
area of a grass coleoptile, an “influence is transported”
that causes bending towards the light in another
2
.
Forty-five years later, in 1926, this messenger was
separated from plant tissues simply by being allowed to
diffuse into agar blocks, which then retained a growth-
promoting activity
3,4
. Three kinds of auxin were ini-
tially found in plants, of which one was also found in
human urine. Subsequently, the first published reports
began to appear on the crystallization and structural
characteristics of auxin. In those early days, only one of
the structures, that of indole-3-acetic acid (IAA), was
correctly identified.
Auxin is now used as the generic name for a group
of important molecules in plants, which can also be
found in humans, animals and microorganisms. IAA
is the predominant auxin in plants
(TABLE 1), and is an
indispensable phytohormone with a well-documented
ability to regulate many aspects of plant development.
Synthetic auxin derivatives are still important herb-
icides; for example, 2,4-dichlorophenoxyacetic acid is
one of the world’s most widely used weed-killers. The
effect of auxin on a growing plant depends on the type
of auxin applied and its concentration. Endogenous IAA
has been implicated in embryonic and post-embryonic
development, and tropisms such as movement in rela-
tion to light and gravity. So, auxin influences aspects
of cell division, cell elongation and cell differentiation,
although exactly how it is involved in each process (and
to what extent they are intertwined) is not completely
Institut für Biologie II/Botanik,
Schänzlestrasse 1,
79104 Freiburg, Germany.
Correspondence to K.P.
e-mail: klaus.palme@biologie.
uni-freiburg.de
Published online
20 September 2006
doi:10.1038/nrm2020
Auxin in action: signalling, transport
and the control of plant growth and
development
William D. Teale, Ivan A. Paponov and Klaus Palme
Abstract | Hormones have been at the centre of plant physiology research for more than a
century. Research into plant hormones (phytohormones) has at times been considered as
a rather vague subject, but the systematic application of genetic and molecular techniques
has led to key insights that have revitalized the field. In this review, we will focus on the plant
hormone auxin and its action. We will highlight recent mutagenesis and molecular studies,
which have delineated the pathways of auxin transport, perception and signal transduction,
and which together define the roles of auxin in controlling growth and patterning.
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Regulon
A collection of separate genes,
the expression of which is
controlled as a unit by a
specific signalling compound or
factor.
Degron
A protein element, usually a
sequence motif, that targets
the protein for proteolytic
degradation.
SCF
TIR1
E3 ubiquitin ligase
A multisubunit ubiquitin ligase
that consists of SKP1, CUL1
and an F-box protein (TIR1 in
this case) that confers
substrate specificity, as well as
a RING protein that is also
known as HRT1, RBX1 or
ROC1.
understood. Its diverse effects in plants might also
extend to animals, as photoactivated auxin seems to have
potential as a cytotoxin in cancer therapy
5,6
.
The reason why auxins have attracted so much
attention for almost a century is not only that they
have the capacity to influence growth, but that they have
additional farther-reaching effects on the life cycle of
plants. Recent evidence shows that, through a unique
mechanism of perception and elicitation, the physiological
responses that auxin governs are central to a plant’s
structure and functioning.
Auxin-mediated regulation of gene expression
For a long time it was thought that, like steroid receptors
in animals, dedicated plant-receptor proteins initiate
the transduction of the auxin signal into numerous
physiological responses. Finally, and after decades of
research, two separate approaches yielded valuable
information on the nature of how auxin functions. The
first approach was based on observations made more
than 20 years ago, which showed that auxin alters gene
expression after only minutes in a selective and dramatic
way
7–10
. The second approach was based on the analysis
of a series of auxin-resistant mutations. Many of these
different mutant plants lacked functional components of
the ubiquitin-mediated proteolytic pathway, indicating
that selective protein degradation is a crucial regulator
of many aspects of the auxin response
11–13
.
Whereas the levels of some mRNAs decrease many
fold in response to auxin, those of other mRNAs increase
many fold (for example, Aux/IAA, GRETCHENHAGEN-3
(GH3) and members of the small auxin up RNA (SAUR)
gene family)
10,14,15
. Furthermore, auxin activates regulons
directly and rapidly. The genes that are activated or
repressed during this process are ultimately respon-
sible for the many physiological effects of auxin. The
complex auxin responses are mediated by two groups
of well-studied genes: the Aux/IAA genes, which consist of
29 members, and the auxin response factor (ARF) genes
with 23 members, in Arabidopsis thaliana
16–18
.
Aux/IAA genes. The Aux/IAA family forms a group of
early auxin-response genes. The variation in amino-
acid identity among them is high and ranges from
10% to 83%. However, even poorly conserved family
members seem to have compensatory functions,
which means that despite their distinct induction
kinetics, dose responses and expression profiles,
obtaining conclusive functional information for the
Aux/IAAs using loss-of-function mutants has been
difficult
18
. Each individual Aux/IAA gene might have
a set of non-essential functions, but these functions
combine to form an important regulatory programme.
Similar observations have been made in studies of the
yeast oxysterol-binding protein family
19
(discussed
in
REF. 18), in which a functional analysis of various
sets of different proteins, which individually perform
non-essential functions, revealed that they combine
to perform essential regulatory functions. In the
future, extensive multidimensional expression maps
and genetic studies of Aux/IAAs and ARFs might be
necessary to tackle this difficult problem.
Aux/IAA genes encode proteins that generally have
nuclear localization signals and four conserved domains
(I–IV). Domain III has a predicted ribbon–helix–helix
DNA-binding domain that is found in bacterial tran-
scriptional regulators (although it is not thought that
Aux/IAAs bind DNA in plants)
20
. Aux/IAAs have
indeed been found in the nucleus. Several Aux/IAA
genes are transcribed within minutes of plants or cells
being exposed to exogenous auxin or protein synthesis
inhibitors. Most strikingly, they were shown to form
homo- and heterodimers not only with one another, but
also with members of the ARF family.
ARF genes. ARFs are transcription factors that contain
an amino-terminal B3-like DNA-binding domain, which
binds to an auxin-responsive element (ARE; TGTCTC)
in the promoter of auxin-response genes in an auxin-
independent manner
21,22
. The carboxy-terminal domain
is similar to the carboxy-terminal region of the Aux/IAA
proteins and is likely to promote direct interaction between
both groups of proteins while bound to the ARE
23
.
This interaction blocks ARE-mediated transcription
20
.
Auxin-mediated gene regulation. Aux/IAA proteins
have been shown to function as negative regulators
of gene expression
20
. Semi-dominant alleles (that
show intermediate phenotypes) with mutations in
the GWPPV motif of the conserved domain II cause
severe auxin-related phenotypes as they affect the
stability of these repressors
24
. In some cases, this can
result in contradictory regulatory phenomena in which
the sensitivity of the mutant to exogenously applied
auxin is reduced, whereas the auxin-related pheno-
type is simultaneously enhanced
25
. The core region of
domain II was mapped to a 13-amino-acid sequence,
known as the
degron: a motif that is sufficient to confer
instability, even when fused to other proteins such as
β-glucuronidase or luciferase
26–28
. Proteins that contain
this degron sequence are an efficient target of the
SCF
TIR1
E3 ubiquitin ligase
complex
29
.
Table 1 | Properties of three commonly used auxins
Properties Natural Synthetic
Chemical structure
N
H
COOH
IAA
Cl
Cl
O COOH
2,4-D
COOH
NAA
Affinity to receptors
TIR1 binding (K
d
) High Low Middle
ABP1 binding (K
d
) Middle Low High
Transport capacity
Influx carriers Yes Yes No (by diffusion)
Efflux carriers Yes No Yes
Indole-3-acetic acid (IAA) is considered to be the most important natural auxin,
1-naphthaleneacetic acid (NAA) is a horticulturally important auxin, and 2,4-dichlorophenoxy-
acetic acid (2,4-D) is a common selective herbicide.
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ARE
Aux/IAA
ARF
ARE
ARF
SCF
TIR1
Auxin
Auxin
Ub
Ub
Ub
Ub
No gene transcription
Gene transcription
1
2
3
4
Aux/IAA
F-box protein
A component of the machinery
for the ubiquitin-dependent
degradation of proteins. F-box
proteins recognize specific
substrates and, with the help of
other subunits of the E3
ubiquitin ligase, deliver them to
the E2 ubiquitin-conjugating
enzyme.
26S proteasome
A protein complex that is
responsible for degrading
intracellular proteins that have
been tagged for destruction by
the addition of ubiquitin.
COP9 signalosome
An eight-subunit protein
complex that regulates protein
ubiquitylation and turnover in
a range of developmental and
physiological contexts.
Extensively characterized in
plants but fundamental to all
eukaryotes, this complex post-
translationally modifies the
cullin subunit of E3-ubiquitin
ligases by cleaving off the
covalently coupled peptide,
Nedd8/RUB1.
T-DNA mutation
A mutation that is the result of
the integration of DNA from
Agrobacterium tumefaciens
into plant genomes. The
insertion is random and might
therefore disrupt genes,
causing a mutation at the
insertion point.
The SCF complex, which is well characterized in
many organisms including yeast and mammals, has an
important role in a wide range of signal-transduction
processes by ubiquitylating target proteins that are selected
by
F-box proteins
30,31
. These F-box-containing SCF com-
plexes not only specifically select, but covalently modify
their target proteins through the addition of several ubiq-
uitin peptides, a process that marks the target proteins for
degradation by the
26S proteasome. TIR1, an F-box pro-
tein that was identified in a screen for mutants that were
resistant to a chemical inhibitor of active auxin transport,
was shown to have a relatively mild auxin-insensitive phe-
notype when mutated
156
, which indicated that other F-box
proteins (subsequently termed AFB1, 2 and 3) also control
the ubiquitylation of Aux/IAA proteins. Degradation of
Aux/IAA proteins reduces the proportion of Aux/IAA-
bound ARFs, thereby allowing ARE-mediated gene
transcription to elicit an auxin response
(FIG. 1).
The regulation of SCF activity is complex, and depends
on a finely balanced network of post-translational modi-
fications and protein–protein interactions. Empirical
evidence for the influence of such components is
occasionally counter-intuitive. For example, the
COP9
signalosome
(CSN) is a protein complex that removes
RUB1 (a small ubiquitin-like protein that regulates SCF
E3 ligase activity
32
) from the SCF complex. The inacti-
vation of the CSN complex results in the stabilization
of certain Aux/IAA proteins, which indicates that the
auxin response can be regulated
33
. Furthermore, CAND1
(CULLIN-ASSOCIATED AND NEDDYLATION-
DISSOCIATED), an SCF-binding protein, prevents F-box
proteins from also binding to the SCF complex. Para dox-
ically, a cand1 mutant causes the stabilization of Aux/IAA7
(REF. 34). It has been proposed that the rate of F-box recy-
cling on the SCF is an important factor in controlling
the proteasome. Several reviews illustrate the biological
relevance of auxin-mediated protein degradation
29,35–42
.
F-box protein TIR1 is an auxin receptor
In one of the most important advances in plant biology of
recent years, the F-box protein TIR1 has been identified
as an auxin receptor
43,44
. Two crucial observations that
led to this discovery were that auxin enhances the inter-
action between TIR1 and Aux/IAAs, as shown in a cell-
free system
45
, and that pretreatment of TIR1 with auxin
enhances its binding to Aux/IAAs
27,46
. Two other recent
studies demonstrated that auxin stabilizes the interaction
between TIR1 and the Aux/IAAs, that auxin is continu-
ously required for this effect, and that SCF
TIR1
binds auxin
directly with a dissociation constant of between 20 and
80 nM
(REFS 43,44).
Although the mechanism by which the binding of
auxin to TIR1 promotes its interaction with Aux/IAAs is
not yet known, it is safe to conclude that SCF
TIR1
and the
associated protein-degradation machinery, together with
Aux/IAAs and ARFs, represent the full signal-transduction
cascade from the auxin signal to gene expression, and that
these F-box proteins represent a new class of receptors
(FIG. 1). In A. thaliana, ~700 different F-box proteins are
encoded by the genome, many more than have been found
in any other non-plant eukaryotic organism. This creates
an extremely large range of specificity for recognition
and proteolytic targeting
47–49
. TIR1 itself is a member of
a small clade of F-box proteins, other members of which
also seem to mediate auxin-dependent developmental
regulation
50
. Genetic studies indicate that AFB1, AFB2
and AFB3 (which are encoded by the three genes that are
most closely related to TIR1 in the A. thaliana genome)
function in a partially redundant manner in mediating the
auxin response. They are expressed in overlapping patterns
in seedlings, leaves and flowers
50
. All three contribute to
the auxin response as shown by
T-DNA mutations. Analysis
of two of the T-DNA alleles (afb2 and afb3) showed that
the transgenic seedlings were more resistant to auxin.
Higher-order mutants result in a progressive decrease
in diverse auxin responses such as root elongation and
lateral root formation
50
. A tir1 afb2 afb3 triple mutant
either arrested shortly after germination or developed a
root that was partially resistant to auxin.
F-box proteins seem not only to be important receptors
for the specific degradation of Aux/IAA proteins
51
, as
closely related family members also mediate the response
to jasmonic acid (a hormone that is involved in wound
healing and pathogen defence)
52
and gibberellin
53
.
These results place the 26S proteasome at the hub of plant
growth and development. Assigning functions to the rela-
tively large number of F-box proteins that are encoded
by the A. thaliana genome is a daunting task, but will be
one that could potentially revolutionize the way we think
about signalling and development in plants.
Figure 1 | SCF
TIR1
-mediated auxin signalling. There are
four distinct layers of regulation in auxin-mediated gene
expression (steps 1–4). Members of the auxin response
factor (ARF) family are transcription factors that bind to
auxin-responsive elements (AREs) in the promoters of
primary auxin-responsive genes, mediating their
transcription (step 1). Aux/IAAs are early auxin-response
proteins that bind ARFs, thereby inhibiting ARE-mediated
gene transcription (step 2). Aux/IAAs are targets of 26S
proteasome-mediated degradation, and this degradation
is directed by the ubiquitylation (Ub) of Aux/IAAs; a
reaction that is catalysed by an SCF E3 ubiquitin ligase
(step 3). Ubiquitin-mediated proteolysis of Aux/IAA is
stimulated by the binding of auxin to the F-box protein
TIR1, the component of the SCF E3 ligase that specifically
recognizes the proteins to be degraded (step 4).
IAA, indole-3-acetic acid.
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ARFs, Aux/IAAs and the auxin response
With the potential involvement of many F-box proteins,
proteasome-mediated auxin signalling is unexpectedly
complicated. This auxin-receptor-binding signal is then
relayed to the subsequent phase of auxin-responsive
gene expression, which is anticipated to be an intricately
intertwined set of processes. The effects of auxin are
thought to depend on its concentration, with high and
low doses eliciting different responses. A framework for
understanding how auxin can have such different roles
in plant development is now in place. At basal auxin
levels, Aux/IAAs are relatively stable, homodimerize
and heterodimerize with ARFs that can bind to AREs
in the promoters of auxin-responsive genes
20,54
. The
ARF-bound Aux/IAA proteins block transcription from
auxin-responsive promoters by controlling the amount
of free ARF transcription factors to the promoters
23
.
An increase in auxin levels causes the proteasome-
mediated degradation of Aux/IAAs, which in turn
allows for a gradually increasing number of functionally
active ARF proteins and the transcriptional activation of
auxin regulons. ARFs can be grouped into three subsets
and vary between 57 and 129 kDa in size. The amino-
acid content in the variable middle region determines
whether a particular ARF functions as a repressor or
an activator
17,55
. Glutamine-rich ARFs such as ARF5
and ARF7 activate transcription. When mutated, they
often give remarkable phenotypes; for example, those
seen in monopteros (ARF5) (rootless) and those seen
in nonphototropic hypocotyl4 (ARF7) (unable to bend
towards light)
56
. There is emerging evidence that ARFs
that lack a glutamine-rich middle region function as
transcriptional repressors
55
.
The expression patterns of ARF and Aux/IAA genes
vary and depend on the tissue and stage of development
(FIGS 2,3). Moreover, some of the most related ARF and
Aux/IAA proteins (ARF3 and
ARF4, ARF6 and ARF8,
ARF10 and ARF17, and
ARF11 and ARF18; IAA6
and
IAA19, IAA8 and IAA9, and IAA32 and IAA34)
(REF. 16) also share similar expression patterns. ARFs are
specific to particular plant responses; for example,
ARF1
and
ARF2 regulate floral senescence
57,58
, and ARF7 and
ARF19 regulate leaf expansion and lateral root devel-
opment
17,59
. The picture is further complicated by the
discovery that specific pairs of ARFs and Aux/IAAs
can preferentially bind to each other and can also
mediate specific processes
60
. Unravelling these further
layers of regulation, analysing the possible combina-
tions of ARF and Aux/IAA interaction, and assigning
specific pairs of ARF and Aux/IAA proteins that have
a functional significance in planta promises to take our
understanding of auxin signalling to unprecedented
levels of detail.
Why do plants need so many different ARF proteins,
and what are their specific regulatory targets? As has
been observed in the case of Aux/IAA loss-of-function
mutants, systematic forward-genetics approaches with
ARF genes has also failed to reveal additional growth
phenotypes, which indicates that ARFs have redundant
and probably compensatory functions. Direct evidence
for this was provided by microarray studies, which
showed that different ARF proteins as well as different
ARF–ARF and ARF–Aux/IAA combinations function in
particular developmental windows to form a regulatory
code that programmes the expression of not only auxin-
sensitive genes but also genes that are regulated by auxin
in a more indirect manner
17,18
. Further global expression
studies with multiple higher-order (for example, double
or triple) mutants are likely to provide detailed expres-
sion maps of distinct tissues and cells that might point
to the regulatory targets of these genes.
Auxin concentration also influences cell patterning,
as the highest levels of auxin signalling proteins corre-
spond to the sites of new lateral roots and leaves, which
has been shown to be functionally significant
61,62
. In the
A. thaliana root tip, the relationship between the forma-
tion and structuring of new organs has been studied in
detail. Here, the Aux/IAA–ARF signalling pathway is
necessary for the expression of transcription factors that
determine the correct differentiation of the various cell
types that are present in roots. PLETHORA proteins, for
example, provide positional information that is neces-
sary for proper root development and depend on auxin
(through ARF5 and ARF7) for their expression
63
.
Alternative signalling pathways
Aux/IAA–ARE-mediated signalling is probably not
the only pathway through which auxin functions.
Compelling reasons come from many studies: those in
which impermeable auxins show their effects without
entering the cell
64
, or in which auxin responses are
simply too fast to be mediated by gene transcription,
for example, in membrane depolarization
65
. Decades of
auxin research have shown that auxin controls numer-
ous cellular processes, although the features of some
responses indicate that they might not be the subject
of transcriptional control (see also
BOX 1). The effects of
auxin on the abundance and activity of the plasma-
membrane-located H
+
-ATPase are particularly well
studied
66–69
. Other regulatory targets are the potassium
and chloride channels and chloride-uptake transport-
ers
70–73
. This auxin-mediated stimulation of ion uptake
correlates with the idea that auxin either controls or sus-
tains the intracellular turgor pressure that is necessary
for plant cell growth
74
.
In addition to the chain of events that is initiated by
the binding of auxin to F-box proteins in the nucleus,
alternative modes of auxin perception have been
proposed. Many of these mechanisms are based on
proteins that have been shown to bind auxin directly.
Many auxin-binding studies have been carried out,
and more than 30 years ago several binding sites were
identified in the cellular membrane system (in line with
the classical idea that a signal-transduction pathway
begins with ligand binding to a membrane-bound
receptor) — in the plasma membrane, the endoplasmic
reticulum (ER) and the vacuole
75,76
. Although several
proteins with clear binding specificities were identi-
fied, the functional characterization focused on one
of them, AUXIN-BINDING PROTEIN-1 (
ABP1), as
it binds auxins with high specificity and affinity (K
d
in
the 10
–8
M range)
77
.
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ARF1
ARF18
ARF11
ARF3
ARF4
ARF2
ARF5
ARF6
ARF8
ARF7
ARF16
ARF19
ARF9
ARF10
ARF17
ARF12
ARF13
ARF23
ARF21
1
12
13
21
100
101
91
96
97
10
5
17
14
15
16
20
22
23
24
25
26
77
78
79
81
82
83
84
7
87
89
90
73
76
19
40
34
39
42
43
35
2
27
28
3
9
93
94
95
98
99
41
28
8
31
32
92
4
6
33
37
45
36
Leaves Seeds Flowers FlowersRoots
ABP1 is a soluble, ER-located, dimeric glycoprotein,
which forms a β-jellyroll barrel that carries auxin in
a central hydrophobic pocket. It resembles the 7S
seed-storage proteins of the ancient family of
cupin
proteins
that is functionally highly diversified
78
. Cupins
and triose isomerase barrel proteins form a superfamily
Figure 2 | The ‘auxin code’ as determined by hierarchical cluster analysis of the expression patterns of ARF genes.
This figure illustrates the wide variety of expression patterns observed within the auxin response factor (ARF) gene family
(
FIG. 3 illustrates the wide variety of patterns observed within the Aux/IAA early auxin-response gene family). The x axis
represents different tissues at different stages. Genes are listed on the y axis. In any specific tissue at any one time,
a unique set of ARF genes are expressed. This heterogeneity has the potential to deliver an extremely specific signal and
discriminate among all of the possible downstream effects that auxin can elicit. Bright green indicates low expression and
bright red indicates high expression. Three replicates of 63 experiments were taken in different tissues and developmental
stages of wild-type Arabidopsis thaliana (ecotype Columbia-0). The data file used for the expression analysis comes from
the
Developmental Affymetrix Gene Expression Atlas supplied by the Max Planck Institute, Tübingen (M. Schmid, J.
Lohmann and D. Weigel laboratory ). The experimental design can be downloaded from
TAIR (The Arabidopsis Information
Resource). The data were clustered using a Pearson correlation for the condition tree and standard correlation for the gene
tree (GeneSpring 7.2). When four or more of the same tissues cluster together they are labelled. IAA, indole-3-acetic acid.
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IAA1
IAA7
IAA2
IAA3
IAA6
IAA19
IAA4
IAA16
IAA18
IAA11
IAA8
IAA9
IAA12
IAA26
IAA27
IAA5
IAA29
IAA10
IAA30
IAA34
IAA32
IAA20
IAA33
IAA13
IAA28
IAA31
IAA14
IAA17
1
10
5
91
87
89
90
7
29
8
6
4
100
101
96
97
26
12
13
14
15
22
16
20
21
23
24
17
39
42
43
25
41
34
36
79
81
82
83
84
73
31
92
32
33
35
19
2
3
9
93
95
94
98
99
27
28
40
37
45
76
77
78
Leaves Leaves Flowers RootsSeeds Seeds
in plants with possibly the widest range of biochemi-
cal functions known for any superfamily described
to date
79
. It is thought that many of these proteins are
important for cell survival through their involvement
in cell-wall structure, modification or maintenance. In
line with structural data, numerous observations show
an involvement of ABP1 in cell expansion, stomatal
closure, plasma-membrane hyperpolarization and
cell division
68,80–84
. Although most of these responses
are mild, some effects seem striking, with a complete
loss-of-function mutation of the A. thaliana ABP1 gene
conferring embryo lethality, which indicates an essential
Figure 3 | The ‘auxin code’ as determined by hierarchical cluster analysis of the expression patterns of Aux/IAA
genes. This figure illustrates the wide variety of expression patterns observed within the Aux/IAA early auxin-response
gene family (
FIG. 2 illustrates the wide variety of patterns observed within the auxin response factor (ARF) gene family).
The x axis represents different tissues at different stages. Genes are listed on the y axis. In any specific tissue at any
one time, a unique set of Aux/IAA genes are expressed. Bright green indicates low expression and bright red indicates
high expression. IAA, indole-3-acetic acid. For further details, see
FIG. 2.
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Cupin proteins
A diverse family of plant
proteins, all of which contain at
least one double-stranded
α-helix or jelly-roll structural
motif. This motif is also present
in all structurally characterized
2-oxoglutarate-dependent
oxygenases, including the
hypoxia-inducible factor (HIF)
hydroxylases, and is
characteristic of the Jumonji
transcription factors.
Root cap
The layers of protective cells
that cover the tip of the
growing root.
Expansins
A class of proteins that are
able to catalyse the loosening
of the cell wall, enabling cell
expansion.
Extensins
Proline-rich proteins that
connect the cell wall and the
plasma membrane.
function for this protein in plant development
85
. There is
no direct evidence for any downstream signalling events
that are thought to occur after the binding of auxin to
ABP1, or regarding the extent to which this signalling
pathway mediates different auxin responses compared
with the SCF
TIR1
pathway. However, given the almost
instantaneous auxin responses that ABP1 can mediate,
it is clear that gene expression need not be involved in
certain aspects of auxin signalling.
Downstream auxin signalling
The effects of auxin are many and diverse, and have been
difficult to separate. They can, however, be divided into
two broad categories: effects on cell expansion and effects
on cell division. There is also evidence that auxin might
have morphogenetic properties that are analogous to
chemicals found in the animal kingdom, but the ability
of auxin to change directly the developmental fate of cells
has not yet been conclusively demonstrated.
Auxin and cell expansion. Consequences of ABP1-
mediated auxin signalling, such as cell expansion and
membrane hyperpolarization, are evident at the cell
periphery. Furthermore, genes that encode extracellu-
lar proteins — for example, those involved in cell-wall
degradation,
expansins and arabinogalactans (which are
cell-wall proteins),
extensins, Pro-rich proteins (which
function as links between the cell wall and the plasma
membrane), and class III peroxidases (which are involved
in lignification, pathogen defence and wound healing)
— are affected in a gain-of-function iaa17 mutant
18
. This
implicates at least two distinct auxin signalling pathways
in plant structure and function through changes to the
cell wall and the plasma membrane.
Less clear, but probably just as significant, are the
responses that might be mediated by other, less well-
studied groups of enzymatic early auxin-response genes.
Two of these groups are the glutathione-S-transferases,
which are involved in the metabolism and detoxification
of xenobiotic compounds, and the quinone reductases,
which protect cells directly against oxidative stress by
decreasing the formation of reactive oxygen species
(ROS)
86
. ROS species mediate cell-wall loosening and
extension growth
87
in a process that has been linked
with auxin for a long time
88
. The effects of ROS could
be caused either directly as a consequence of the oxida-
tive effects of ROS on cell-wall proteins and structures
88
,
or indirectly through the activation of signalling path-
ways and intermediate kinases and phosphatases: these
would in turn regulate gene expression
89
. It has been
suggested that the translation of enzymes such as the
quinone reductases protects the cell against damage
from auxin-induced oxidative stress
86
.
Auxin and cell division. Auxin also promotes cell divi-
sion. Although this relationship is well known, its exact
molecular basis is not understood. It is not yet clear
how closely auxin is linked to progression of the cell
cycle, even though the expression of many cell-cycle
genes is induced by auxin
90,91
. There is evidence that
this induction is mediated through both proteasome-
dependent
92
and ABP1-dependent
85
pathways, and that
auxin has many potential targets. These targets include
proteins that are involved in transitions throughout
the cell cycle, for example, entry into S phase
92
and the
G2–M transition
93
.
Biosynthesis and distribution of auxin
The fate of developing tissue can be determined by the
sensitivity of the growing cells to auxin (as indicated by
the relative expression of the various components of its
signalling machinery), the concentration of active auxin
and the relative concentrations of other phytohormones.
This can also vary widely in different tissues at different
developmental stages. Auxin is readily conjugated to a
wide variety of larger molecules, rendering it inactive.
Indeed, the majority of IAA in the plant is in the form of
inactive conjugates. Auxin conjugation and cata bolism
can therefore decrease active auxin levels. De novo
synthesis and hydrolysis of conjugates contribute to
Box 1 | Post-transcriptional control of auxin signalling
There are several points at which a plant’s response to auxin is controlled. Transcriptional control is by far the best
understood (see main text), but it is emerging that post-transcriptional control through microRNAs (miRNAs) and small
interfering RNAs (siRNAs) also has a significant role by modulating the levels of auxin signalling proteins. The connection
between RNA-mediated gene silencing and the auxin response was made after the mRNAs of five auxin response factors
(ARFs) were predicted to be targets for miRNAs
147
. It has since emerged that, in the context of auxin signalling, ARFs seem
to be particularly prevalent targets for small-RNA-mediated degradation. The potential of miRNAs to affect plant
development through the stimulation of ARF RNA degradation was subsequently shown for a number of specific
transcripts. For example, ARF10 and ARF16 are targeted by miR160 during
root cap development
148
, and ARF3 is targeted
by TAS3 (a trans-acting siRNA) in the juvenile-to-adult-phase transition
149
. Significantly, miR164 is induced by auxin and
targets NAC1, an mRNA that is involved in downstream auxin signalling
150,151
. This regulation forms a homeostatic
mechanism that controls the auxin response. However, as yet, the extent and exact developmental significance of
specific mRNA degradation in auxin signalling is not fully understood.
miRNAs have also been shown to target mRNAs of the F-box auxin receptors TIR1, AFB2 and AFB3 in a study that
gives our first functional insights into the relationship between auxin and miRNAs
152
. In this case, it was shown that
exposure to a specific bacterial peptide results in downregulation of the auxin receptors. This is achieved by the
induction of specific miRNAs by the peptide. Certain bacterial pathogens synthesize indole-3-acetic acid (IAA), which
indicates that auxin aids the infection process. An attenuation of the auxin response by miRNA has therefore been
described as part of a plant’s natural immune response
152
.
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Meristem
A zone (for example, the apex
of the shoot) that contains
undifferentiated cells that
continue to divide, providing
cells for further growth and
differentiation.
Phloem
Vascular tissue that carries
organic nutrients as well as
information molecules such
as hormones throughout the
plant.
Xylem
Vascular tissue that delivers
water and mineral nutrients,
which are taken up by the root
system, to aerial organs. It also
provides mechanical support.
Agravitropic
When a plant is unable to
either perceive or respond
to gravity. Typically, the roots of
agravitropic plants grow in all
directions.
the developmental regulation of auxin homeostasis by
increasing active auxin levels
94–98
. There is a high capacity
for auxin biosynthesis not only in young aerial tissues,
but also in roots, particularly in the
meristematic primary
root tip
98
. Auxin is synthesized from indole through
tryptophan-dependent and tryptophan-independent
pathways, and has been recently reviewed
99
. The fact
that no fully auxin-deficient mutant plants have been
identified reflects the importance of auxin in plant
development.
Distribution of auxin. Auxin is unusual among phyto-
hormones in that it has been shown to be specifically
and actively transported. Indeed, the pattern of a plant’s
response to auxin is not so much determined by the
relative rates of auxin synthesis and catabolism as by
the cells’ capacity for auxin influx and efflux. Although the
rates of synthesis and conjugation are undoubtedly
important for the overall auxin status of the plant, it is
the fine concentration gradients across only a few cells
that have powerful effects on plant development. These
observations have made auxin transport one of the most
studied topics in plant development.
Auxin redistribution involves many proteins
100–110
;
among them, the most investigated belong to a family
of polarly localized plasma-membrane proteins, the PIN
proteins. PINs are found throughout the plant kingdom
111
and mediate auxin efflux
105–108,111–114
. Over short dis-
tances, PIN-dependent auxin transport mediates many
developmental processes, including root development
106
and organogenesis
61,109
.
Sites of auxin transport. Ever since its discovery, auxin
has been considered a highly mobile signalling molecule.
Implicit in its role in tropic growth is a requirement for
directional and responsive transport. Such a mechanism
needs specific and active transporters, the existence of
which was predicted long before their discovery. However,
as the involvement of auxin in root specification illus-
trates, it is not limited to growth in response to external
cues such as light and gravity, as was observed by Francis
and Charles Darwin
2
— its capacity for redistribution is
at the heart of plant development.
In trees, as well as moving passively in the bulk
flow, auxin is transported actively through the vascular
cambium — a cylinder of meristematic tissue that gives
rise to (and is sandwiched between) the
phloem and the
xylem. In A. thaliana, a plant that normally contains no
woody tissue, auxin is actively transported through the
vascular parenchyma. This conclusion is supported by
the cellular location of
AUX1, an auxin cellular influx
carrier that is seen in positions that are consistent with
vascular loading (in the leaves) and unloading (in the
roots)
115–117
. AUX1 transports auxin directly, and this
capacity was demonstrated in Xenopus laevis oocytes at
physiologically significant concentrations
118
. The asym-
metrical subcellular localization of AUX1 (a member
of the LAX family of transporters) is thought to have
functional significance, and its localization is depend-
ent on an ER protein, AXR4, which has been shown
to be responsible specifically for AUX1 distribution
119
.
AUX1 has a strongly
agravitropic phenotype and has a
role in auxin influx
110,117
, but exactly how the phenotype
is caused is unknown. The characterization of other
members of the LAX gene family should improve our
understanding of auxin influx.
PIN proteins are related to bacterial transporters and
are commonly distributed polarly in the plasma mem-
brane, which correlates with the expected direction of
polar auxin flux
105–108,120
. Loss-of-function pin1 mutants
show a distinctive phenotype after the floral transition:
they often grow a single pin-shaped stem, with none of
the flowers or characteristic branching of the wild-type
plant
112
. It has subsequently been shown that this pheno-
type is due to lower rates of auxin transport
107
. Indeed, if
auxin is applied to this pin-shaped stem, lateral growth
is stimulated
61
.
PIN-mediated auxin transport
There are eight PIN proteins in A. thaliana, and they
are expressed at distinct times and locations
111
. Five
have been well characterized and show a distinctive
polar subcellular localization. Although data for
PIN1
(the knockout of which gives rise to the most dramatic
phenotype) have not been reported, when expressed
in heterologous systems, two other PINs — PIN2
and
PIN7 — transport IAA
121
. Therefore, it has been
suggested that these proteins at least do not need any
other plant-specific co-factors for auxin efflux. However,
heterologous expression results in a reduction of their
substrate specificity, which implies that plant-specific
co-factors might still have a role. Furthermore, it has
been shown that at least four PINs (PIN1, PIN4, PIN6
and PIN7) are rate-limiting for auxin efflux from plant
cells
121
.
The localization of PINs is extremely dynamic and
can change rapidly; such changes and the associated
repositioning of peaks of auxin concentration are asso-
ciated with important developmental events, such as
embryonic development
109
and the response to gravity
105
.
The mechanism that underlies the polar localization of
PIN proteins and their ability to relocalize rapidly is
the recycling of PIN-containing endocytotic vesicles
to and from the plasma membrane. There is evidence to
indicate that auxin can directly influence endocytosis.
After blocking the return of endosomes to the plasma
membrane
122
, visible changes in the distribution of PINs,
as well as more general endosome markers, between the
endosome and the plasma membrane could be observed
at 5 μM auxin
123
(although this is a high concentration
for root growth inhibition
99
). The discovery that the
application of inhibitors of endosomal trafficking also
inhibited polar auxin flux revealed a close relationship
between the two processes
122
. It is still not clear to what
extent these processes can be separated.
The relationship between PINs and other auxin
transporters is still unclear. For example, in A. thaliana
two multiple drug resistance/P-glycoprotein-like pro-
teins (PGPs), PGP1 and MDR1, transport auxin
100
.
It has been reported that PIN1 is mislocalized in a pgp1
mdr1 double mutant, which indicates a certain extent of
control over PIN function
124
. However, this finding has
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SCF
TIR1
Auxin
ARE
ARF
Cell specification
Other signals
Aux/IAA
ARF
Auxin transport,
PINs
Positional clues
(SHR, SCR)
3
2
1
4
5
6
Guanine nucleotide
exchange factor
A protein that facilitates the
exchange of GDP (guanine
diphosphate) for GTP
(guanine triphosphate) in the
nucleotide-binding pocket of a
GTP-binding protein.
GTPase-activating protein
(GAP). A protein that
stimulates the intrinsic ability
of a GTPase to hydrolyse GTP
to GDP. Therefore, GAPs
negatively regulate GTPases by
converting them from active
(GTP-bound) to inactive
(GDP-bound).
been questioned by results showing that PIN1 function
is not dependent on the presence of PGPs
121
.
Although distinct roles for the PIN proteins have
been described, there remains significant functional
overlap among them. The most similar family members
can complement one another in knockout mutants, and
display some degree of adaptability in the cells in which
they are expressed. For example, in the A. thaliana pin1
mutant, PIN4 is seen to extend into the cells where PIN1
would have been present
114
. Auxin induces the expres-
sion of many PIN proteins in an Aux/IAA-dependent
manner
125
. However, during the regeneration of a dam-
aged root tip, the correct expression of PIN proteins has
recently been shown to be dependent on pre-existing
cell patterning rather than auxin concentration
126
. In
addition, the presence of auxin alone is not sufficient for
root specification in the absence of PIN-mediated auxin
transport
127
. These two observations therefore make it
unlikely that PINs alone directly determine either cell
specification or polarity; instead, they indicate that
PINs mediate distinct developmental signals as part of
a wider developmental programme
126
(FIG. 4).
Regulation of PIN expression. Of the many factors that
have been shown to control PIN localization and func-
tion, there are two broad groups that have received much
attention. The first regulates the vesicle-cycling machin-
ery, and the second determines the phospho rylation
status of the cell.
ADP ribosylation factors are monomeric GTPases
that are involved in vesicular trafficking. Their activity
is regulated by ADP ribosylation factor
guanine nucleotide
exchange factors
(ARF GEFs) and ADP ribosylation
factor
GTPase-activating proteins (ARF GAPs). Loss-of-
function alleles of these regulators, as well as of ARF1
(
ADP RIBOSYLATION FACTOR-1) itself, can lead to
defects in PIN function
128,129
. In particular, GNOM, an
ARF GEF, controls many general aspects of polar auxin
transport
130
and is involved in the mechanism that
targets polar PIN1 to the appropriate end of the cell
129
.
PIN expression is regulated by phosphorylation. The
ROOTS CURL IN NPA-1 (
RCN1) gene encodes a regula-
tory subunit of protein phosphatase-2A, a heterotrimeric
serine/threonine protein phosphatase
131
. Loss-of-function
mutations in the RCN1 gene exhibit an elevated level of
root basipetal auxin transport
132
. Furthermore, treatment
with inhibitors of protein phosphatases-1 and 2A mim-
ics the phenotypes of rcn1 mutants, including increased
root basipetal auxin transport and an altered root gravity
response
132,133
. However, at higher concentrations a sig-
nificant reduction in root basipetal auxin transport was
observed, which indicates a biphasic response of the root
to dephosphorylation.
Loss-of-function mutations in PINOID (PID), a gene
that encodes a protein kinase, cause flower stems similar
to those of pin1 mutants
134–136
and roots that are unable to
respond to gravity
135,136
. Crucially, pid displays an apical–
basal shift in the polarity of several PIN proteins
137
,
depleting the primary root meristem of auxin and caus-
ing a root meristem collapse. PID directly controls PIN
polarity. It functions as a binary switch, with subthreshold
PID levels leading to basal PIN localization and above-
threshold PID levels leading to apical PIN localiz ation.
Although PID interactors have been identified, it is
unclear whether they are targets for phosphorylation
or whether they are upstream signalling components
138
,
although the central regulator PDK1 has recently been
shown to bind and phosphorylate PID directly
139
. The
elusive mechanism of PID in determining cell polarity
makes it one of the most mysterious proteins in plants.
The auxin signalling–transport relationship
Active auxin transport mediates cellular auxin con-
centration and is therefore a crucial component in the
coordination of plant development
(FIG. 5). However,
the specific relationship between auxin signalling and
auxin transport is poorly understood. Various kinases
are regulated by auxins
140–142
; recently, elements of the
oxidative stress MAP kinase cascade including MAP
KINASE KINASE-7 (MKK7) have been identified
that control plant architecture through the negative
regulation of polar auxin transport
143
. It has been sug-
gested that the auxin transporter also serves as an auxin
receptor, but the suggestive correlative data currently
Figure 4 | The developmental feedback loop of auxin signalling and auxin
transport in roots. The Arabidopsis thaliana root tip is a commonly used model system
in plants. Here, we describe the relationships between auxin signalling, auxin transport
and cell specification. In step 1, auxin leads directly to the destabilization of Aux/IAA
proteins, allowing auxin response factors (ARFs), which are transcription factors, to
control the transcription of auxin-regulated genes (see also
FIG. 1). In step 2, the
subsequent expression of certain auxin-inducible genes, such as PLETHORA, initiates a
complex chain of cell-specification events. However, as in step 3, cell specification is also
dependent on self-generated positional cues, for example SHORTROOT (SHR) and
SCARECROW (SCR), which mark the central cylinder and the endodermis/quiescent
centre, respectively. In step 4, the presence of auxin is not sufficient for cell specification.
For example, even though it is required for the first events in root specification, auxin
alone cannot induce root formation. Therefore, interactions
between this and other
signalling pathways define the final cell specification. In step 5, the correct polarization
of the auxin-transport machinery (the PIN proteins) is a result of as yet unknown
polarized markers that are laid down by cell specification. In step 6, changes in auxin
concentration that are a result of auxin transport control the expression of the early
auxin-responsive Aux/IAA genes and the regulation of auxin-inducible transcription by
ARF transcription factors. IAA, indole-3-acetic acid.
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a Lateral root b Embryo
c Shoot apical
meristem
d Leaves
e Main root
P2
P1
I1
only underline the gaps in our understanding of these
processes
144
. Another surprising link indicates a role
for Rac-like (ROP) GTPases in auxin action. Auxin
activates ROP3
(REF. 145); and both auxin and active
ROP3 (or RAC1) cause Aux/IAA proteins to aggre-
gate into discrete nuclear bodies. These structures are
proteolytically active, resulting in the 26S proteasome-
mediated degradation of Aux/IAA proteins. This
potentially provides a link between auxin perception
at the plasma membrane and the regulation of genes in
the nucleus
145,146
. Proteasome-dependent auxin signal-
ling does not seem to be in exclusive control of auxin
transport. As previously discussed, synthetic auxins
have been shown to slow the rate of PIN endocytosis,
thereby increasing the amount of PIN at the plasma
membrane. This mechanism outlines a potential
negative-feedback loop for the maintenance of
cellular auxin concentration but, significantly, has not
been shown to be affected by SCF
TIR1
(REF. 123). The
regulation of auxin transport, and specifically the role
of auxin itself in its own transport, is beginning to be
uncovered. This area of research promises to teach us
much about how plant growth is controlled.
Concluding remarks
Almost 40 years ago it was proposed that the auxin
efflux carriers and auxin receptors were closely linked,
if not identical
157
. And although this proposition has
not found widespread support, the fact that it cannot
be fully dismissed illustrates just how closely auxin
signalling and auxin transport are intertwined. Indeed,
many data now point to a crucial and central role of
Figure 5 | The developmental processes that are controlled by auxin flux. a | Lateral root. PINs conduct auxin from
the centre of the root (stele) to the new root tip (auxin is indicated in green and auxin transport is indicated by red
arrows), and then away again through the epidermis. This forms the basis of the ‘fountain’ model of lateral root
formation
62
. b | Embryo. Auxin is taken to the very young embryo by PIN7 (left). At a later stage (right), the auxin flux is
reversed as PIN1, PIN4 and PIN7 conduct auxin out of the embryo. Transport by PIN1, PIN4 and PIN7 is indicated by
blue, green and red arrows, in corresponding order. c | Shoot apical meristem. Auxin is redirected towards the site of
new leaf formation (primordial P1 and P2 and the incipient primordium I1) in the epidermal layer. The shoot apex is
indicated in blue. d | Leaves. Auxin mediates vascular tissue development (indicated as uninterrupted green lines) and
patterning in the developing leaf through non-polar PIN1. The arrows indicate sites of auxin production and the red
circles indicate auxin accumulation.
e | Main root. PINs determine the flux of auxin towards the root tip in the centre of
the root, and back again in the epidermis. This movement forms the basis of the root’s ability to respond quickly to
gravity. Parts a–e adapted, with permission, from
REFS 62,125,153–155 © (2003) Cell Press, (2005) Company of
Biologists Ltd, (2005) Current Biology Ltd, (2004) Kluwer Academic Publishers, and (2005) Scandinavian Society for
Plant Physiology, in corresponding order.
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Acknowledgements
We thank W. Hartung, R. Hertel, S. Kepinski, J. McWilliams
and P. Schopfer for critical reading of the manuscript. Our
work was supported by the Deutsche Forschungsgemeinschaft
(DFG), the Bundesministerium für Bildung und Forschung,
Fonds der chemischen Industrie, the European Union and the
Landesstiftung Baden-Württemberg GmbH. K.P. is particu-
larly grateful for having had the opportunity to contribute to
the DFG network’s Molecular Mechanisms of Phytohormone
Action.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
TAIR: http://www.arabidopsis.org
ABP1 | ADP RIBOSYLATION FACTOR-1 | ARF1 | ARF2 | ARF4 |
ARF6 | ARF8 | ARF11 | ARF19 | AUX1 | CAND1 | IAA6 | IAA9 |
IAA19 | IAA32 | IAA34 | PIN1 | PIN7 | RCN1 | RUB1 | TIR1 |
FURTHER INFORMATION
Developmental Affymetrix Gene Expression Atlas:
ftp://ftp.arabidopsis.org/home/tair/Microarrays/Datasets/
ExpressionSet_ME00319/
Klaus Palme’s homepage: http://www.biologie.uni-freiburg.
de/data/bio2/palme/index.html
Access to this links box is available online.
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