Abstract. The plant hormone auxin plays crucial roles
in regulating plant growth development, including embryo
and root patterning, organ formation, vascular tissue
differentiation and growth responses to environmental
stimuli. Asymmetric auxin distribution patterns have
been observed within tissues, and these so-called auxin
gradients change dynamically during different developmental
processes. Most auxin is synthesized in the shoot
and distributed directionally throughout the plant. This
polar auxin transport is mediated by auxin influx and
efflux facilitators, whose subcellular polar localizations
guide the direction of auxin flow. The polar localization
of PIN auxin efflux carriers changes in response to developmental
and external cues in order to channel auxin
flow in a regulated manner for organized growth. Auxin
itself modulates the expression and subcellular localization
of PIN proteins, contributing to a complex pattern of
feedback regulation. Here we review the available information
mainly from studies of a model plant, Arabidopsis
thaliana, on the generation of auxin gradients, the regulation
of polar auxin transport and further downstream
cellular events.
Keywords. Indoleacetic acid, auxin transport, polar protein targeting, patterning, tropism, Arabidopsis.
Introduction
Auxin is a prominent plant hormone that plays critical
roles in plant growth and development, including regeneration
and adaptation responses.While the most common
naturally occurring auxin is indole-3-acetic acid (IAA),
other species include indole-3-butyric acid (IBA) and 4chloroindole-3-acetic
acid (4-Cl-IAA) [1]. Functionally
analogous, synthetic auxins such as 1-naphthylacetic acid
(1-NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) are
metabolically more stable and, thus, they are more widely
used in agricultural applications and biological studies.
Interestingly, the exogenous application of each auxin
species leads to a distinct but partially overlapping spectrum
of effects [1]. This is possibly due to the specific
transport properties for each type of auxin, and differences
in tissue permeability and metabolic stability.
IAA is structurally related to and biochemically derived
from tryptophan (Trp). However, Trp is not absolutely
necessary for the synthesis of IAA, and several branching
pathways, both dependent or independent ofTrp, have
been proposed for auxin biosynthesis [1,2]. As these biosynthetic
pathways are redundant and show strong species-
and tissue-specific differences, the contribution of
each pathway is unclear. In addition, IAA can be inactivated
or stored via conjugation to amino acids, sugars and
peptides [1, 2]. Enhanced production of auxin in planta
by the overexpression of bacterial [3, 4] or endogenous
plant [5, 6] genes for auxin metabolism resulted in phenotypes
similar to those induced by the exogenous application
of auxin, indicating that the regulation of auxin biosynthesis
and metabolism is important for plant growth
and development.
Review
Spatiotemporal asymmetric auxin distribution:
a means to coordinate plant development
H. Tanaka, P. Dhonukshe, P. B. Brewer and J. Friml*
Zentrum für Molekularbiologie der Pflanzen, Universität Tübingen, 72076 Tübingen (Germany),
e-mail: jiri.friml@zmbp.uni-tuebingen.de, Fax: +49 7071 295797
Received 10 March 2006; received after revision 26 June 2006; accepted 9 August 2006
Online First 29 September 2006
* Corresponding author.
Cell. Mol. Life Sci. 63 (2006) 2738–2754
1420-682X/06/232738-17
DOI 10.1007/s00018-006-6116-5
© Birkhäuser Verlag, Basel, 2006
Cellular and Molecular Life Sciences
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2739
In addition, the intercellular transport of auxin has been
shown to be crucial for most of the known effects of
auxin. It is increasingly clear that polar auxin transport
(PAT) is an important mechanism in plants, whereby
auxin is channeled into specific plant tissues in order to
trigger or maintain certain developmental responses. The
effect of auxin overproduction or degradation, at least in
some tissues such as embryonic cotyledons and root tips
in Arabidopsis thaliana, can, to a large extent, be compensated
by PAT [7]. Thus, the overall distribution of
auxin in planta is determined by the net effects of auxin
biosynthesis, metabolism and, by its directional transport.
In this review, we discuss recent advances in the understanding
of auxin distribution, its regulation by upstream
elements and the induced downstream signaling cascades
that together lead to diverse developmental responses.
Local auxin distribution patterns
The asymmetric distribution of auxin across plant tissues
accompanies and mediates developmental responses
from the earliest changes in embryo shape to large tropic
responses in adult plants. However, the discovery of the
importance of these auxin gradients first relied on the
careful documentation of auxin distribution. A variety
of methods have been successfully used, including immunolocalization
with an anti-IAA antibody [8], direct
measurements of endogenous auxin [9–12], by monitoring
radioactively labeled auxin [13–16] and observation
of the activity of auxin-responsive marker genes
(e.g. synthetic promoter DR5; [10, 17–20]). Finding the
consensus sequence TGTCTC (auxin response element,
AuxRE) within the promoter regions of genes that are
rapidly upregulated by auxin [21] (reviewed by [22]) was
an important breakthrough for testing auxin distribution
in planta with cellular resolution. The most widely used
synthetic promoter, which is made up of multiple AuxRE
repeats, is derived from the promoter of a primary auxin
response gene from soybean, GH3, and is termed DR5
or DR5rev (another variant with inverse repeats). DR5
promoters are highly responsive to active auxins, and,
therefore, the promoter activity can be used as an indirect
indicator of endogenous auxin levels. The main advantage
over natural auxin-inducible promoters is the lack
of additional endogenous promoter elements that could
potentially confer tissue-specific regulation. Expression
of DR5 can be induced by exogenously applied auxin
(e.g. 2,4-D) [8, 17, 20], and the DR5 pattern is altered by
inhibition of PAT [8, 17–20]. Importantly, the activity of
DR5 correlates well with the results obtained by direct
measurements of auxin content within wild-type Arabidopsis
roots [23]. Furthermore, the accumulation pattern
of auxin, as inferred from anti-IAA immunolocalization
patterns, reflects the pattern of DR5 activity [8]. Thus,
despite limitations for the use of such synthetic promoters,
including dependence on induction through a transcription
pathway and a limited threshold of detection, it
seems that DR5 activity can be used as a reasonable measure
for auxin accumulation patterns, at least in embryos
and root meristem. Nevertheless, more direct measurements
of auxin in vivo (e.g. real-time monitoring using
fluorescence resonance energy transfer (FRET)-based
reporters [24]) would be an important subject for future
study in this field. In the following sections of this review,
auxin distribution patterns in Arabidopsis thaliana,
inferred from the pattern of DR5 expression and detection
by anti-IAA antibodies, are discussed.
Auxin distribution during embryogenesis
Embryogenesis is a fundamental process in plant development,
in which the basic body plan, including apicalbasal
axis, radial and bilateral symmetries, as well as
apical root and shoot meristems are established. Interestingly,
auxin distribution dynamically changes at key
steps of embryo development [20]. After fertilization, the
single-celled zygote divides to form an apical cell, which
develops into the embryo proper, and a basal cell, which
develops into the suspensor. DR5 activity is higher in the
apical cell and its daughter cells until the 32-cell-embryo
stage. At later stages an inverse pattern of auxin maxima
is observed [20] (Fig. 1a), whereby the highest activity
is found in the uppermost suspensor cells, including the
precursor to the root meristem – the hypophysis. Later
still, DR5 activity is detected in cotyledon apices and in
provascular cells. This pattern of auxin response maxima
mirrors the dynamic IAA accumulation pattern verified
by immunolocalization with an anti-IAA antibody [8,
20] and is abolished either by pharmacological or genetic
interference with PAT [20], indicating that proper PAT
underpins the generation of auxin asymmetry during em-
bryogenesis.
Auxin distribution during post-embryonic organ
formation
During post-embryonic development, the local application
of auxin is sufficient to trigger leaf or flower formation
in the shoot apex [25] or lateral root initiation [26],
suggesting that auxin plays a crucial role in organ initiation.
Under normal conditions, places of increased auxin
levels also correlate with the initiation sites of organ primordia
in both root and shoot [8, 27].
In the primary root of Arabidopsis thaliana, a DR5 maximum
is detected in columella initials and in the quiescent
center (QC), with lesser activity in the mature columella
root cap [17] (Fig. 1b). DR5 activity decreases gradually
as the cells in the root differentiate and are displaced into
maturing root tissues. However, the activity increases
2740        H. Tanaka et al. Auxin gradients, upstream and downstream
Figure 1. Schematic representations of PIN-mediated polar auxin transport. (a) Three subsequent stages of plant embryo development
from the two-cell stage to the triangular stage, each of which exhibits distinct localizations of PIN proteins. (b) PIN-mediated polar auxin
transport in the mature root and for gravitropic response. Auxin is transported in a radially symmetrical manner when the root grows along
gravity vector (left). After a change in the gravity vector, auxin redistributes asymmetrically, allocating more to the lower side (right). (c)
PIN-mediated polar auxin transport during lateral root primordial development. The polarity of PIN proteins and presumptive directions of
auxin flow are indicated by arrows. The place of auxin accumulation is highlighted in green. lrc, lateral root cap; e, epidermis; c, cortex;
en, endodermis; v, vascular bundle.
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2741
again in the presumptive founder cells of lateral root
primordia in the pericycle [8]. Upon formation of initial
cells by anticlinal cell divisions, DR5 activity is observed
in all these cells. After periclinal divisions have occured
to form inner and outer cells, the DR5 activity pattern
gradually changes and is confined to central cells in both
cell layers [8]. Later, a gradient with its highest activity at
the tip is established (Fig. 1c).
The shoot apical meristem generates successive lateral
organs in a predictable phyllotactic pattern. The site-specific
microapplication of auxin can induce the generation
of primordia in peripheral zone tissue closest to the site of
application, suggesting that auxin can regulate the position
and emergence of organ primordia in the shoot apical
meristem [25]. Interestingly, under normal conditions,
DR5 activity is detected in incipient primordia before it
starts to bulge (e.g. position I2), and then again later in
young primordia at their apices [8, 27]. Similarly, DR5
activity is detected at the tips of developing floral organs
[8]. Thus, despite the large morphological variety of lateral
organs, a similar pattern of auxin distribution with its
apex-localized maxima appears to underlie primordium
development in various organs.
Once established, organs gradually become connected to
the preexisting vascular tissue by newly formed vascular
strands. In large organs such as leaves, a dynamic but typical
pattern of vascular strands emerges ensuring an efficient
exchange of nutrients, and assimilates throughout
the organ. Interestingly, when vascular strands are disrupted
by wounding, new vascular tissue can form, a process
which can be also initiated by the local application
of auxin [28]. In all tested instances, an increase in DR5
activity precedes and accompanies the formation of new
vascular strands, thus suggesting that auxin may also play
a role in the differentiation of vascular tissue [8, 29].
Auxin distribution during tropisms
Plants can change the direction of their growth according
to the vectorial input of light (phototropism), gravity (gravitropism)
or other signals (hydrotropism, thigmotropism).
More than hundred years ago, Darwin’s classical experiments
on the phototropism of grass coleoptiles showed
that while light is perceived at the tip, the differential elongation
response occurs in the tissues spatially separated
below. This indicated that there could be a mobile signal
that regulates cell activity [30]. These observations eventually
led to the emergence of the Cholodny-Went theory,
which predicts that organ bending results from differential
growth facilitated by the asymmetric distribution of auxin
(summarized in [31]). Indeed, increased auxin levels were
detected in various plant organs, including gravity-stimulated
tobacco shoots (at the lower side) [32], light-stimulated
maize coleoptiles (at the shaded side) [11], light- and
gravity-stimulated Arabidopsis and Brassica hypocotyls
[12, 18], and gravity-stimulated Arabidopsis roots (at the
lower side) [19, 33, 34]. Auxin transport inhibitors interfere
with both asymmetric auxin distribution and tropic
growth, suggesting that PAT underlies plant tropism [18,
35]. In support of this, various mutants in auxin influx and
efflux facilitators show defects in photo- and gravitropism
[18, 36, 37].
Upstream of local auxin distribution: polar auxin
transport
Genetic and pharmacological studies collectively suggest
that asymmetric auxin distribution results mainly from
intercellular auxin transport. Auxin is unique among
plant hormones in that it is transported in a polar manner
through the plant tissues. Experiments using radioactively
labeled auxin showed that the translocation of auxin involves
two physiologically distinct pathways: first, auxin
is translocated rapidly with the mass flow of other metabolites
in mature phloem, and second, auxin is transported
from the shoot apex by a much slower, carrier-dependent,
cell-to-cell polar transport (for overview, see [38]). PAT
is specific for auxins and is predominantly unidirectional,
running from the apical aerial tissues through vascular
tissues towards the root tip. In the root apex, some of this
auxin is then transported away from the tip through the
outer lateral root cap and epidermis tissues [13, 16]. Once
in the PAT system, two distinct carrier mechanisms control
cellular influx and efflux separately. Both influx and
efflux have been independently demonstrated in single
plant cells and tissue segments [39–41]. In comparison
to IAA, the other major naturally occurring auxin, IBA,
is also transported in a polar manner, which can be uncoupled
from polar transport of IAA [2]. It is interesting
that the synthetic analogue, 1-NAA, while being an excellent
substrate for cellular efflux, is only poorly actively
transported by influx. The lack of influx is compensated
by the passive diffusion of 1-NAA into cells (see next sections).
On the other hand, another synthetic auxin, 2,4-D,
is transported by influx but only poorly by efflux, resulting
in its retention within cells [41]. The combination of
greater metabolic stability and cellular retention probably
accounts for the greater ability of 2,4-D to affect plant
growth.
Cellular influx and efflux of auxin has also been discriminated
pharmacologically using specific inhibitors
for each pathway. Auxin efflux inhibitors [42] such as
1-N-naphthylphthalamic acid (NPA) and 2,3,5- triiodobenzoic
acid (TIBA) have been used for more than half a
century to study auxin-dependent plant development.The
use of auxin efflux inhibitors has facilitated the physiological
characterization of PAT and has established that
auxin efflux is important for many aspects of plant development.
Exogenously applied auxin efflux inhibitors
2742        H. Tanaka et al. Auxin gradients, upstream and downstream
affect a number of processes, such as embryonic axis
formation [43, 44], the initiation of lateral roots [23] and
aerial organs [14, 25], root meristem patterning [17, 45,
46], vascular patterning [47], hypocotyl and root elongation
in light [48], apical hook formation [35] and tropic
responses [32, 42, 49]. On the other hand, inhibitors of
auxin influx such as 1-naphthoxyacetic acid (1-NOA) and
3-chloro-4-hydroxyphenylacetic acid (CHPAA) were discovered
only recently [50]. These auxin influx inhibitors
have been used to demonstrate the roles of auxin influx
in root gravitropism [51] and in correct leaf positioning
[52]. Thus, cellular influx and efflux contribute to PATdriven
auxin distribution and are critical for many plant
developmental processes.
Chemiosmotic hypothesis
Based on the chemical characteristics of auxin and the
physiological nature of PAT, the chemiosmotic hypothesis
was formulated to explain the cellular mechanism of
auxin transport (for overview, see [38]). According to the
model, plasma membrane-localized proton pumps generate
a proton gradient across the plasma membrane. As
some IAA in the acidic environment of the apoplast (e.g.
approximately 16% at pH 5.5) exists in the protonated,
hydrophobic form (IAAH), it is able to pass passively
through the plasma membrane. However, as the cytosol
is more basic, the majority of auxin there (e.g. more than
99% at pH 7.0) exists in the dissociated form (IAA– anion),
and is unable to pass through the plasma membrane
by a simple diffusion. As a result, IAA becomes trapped
within cells and can leave only by the action of efflux carriers.
The directionality of auxin transport was explained
by assuming that auxin efflux carriers are asymmetrically
localized. While not accounted for in the original model,
physiological experiments suggest that the cellular influx
of auxin also participates in PAT [39, 41], presumably by
increasing the efficiency of auxin transfer and/or direc-
tionality.
In support of the chemiosmotic hypothesis, Li and colleagues
recently demonstrated that transgenic Arabidopsis
plants overexpressing the H+-pyrophosphatase AVP1
(for Arabidopsis vacuolar pyrophosphatase) have an decreased
apoplastic pH and exhibit an increased basipetal
transport of IAA [53]. Conversely, in the avp1 loss-offunction
mutant, the apoplastic pH was more alkaline
than in wild-type plants, and basipetal IAA transport was
reduced [53].These studies provide an important molecular
support for the chemiosmotic hypothesis.
Molecular components
The chemiosmotic hypothesis proposed the existence of
auxin carrier proteins. Mainly through molecular genetics
using Arabidopsis thaliana, several classes of genes
required for auxin influx, efflux and/or PAT have been
identified.
AUX1
The AUXIN1 (AUX1) gene encodes a transmembrane protein
sharing significant similarity with plant amino acid
permeases and was proposed to be involved in the influx
of tryptophan-like IAA [37]. Plants mutant for aux1 exhibit
two major auxin-related phenotypes: resistance to
2,4-D-mediated inhibition of root growth, and agravitropic
root growth. Several lines of evidence support the
role of AUX1 in auxin influx in the root: First, resistance
to the inhibition of root elongation by auxin is not observed
with the membrane permeable auxin 1-NAA [49].
Similarly, the agravitropic root phenotype of aux1 mutants
can be rescued by exogenously applied 1-NAA [49,
54]. The restoration of gravitropism by 1-NAA seems to
correlate with the recovery of basipetal auxin transport
[55], which is defective in aux1 [56]. Second, aux1 roots
incorporate approximately twofold less labeled 2,4-D
than wild-type roots [49]. Third, auxin influx inhibitors,
1-NOA and CHPAA, phenocopy the root phenotype of
aux1 [51]. Recently, experiments that included the heterologous
expression of AUX1 in Xenopus oocytes directly
demonstrated thatAUX1 functions as a high-affinity, specific
auxin uptake carrier [57]. AUX1 is expressed in various
tissues, including protophloem cells, root columella
cells, the lateral root cap and the shoot apical meristem
[55, 58]. The aux1 mutant root tip contains less auxin
compared with wild-type, indicating that auxin supply to
the root apex is defective, despite normal AUX1 expression
being limited to the root tip [55]. These data suggest
that AUX1 could be involved in the loading of auxin into
the root tip from the phloem [55], providing a potential
mechanism for the connection between phloem-based,
non-polar auxin transport and PAT pathways, which was
previously demonstrated physiologically [59]. In the Arabidopsis
genome, several Like-AUX1 (LAX) genes were
identified, but their functional characterization has not
yet been established [60].
PIN proteins
The function of the PIN1 gene in auxin efflux and transport
was primarily surmised from the phenotypic analysis
of Arabidopsis pin-formed1 (pin1) mutants.The pin1 mutants
are progressively defective in organ initiation and
phyllotaxy, which, in the end, results in pin-shaped inflorescence
meristems devoid of flowers [14]. Similar defects
can be phenocopied by culturing wild-type plants in
the presence of auxin efflux inhibitors, and it was shown
that PAT is dramatically reduced in pin1 stem segments
[14]. The PIN1 gene encodes a protein with 10–12 putative
transmembrane domains sharing a limited similarity
to a group of bacterial transporters [61]. Mutations in a
homologous gene PIN2 (concurrently published as, ETH-
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2743
YLENE INSENSITIVE ROOT 1 and AGRAVITROPIC 1)
cause a defect in root gravitropism [15, 36, 62, 63]. The
other homologues, PIN3, PIN4 and PIN7, are required for
tropism, root meristem patterning and patterning in early
embryogenesis, respectively [10, 18, 20]. The Arabidopsis
PIN gene family consists of eight members in total.
The functions of the remaining three members, PIN5, 6,
8, remain unknown.
Remarkably, all the reported phenotypes of pin mutants
can be mimicked by auxin efflux inhibitors, suggesting
that PIN proteins are involved in some aspect of
auxin efflux. The pin1 and pin2 phenotypes are associated
with reduction of PAT in the inflorescence stem
and root, respectively [14, 16]. In addition, examination
of auxin distribution based on DR5 activity and/or by
direct measurement has revealed that the loss of PIN2,
PIN4 or PIN7 activity affects local auxin distributions
[10, 20, 36]. Furthermore, when PIN2 was overexpressed
in yeast, it retained less radioactively labeled auxin than
control yeast and showed enhanced resistance to the cytotoxic
IAA analogue 5-fluoro-indole [15, 36]. Recently
presented experiments based on the heterologous expression
of PINs in tobacco, mammalian and yeast cultured
cells provide strong evidence for a direct role for PIN
proteins in auxin efflux [64]. In addition, manipulation
with PIN polar localization showed that, at least in meristematic
tissues and for short-range transport, the polarity
of PIN localization determines the direction of auxin
flow [65]. Thus, a network of distinctly expressed and
polarly localized PIN proteins provides a molecular basis
for PAT in different parts of the plant [10, 18, 20, 61, 62]
(Fig. 2).
MDR/PGP subfamily of ABC transporters
Besides AUX1 and PIN protein families, members of the
ATP-binding cassette (ABC) protein superfamily have
been suggested to play a role in cellular efflux and influx
of auxin. Members of the multi-drug-resistant/P-glycoprotein
(MDR/PGP) subfamily, AtMDR1 (also know as
AtPGP19), AtPGP1, AtPGP2, AtPGP4 and AtPGP10,
have been isolated as proteins binding to the auxin transport
inhibitor NPA [66, 67]. Loss-of-function mutations
in several PGP genes cause diverse developmental defects,
which can be interpreted as a consequence of altered
auxin signaling or transport, but in most cases
cannot be mimicked by treatment with auxin transport
inhibitors. In addition, some of these mutants show aberrant
auxin transport, including defective basipetal auxin
transport in pgp1, pgp4 and pgp19 roots, reduced net efflux
from pgp1 and pgp19 leaf protoplasts, and reduced
auxin uptake into the pgp4 root tip [67–69]. Furthermore,
heterologous expression of PGP1 in HeLa cells and yeast
results in the cellular export of auxin and the toxic IAA
analogue, 5-fluoro-indol [68]. Conversely, expression of
PGP4 in HeLa cells and yeast mediates cellular import of
auxin and 5-fluoro-indol [67, 69]. PGP1 and PGP4 proteins
are uniformly distributed at the surface of root apex
cells with asymmetric localization in some other tissues,
including the endodermal and cortex cells of the root
elongation zone [67, 68]. However, the role of this polar
PGP localization for directional auxin movement remains
to be addressed. In summary, these data suggest that some
plant ABC transporters are involved in both efflux and
influx of auxin in different plant tissues. Whether PGPmediated
auxin transport is in any way related to PINdependent
auxin transport, and what their possible functional
interaction is, remains unknown.
Feedback regulations in auxin distribution
Numerous observations of the physiological effects of
auxin, including its inductive effect on organ formation
and vascular tissue differentiation, led to the formulation
of the canalization hypothesis, which proposed that
auxin has a feedback effect on both its own transport and
the directional polarization of auxin flow [28]. A positive
feedback effect on auxin transport could theoretically be
driven by upregulation of auxin transport capacity and
polarity, which could occur through auxin regulating the
transcription, degradation and/or subcellular localization
of its own transport machinery.
Figure 2. Polar localization of PIN proteins in Arabidopsis tissues.
(a) PIN1 localization in the triangular-stage embryo. PIN1 (green)
localizes basally in inner cells in a medial optical section (left) and
points toward the tips of cotyledons in outermost cells (middle,
right). (b) Localization of various PINs in the plant root. Arrows
indicate the presumptive direction of auxin flow.
2744        H. Tanaka et al. Auxin gradients, upstream and downstream
Regulation of transcription: PIN functional
redundancy
Genetic analyses suggested that PIN proteins exhibit extensive
functional redundancy, even though most members
show only partially overlapping patterns of expression
[8, 20, 70]. For example, pin3 and pin4 mutations
enhance the patterning defects of early pin7 embryos
(e.g. in pin1,3,4,7 quadruple mutants) [20, 71], although
surprisingly the expression of PIN3 and PIN4 is only detected
in later embryo stages in wild-type [10, 20]. These
observations suggested that a special type of functional
redundancy was involved, in which the compensatory
ectopic expression of other PIN genes in pin mutant
backgrounds could occur. Indeed, PIN4 was found to be
expressed in the suspensor cells of young globular-stage
pin7 embryos, whereas in wild-type only PIN7, but not
PIN4, is present at this stage [71]. Similarly, in roots, five
membersofthePINgenefamily(PIN1,PIN2,PIN3,PIN4
and PIN7) are expressed and play a collectively essential
role in root meristem organization [70]. The systematic
analysis of PIN protein expression in various pin mutant
backgrounds has revealed upregulation of compensatory
members [70, 71], providing additional evidence for
extensive cross-regulation between members of the PIN
gene family.
Since mutations in PIN genes disturb the local distribution
of auxin, one possible mechanism for PIN cross-regulation
could be that auxin itself changes the expression patterns
of PIN genes. Indeed, auxin treatment influences the
accumulation of transcripts from all tested members of
the PIN gene family with different tissue specificity [71].
In addition, GUS reporter activity driven by the promoter
from each PIN gene positively responds to auxin [71].
These changes to PIN expression patterns are regulated
by auxin through Aux/IAA-dependent signaling (see below)
[71]. These results show that the expression of PIN
genes is either directly or indirectly regulated by auxin at
the transcription level, providing a plausible explanation
for the ectopic upregulation of PIN genes in pin mutants
(Fig. 3). Interestingly, auxin seems also to affect PIN
protein stability, at least in the case of PIN1, PIN2 and
PIN7 [71, 72], providing an additional means of regulating
cellular PIN levels. Furthermore, microarray analysis
indicated that, in addition to PINs, expression of AUX1,
PGP1 and PGP19 is induced by auxin [73]. These observations
collectively indicate that the auxin-dependent
transcriptional and post-transcriptional control of auxin
transport components is involved in the fine tuning of
auxin flow.
Constitutive protein cycling: control of PIN
localization
Not only is the expression of PIN genes exceptionally
adaptable, several studies have indicated that the subcellular
localization of PIN proteins is also very dynamic.
In response to the treatment of Arabidopsis roots with
the fungal toxin brefeldin A (BFA), which inhibits exocytosis
(movement to the plasma membrane) but allows
endocytosis, PIN1 protein rapidly internalizes from the
plasma membrane and accumulates into the so-called
BFA compartments [74]. After BFA washout, PIN1 relocalizes
back to the plasma membrane. PIN1 internalization
occurs in this reversible manner even when new
protein synthesis is inhibited, indicating that PIN1 protein
constitutively cycles between the plasma membrane
and endosomes [75]. In support of this, antibodies raised
against a conserved peptide of Arabidopsis PIN1 protein
not only labels the plasma membrane but also the Golgi
network and endosomes, although the exact specificity of
this intracellular labeling remains to be proved [76].
The primary cellular target of BFA, in terms of its effect
on PIN1 localization, is GNOM [77], a GDP/GTP exchange
factor for a small G protein of the ARF (adenosyl
ribosylation factor) class [74], which is involved in the
exocytosis of PIN1 [77]. Furthermore, PIN1 is mislocalized
in gnom mutant embryos, such that cellular PIN1 polarity
does not align with the embryonic axis [74]. These
results indicate that proper vesicle trafficking plays an
essential role in the normal localization of PIN1. In addition
to PIN1, PIN2 and PIN3 cycle between the plasma
membrane and endosomal compartment(s) in an actindependent
manner [18, 75, 78]. It is now not so surprising
that, consistent with the loss of PINs from the plasma
Figure 3. Possible feedback regulations in the generation of auxin
gradients and auxin-related downstream events. Auxin modulates
its own transport by upregulating PIN gene expression, PIN protein
degradation and the redistribution of PIN proteins.
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2745
membrane, BFA rapidly interferes with auxin efflux [79,
80] and can also mimic the physiological effects of auxin
efflux inhibitors such as inhibition of root gravitropism
and lateral root initiation [75].
The function of the constitutive cycling of PIN is not yet
clear. It is possible that it facilitates the redirection of
auxin flux by rapidly repositioning PIN proteins to the
required places at the plasma membrane in response to
environmental stimuli. Indeed, PIN3 was shown to rapidly
relocate in response to gravity stimulation [18]. The
dynamics of the cycling of PIN proteins also potentially
regulates protein localization at the membrane by controlling
endo- and/or exocytosis themselves. Three key
pieces of evidence support the idea that auxin modulates
PAT by temporarily inhibiting the endocytosis of PIN
proteins [33]. First, an increased level of auxin either by
exogenous application, genetic manipulation or gravity
stimulation reduced the endocytosis of proteins, including
PIN1 and PIN2, in Arabidopsis roots [33]. Second,
consistent with the stabilization of PINs at the plasma
membrane, the efflux of NAA from tobacco BY-2 cells
was increased by pretreatment with 2,4-D [33]. Third,
superroot1 mutant plants, which have an elevated level
of auxin, are defective in PIN internalization [33] and exhibit
increased PAT [81]. This novel effect supports the
canalization hypothesis, where auxin, by inhibiting endocytosis,
seems to positively regulate its own transport.
The molecular mechanism by which auxin displays this
effect is as yet unknown, and further dissection of this
process by the analysis of proteins involved, including the
Callosin-like protein BIG [33], is keenly awaited.
PIN polarity and direction of auxin flow
Increasing lines of evidence suggest that the asymmetric
cellular distribution of PIN proteins is important for
the directional guidance of auxin transport [82]. Notably,
changes in the polarity of engineered PIN proteins are
sufficient to determine the direction of auxin flow in the
root epidermis [65]. In addition, the polarity of PIN proteins
can be modified in response to external and developmental
cues [8, 18, 20, 83]. Several factors required for
the polar localization of PINs are described below.
TheArabidopsis mutant,orc,exhibitscellpolaritydefects,
and the localization of PIN1 and PIN3 is disturbed in orc
seedling roots [84]. Mutation in the STEROL METHYLTRANSFERASE1
(SMT1) gene was found to cause the
orc phenotype, providing a link between sterol biogenesis
and the polarity of PIN localization [84]. Another link
between sterols and PIN localization came to light from
the finding that the sterol-specific fluorescent dye filipin
co-localizes with the PIN2 protein [78]. These observations
suggest that the sterol composition of membranes is
important for the proper localization of PIN proteins.
To date, the only gene directly involved in the regulation
of polar localization of PINs emerged from the analyses
of pinoid (pid) mutants, which share phenotypic characteristics
with pin1 mutants [85]. The PID gene encodes a
serine-threonine protein kinase [86], and its overexpression
affects root development and tropism, seemingly
due to altered auxin transport [87, 88]. Ectopic expression
of PID causes a basal-to-apical shift in PIN1, PIN2
and PIN4 localization in seedling roots and developing
embryos, leading to the loss of auxin gradients. This
supports a direct role of PID in the regulation of PAT.
Conversely, the loss of PID function causes an apical-tobasal
shift in PIN1 localization at the inflorescence apex
[88] and embryonic cotyledons [89]. This suggests that
the activity levels of the PID kinase function as a binary
switch for PIN polar localization and the regulation of
PAT during organ formation. Interestingly, expression of
PID itself is rapidly induced by auxin [73, 86, 87], indicating
a potential feedback regulation of PIN polarity by
auxin-mediated PID expression.
Thus, auxin itself modifies the direction of its own flow
(Fig. 3). However, it remains to be addressed to which extent
such feedback regulations contribute to auxin distribution
in different tissues, including those in shoot and root
meristem. For example, in regenerating root meristems,
auxin affects the localization of PIN proteins only after
cell-type specification or in the absence of developmental
regulators responsible for cell differentiation [83].
Downstream: cellular auxin signaling
It seems that auxin accumulation in certain cells initiates
a plethora of cell-specific developmental responses. It is
increasingly apparent that the conversion of auxin accumulation
patterns into specific physiological responses
occurs through downstream auxin signaling.
For decades, substantial efforts were made to identify
the auxin receptor and its downstream signaling components.
In a biochemical approach, proteins that bind to
auxin have been screened by affinity labeling and affinity
purification. An auxin binding protein (ABP1) has been
repeatedly identified in plant species, including maize
and in Arabidopsis. Evidence for the role of ABP1 in cell
elongation has been provided through functional analyses
involving anti-ABP1 antibodies and by a genetic approach
(for overview, see [90]). From studies in tomato,
an additional member of theABP1 pathway has been possibly
identified as a protein of the cyclophilin class – diageotropica
[91, 92]. However, to date, the role of ABP1 in
a specific cellular process such as auxin-mediated regulation
of gene expression has not been shown.
Pathway for auxin-mediated gene expression
Genetic approaches have been used to identify the molecular
components of the auxin pathway. From screens
2746        H. Tanaka et al. Auxin gradients, upstream and downstream
for auxin-resistant mutants, a number of auxin-related
factors, such as AUXIN-RESISTANT1 (AXR1) and
TRANSPORT INHIBITOR RESPONSE1 (TIR1), have
been identified as being involved in ubiquitin-related
modification of proteins, required for the regulated degradation
of proteins [93]. TIR1 encodes an F-box protein
that forms a complex with AtCULLIN1 (AtCUL1) and
a SKP1-like protein to form an SCF ubiquitin protein ligase
E3 (SCFTIR1) [94, 95] complex, which specifically
ubiquitinates substrates for proteasome-dependent degradation.
Substrates for the SCFTIR1-mediated ubiquitination
were originally identified from molecular biological
techniques. The whole story began with a finding that
auxin rapidly regulates the expression of multiple genes.
Many of these genes were shown to be primary auxinresponse
genes, such as GH3, SAUR and Aux/IAA genes
[22]. This led to the identification of an auxin-responsive
cis-regulatory element (AuxRE) in the promoters of many
auxin-responsive genes. Subsequently, AuxRE-binding
proteins – the ARFs (auxin response factors) – and small,
short-lived Aux/IAA proteins that bind to ARFs and repress
their activity, have been isolated [22]. ARF proteins
contain an N-terminal DNA-binding domain, middle
region and C-terminal dimerization domain. The Aux/
IAA proteins contain homologous dimerization domains
(designated domains III and IV), which are used for their
interaction with ARFs [96, 97]. Specific mutations in
other domains (domain II) of Aux/IAAs leads to the stabilization
of Aux/IAA proteins, often resulting in severe
developmental defects and auxin-resistant phenotypes.
Strikingly, Aux/IAAs directly interact with SCFTIR1 in an
auxin-dependent manner, which provides a direct connection
to the ubiquitination pathway [98]. Auxin enters this
pathway by binding directly to theTIR1 F-box protein [99,
100]. Thus, the pathway for auxin-dependent regulation
of gene expression appears to be remarkably straightforward:
auxin binds to SCFTIR1, facilitating the interaction
of SCFTIR1 with Aux/IAAs. This leads to ubiquitination
and degradation of Aux/IAA repressors and the release of
ARF transcriptional regulators for the induction of auxindependent
gene expression (overviewed by [101]).
The activity of the SCFTIR1 pathway is tightly regulated
by several additional factors including AXR1, a protein
related to the N-terminal half of ubiquitin-activating enzyme
E1. AXR1, in concert with another activating enzyme,
E1 C-terminal Related1 (ECR1), and RUB-conjugating
enzyme1, RCE1, conjugate a ubiquitin-related
molecule (RUB, also referred to as nedd8) to AtCUL1,
a component of SCFTIR1 [102, 103]. AtCUL1 is also associated
with a protein complex called the COP9 signalosome,
which removes the conjugated RUB1 from
AtCUL1 [104]. The cycle of RUB1 conjugation and its
removal appears to be critical for the function of SCFTIR1
and might limit the capacity of auxin responsiveness in
cells.
Auxin signaling can also be switched off, following its
activation, by Aux/IAA repressors, which themselves
are early auxin-inducible genes [22]. In addition, a microRNA-dependent
mechanism seems to be involved in
auxin signaling.The NAC1 gene (encoding a NAM/ATAF/
CUC-domain-containing transcription factor), which acts
downstream of TIR1 and positively regulates lateral root
formation [105], is downregulated by auxin-inducible
miR164 [106]. Increasing lines of evidence point to the
importance of auxin-mediated gene expression mediated
by TIR1-related F-box proteins. However, in part because
of redundancy in each component and early lethality associated
by complete inhibition of the pathway [107], it
remains unclear whether this pathway governs the whole
of auxin-inducible gene expression.
Specificity of ARF- and Aux/IAA-dependent
signaling
An important question remains as to how the rather
simple cellular signaling of auxin, consisting of TIR1-,
Aux/IAA- and ARF-dependent steps, can confer so many
different developmental responses in different cell types
[108]. There are 29 Aux/IAA genes and 23 ARF genes
in the Arabidopsis genome [109, 110], theoretically providing
sufficient molecular complexity, which could further
be dramatically enhanced by specific combinations
of ARF and Aux/IAA interactions. Accordingly, various
cellular auxin responses could be facilitated through the
specific expression of Aux/IAAs and ARFs in particular
cells, specific interactions between particular Aux/IAAs
and ARFs, and specialized roles for each ARF. Genetic
analyses using loss-of-function ARF mutants have indeed
revealed distinct although partially overlapping roles for
ARFs in diverse auxin-related processes such as embryonic
pattern formation [ARF5/MONOPTEROS (MP)]
[111], lateral root formation and tropism [ARF7/NONPHOTOTROPIC
HYPOCOTYL 4 (NPH4) ARF19] [110,
112], adaxial-abaxial axis specification in aerial organs
(ARF3/ETTIN and ARF4) [113], seedling apical hook development
(ARF2) [114], senescence (ARF1,2,7 and 19)
[115, 116], floral organ development (ARF1-3, 6 and 8)
[115, 117, 118] and auxin homeostasis (ARF8 and 17)
[119, 120]. At least to some extent, different ARF proteins
seem to have specific functions, since ARF5/MP
rescued the mp mutant phenotype more efficiently than
ARF16 when expressed under the same promoter [121].
The pattern of Aux/IAA gene expression, at least at one
level, contributes to their specific biological roles since
the ectopic expression of stabilized Aux/IAA proteins in
different cells can induce novel phenotypes [121, 122].
On the other hand, the functional specificity of Aux/IAA
genes is also important, as they cause distinct phenotypes
when expressed under the same promoter [121, 123], producing
equivalent protein amounts [121]. For example,
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2747
iaa12/bdl mimicked the mp mutant phenotype more efficiently
than iaa3/shy2 when expressed under the control
of the BDL promoter, suggesting that BDL/IAA12 and
ARF5/MP are an optimized pair [121]. Thus, it appears
that the specificity of auxin response in a specific tissue
can be determined mainly by three levels: transcriptional
regulation to limit the maximal action of ARFs and Aux/
IAAs, the intrinsic function of ARFs and repression of
ARF functions by an optimized Aux/IAA-ARF pair.
Downstream components of the ARF-dependent
signaling cascade
The question arises next is, what are the factors present
downstream of the ARF-dependent cellular auxin signaling
that ultimately execute various developmental events?
Some of the key factors converting auxin-regulated gene
expression into developmental responses have been
elucidated through genetic analyses and searching for
auxin-inducible genes. For example, during organ formation
and tropic responses, auxin induces the expression
of genes regulating cell-fate determination [124] and cell
elongation, respectively [12].
After tropic stimuli, auxin is redistributed asymmetrically,
resulting in the differential expression of auxin-inducible
genes. Remarkably, included are genes coding for expansins,
which loosen the cell wall and induce cell expansion
[12]. In agreement with the proposed role of expansins in
cell expansion, their transcript accumulation increases in
the shaded or bottom sides of hypocotyls after phototropic
or gravity stimulation, respectively. Induction of their
expression depends on PAT and ARF7/NPH4, suggesting
that auxin distribution generated by tropic stimulation
triggers the expression of proteins that can directly regulate
tropic growths [12].
During root patterning, the PLETHORA1 (PLT1) and
PLT2 genes, which encode AP2-domain-containing transcription
factors required for stem cell specification, are
upregulated by prolonged treatment with auxin [124].
PLT expression spatially correlates with DR5 expression
and depends on ARF activity [124], suggesting that
auxin accumulation either directly or indirectly regulates
the pattern of PLT transcript distribution. PLT, in turn,
regulates the expression of PINs, possibly providing another
feedback loop in auxin distribution that is essential
for root development [70].
In the inflorescence meristem, expression of both DR5
and PIN1 is detected in incipient and very early stages of
primordia initiation [8, 27]. Marker genes derived from
SHOOTMERISTEMLESS (STM) and CUP-SHAPED
COTYLEDON2 (CUC2), which are involved in the formation
and maintenance of the shoot apical meristem
and organ separation, are expressed in a complementary
domain to the PIN1-expressing region [27]. Furthermore,
Heisler and colleagues demonstrated by live imaging of
PIN1::GFP that a developmentally regulated reversal of
PIN1 polarity occurs in the boundary of primordia that
correlates with the upregulation of STM and CUC2 expression
[27]. Consistently, CUC2 expression is downregulated
in cotyledon boundaries of pin1 and mp/arf5
embryos [125], suggesting that auxin transport and response
are involved in the local upregulation of CUC2.
Genetic evidence also suggests that auxin transport is
required for the local downregulation of CUC2 in the inflorescence
meristem [126]. However, whether this regulation
involves ARFs still remains elusive.
Model cases for auxin-dependent development
As the specific developmental processes triggered by
transport-dependent auxin gradients differ considerably,
we now discuss selected examples demonstrating this
complex regulation.
Embryonic axis formation
Embryogenesis is a fundamental step in plant development,
in which shoot and root meristems are generated
along the apical-basal axis. Just prior to key events in
embryonic patterning, the transport-dependent auxin
accumulation pattern changes dynamically [20]. Following
the asymmetric, anticlinal division of the zygote,
PIN7 becomes specifically expressed in basal cell and
its derivatives (suspensor cells). Here, PIN7 localizes
to the apical (upper) surface of the basal cell and directs
auxin flow towards the apical cell, where auxin
accumulates. This accumulation of auxin in the apical
cell persists for several rounds of cell division in the
subsequently formed proembryo cells [20] (Fig. 1a).
From the first periclinal division of the apical cell, PIN1
comes into action, channeling auxin flow. It localizes at
the two facing plasma membranes of the daughter cells
(generated by the division of the apical cell), presumably
to equalize the distribution of auxin between these
cells. At the young globular stage, the developmentally
regulated reversal of PIN polarities takes place. PIN1
starts to localize to the bottom side of the cells in the
embryo proper, which face towards the suspensor cells.
Concomitantly, PIN7 polarity changes in these cells to
shift from the top to the bottom side. This rearrangement
of PIN polarities results in the establishment of an
‘apical-to-basal’flow of auxin. Thus, auxin accumulates
in the basal part of the embryo and the upper suspensor
cells, which later give rise to the root meristem [20]
(Fig. 1a). Mutations in multiple PIN genes [20] as well
as in the components of the auxin signaling pathway,
including TIR1-related genes [107], BDL/IAA12 [127]
and MP/ARF5 [111], cause severe misspecification of
apical and basal embryonic structures. This shows that
2748        H. Tanaka et al. Auxin gradients, upstream and downstream
PIN-dependent auxin accumulation and its cell-specific
interpretation are critical for proper embryo patterning.
As cotyledon initiation begins, PIN1 localization in the
outermost cells of the apical part of the embryo is directed
toward the apices of the incipient cotyledons [8]
(Figs. 1a, 2a). The generation of cotyledons in the apical
region also requires the activities of TIR1-related proteins
[107] and ARFs [128] in addition to those of PINs
[14, 20]. Collectively, these results demonstrate the central
role of cellular auxin transport, auxin distribution
and cellular auxin signaling in the specification of the
apical-basal axis and organ formation during plant em-
bryogenesis.
Phyllotaxis
Aerial organs such as leaves and flowers are generated
by the shoot apical meristem in highly predictable
phyllotactic patterns. For instance, Arabidopsis shoots
produce leaves and then flowers in a spiral pattern. Microapplication
experiments found that auxin can trigger
new leaf and flower formation in the peripheral
region of the shoot meristem [25]. It is now clear that
PIN1-dependent PAT directs available auxin in the shoot
meristem to specific locations for organ initiation. Studies
into the localization patterns of PIN1 and AUX1 at
shoot apices suggest that auxin is transported upwards
into the meristem through the outer L1 layer of the shoot
meristem [8, 58]. In an available space in the meristem,
auxin presumably reaches a threshold concentration
which changes PIN localization. PIN1 then, for the time
being, localizes towards the initiation site, apparently
pumping auxin in towards each new organ position.
This corresponds to a temporary increase in DR5 expression
in the initiation site, and the beginning of organ
growth [8, 27, 58, 129, 130]. These results suggest that
while movement of auxin into an incipient primordium
triggers organ initiation, initiation is also inhibited in
surrounding cells where auxin is temporarily depleted.
Auxin is only able to increase again in cells at a specific
distance from the absorbing influence of preexisting
primordia. Thus, the positions of preexisting primordia,
together with PIN-dependent auxin transport, and the
specific dimensions of the meristem, appear to be sufficient
to generate the distinct phyllotactic patterns that
are seen in plants [27, 58]. It is interesting that computer
simulations can generate phyllotactic patterns of auxin
accumulation and organ formation using experimentally
obtained parameters and the assumption that auxin distribution
also affects PIN polarity [130, 131]. Although
the auxin effect on the polarity of PINs has not yet been
substantiated experimentally, it is possible that auxin
impinges on the PIN polarity through the regulation of
PINOID expression [87, 88] or through the effects of
auxin on PIN trafficking [33].
Gravitropism
Gravitropism is a process which allows plants to sense
the gravity vector and align their growth direction accordingly.
Generally, roots show positive gravitropism
– growing down into the soil – whereas shoots display
negative gravitropism, growing away from the soil. In
many plant species, including Arabidopsis, gravity perception
primarily takes place in the endodermis in shoots
and the columella root cap cells in roots, both of which
contain starch-filled organelles called amyloplasts (also
referred to as statoliths) [132]. As gravity perception and
differential growth response take place in different tissues
of the root (root cap and elongation zone, respectively),
the mobilization of a signal from the perceiving to the
responding tissue has been hypothesized. Similarly, the
transport of such a signal from endodermis to epidermal
cells in the shoot, where the control of shoot elongation
occurs, has been proposed [132].
The classic Cholodny-Went hypothesis proposes that
auxin is the major signal in gravitropic response and
that asymmetric auxin distribution mediates differential
growth, which results in the bending of gravistimulated
organs [31]. It is interesting that after changes to gravity
direction, organs consistently accumulate auxin at their
lower side [12, 18, 32], even though the growth response
may be different. Normally, cell elongation is stimulated
in shoots, whereas in roots it is inhibited, allowing differential
growth upwards and downwards, respectively.
Auxin efflux inhibitors interfere with the generation of
asymmetric auxin distribution and tropic growth, suggesting
that PAT underlies gravitropism [12, 18, 32].
An important insight into the molecular link for gravity
perception and generation of asymmetric auxin distribution
was provided through study of the PIN3 protein [18,
133]. The Arabidopsis pin3 mutant is partially defective
in shoot and root gravitropism. Importantly, PIN3 protein
has been detected in gravity-sensing cells – the columella
cells of the root and endodermis cells of the shoot [18]
(Fig. 2b). In columella cells PIN3 rapidly responds to the
altered gravity vector by relocating to the new bottom
side of the root, suggesting that statolith sedimentation is
translated into changes in PIN3 polarity and PIN-mediated
differential auxin accumulation [18] (Fig. 1b). In addition
to PIN3, root gravitropism involves PIN2, AUX1,
PGP4 and PGP19 [36, 37, 62, 67, 134]. Based on the
capacity of tissue-specific expression of AUX1 to rescue
the agravitropic aux1 root phenotype, AUX1 has been
shown to be required in the lateral root cap and epidermis,
where AUX1 in concert with PIN2 mediates basipetal
auxin transport [34, 62] (Fig. 2b). Loss of PIN2 or AUX1
activities abolishes gravity-stimulated asymmetric auxin
distribution and causes undirected root growth [19, 36,
65]. Signaling events downstream of auxin accumulation
appear to be regulated by Aux/IAAs and ARFs [110, 112,
132]. Targeted expression studies using the dominant
Cell. Mol. Life Sci.   Vol. 63, 2006	 Review Article        2749
mutation of AXR3/IAA17 revealed that auxin response is
specifically required in epidermal cells in the root elongation
zone [34]. It has been reported that pgp19 and
pgp4 mutants exhibit a hypergravitropic hypocotyl phenotype
[134] and reduced gravitropic root bending [67],
respectively. Although PGP4 and PGP19 are involved in
auxin influx and efflux processes, it remains to be unambiguously
demonstrated whether they are involved in
asymmetric auxin distribution [67–68]. In summary, root
gravitropic response involves gravity perception by statoliths
in columella cells, rapid relocation of PIN3 in the
same cells, generation of asymmetric auxin flow towards
the elongation zone through the epidermis by AUX1 and
PIN2, and transcriptional regulation of downstream factors
by ARFs in the elongation zone. However, the causeeffect
relationship between statolith sedimentation and
PIN3 relocation has not been demonstrated so far, and
this would be an interesting topic for future research.
In shoots, PIN3 localizes to the endodermis and exhibits
asymmetric subcellular localization predominantly at the
inner side of cells [18]. The loss-of-function pin3 phenotype
as well as localization of PIN3 protein suggests
that PIN3 plays a similar role in the lateral distribution
of auxin to the lower side of the shoot during the gravitropic
response [18]. Accumulation of auxin at the lower
side of hypocotyls causes expression of a set of genes in
an ARF-dependent manner [12, 112]. The target genes
include those coding for expansins, which can directly
modify the cell wall and induce cell elongation, providing
a likely mechanism connecting auxin distribution and
differential growth [12].
It is possible that a mechanism involving changes in PIN
polarity for asymmetric auxin distribution also underlies
phototropic responses; however, a connection between
light perception and PIN polarity changes has not been
demonstrated so far.
PAT in non-Arabidopsis species
Molecular components potentially involved in PAT, auxin
signaling and downstream events have been also recently
characterized in plant species other than Arabidopsis. In
this section, we summarize selected data with functional
significance from the study of non-Arabidopsis plants
and compare them with what is known from Arabidopsis
research where appropriate.
Early studies of the effect of auxin on plant tropism were
performed using plant species such as canary grass and
oat coleoptiles [30, 31]. This tradition continued with
studies demonstrating that auxin is asymmetrically distributed
preferentially to the elongating side of tissues after
tropic stimulation in both monocotyledonous and dicotyledonous
plants, including maize and rice coleoptiles
[135, 136] and Brassica hypocotyls [12]. Consistently,
differential auxin-inducible gene expression is observed
in various plant species, including light-stimulated maize
coleoptiles [11], gravity- and light-stimulated tobacco
stems [32] and Brassica hypocotyls [12]. In Brassica hypocotyls,
accumulation of transcripts of genes, including
expansins, increase at the bottom and shaded sides of hypocotyls
stimulated with gravity and light, respectively
[12]. Notably, differential auxin accumulation was not
found in the rice mutant coleoptile phototropism1 (cpt1)
[136]. The CPT1 gene encodes a protein orthologous to
Arabidopsis NPH3, a signaling component that physically
interacts with the photoreceptor and is involved in
hypocotyl phototropism [137]. These results demonstrate
that rice and Arabidopsis share a key component for light
signaling involved in phototropism.
A genetic study involving RNA interference indicated
that a rice homologue of PIN1, OsPIN1, is involved in
the formation of adventitious roots [138].The phenotype
associated with suppression of OsPIN1 messenger RNA
(mRNA) accumulation is reminiscent of that of NPAtreated
rice plants [138]. In rice, auxin accumulation
appears to cause adventitious root formation through
the ARF-dependent transcription of CROWN ROOTLESS1,
which encodes a protein of the ASYMMETRIC
LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/
LBD) family [139]. As some genes in the AS2/LBD family
are induced by auxin in an ARF-dependent manner
in Arabidopsis and the overexpression of LBD16 promotes
lateral root formation in the arf7 arf19 double
mutant [110], rice and Arabidopsis appear to share at
least some downstream targets for ARFs. Regulation
of PIN-gene expression appears to be shared between
Arabidopsis and pea, as accumulation of transcripts of
a pea homologue of PIN gene (PsPIN1) is upregulated
by auxin [140].
In summary, increasing lines of evidence suggest that
dicotyledonous and monocotyledonous plants share at
least some key components for light and gravity perception,
auxin transport and downstream factors. So far the
available genetic and physiological data from non-Arabidopsis
species suggest that PAT- and PIN-dependent
asymmetric auxin distribution is a functional module,
which also operates in pattern formation, organogenesis
and tropisms in other higher plant species. Naturally,
this does not necessarily mean that the details of this
mechanism for each particular developmental process
in other plant species are identical to Arabidopsis, as
some processes such as embryogenesis or flower development
are quite diverse in different plant species. It is,
however, clear that growing knowledge from Arabidopsis,
together with the recent availability of genetic and
genomic resources, including rice and maize genomic
DNA sequences [141–144], will accelerate our understanding
of PAT-mediated regulation of plant growth and
development in higher plants in general.
2750        H. Tanaka et al. Auxin gradients, upstream and downstream
Conclusion and outlook
In summary, recent studies using Arabidopsis thaliana
and other species have shown that the transport-dependent
spatiotemporal distribution of auxin serves as
a means to interpret environmental and developmental
cues by modulating the concentration of auxin in order
to trigger cellular growth responses. Such a system,
which involves multiple feedback loops, together with
the combinatorial capacity of downstream auxin signaling
events, could account for the variety of auxin effects
in plant development. However, as the molecular components
that regulate the activity and polarity of auxin
transport components are still poorly understood, identifying
tissue-specific regulators and elucidating mechanisms
that determine the polarity of transport proteins,
including PINs, would be interesting topics to address.
The accumulation pattern of auxin appears to be interpreted
in cells through the binding of auxin with TIR1related
F-box proteins, followed by the degradation of
Aux/IAA proteins and the subsequent release of ARF
proteins, although the possibility of independent parallel
pathways for auxin-inducible gene expression has not yet
been excluded. In various development and growth processes,
overlapping and distinct roles for different ARFs
have been emerging. The increasing knowledge of the
molecular components involved in PAT and downstream
auxin signaling are greatly improving our understanding
of the auxin-related regulation of each developmental
process and how it is connected to the other plant signaling
pathways.
Acknowledgements. We thank Marta Michniewicz for photographs
of PIN protein localization in roots, Michael Sauer, Tomasz Paciorek
and Anne Vieten for helpful discussions. This work was supported
by the Volkswagenstiftung (J. F., P. D.), EMBO YIP (J. F.)
and a long-term fellowship from the Human Frontier Science Program
(HFSP) Organization (H. T.).
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