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Regulation of auxin transport polarity by AGC kinasesHelene S Robert1 and Remko Offringa
The plant hormone auxin controls plant development
through gradients and maxima that are generated by PIN
efflux carrier driven polar auxin transport. PIN proteins direct
this cell-to-cell auxin transport, and thus orient plant
development through their asymmetric subcellular distribution.
PIN polarity is regulated by PINOID and the phototropins,
members of the AGC protein serine/threonine kinase family.
Here we review the signaling pathways of these kinases and the
role of calcium and BTB proteins in translating both internal and
external signals into developmental responses via PIN
relocalization, to adapt plant development to changing
environmental conditions.
Addresses
Department of Molecular and Developmental Genetics, Institute of
Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The
Netherlands
Corresponding author: Offringa, Remko
(r.offringa@biology.leidenuniv.nl)1Current address: Department of Plant Systems Biology, VIB, Ghent
University, Technologiepark 927, 9052 Ghent, Belgium.
Current Opinion in Plant Biology 2008, 11:1–8
This review comes from a themed issue on
Cell Signalling and Gene Regulation
Edited by Jason Reed and Bonnie Bartel
1369-5266/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2008.06.004
IntroductionAs sessile organisms, plants have developed various
mechanisms for perceiving and responding to changes
in environmental conditions, such as light, temperature,
and drought. Studies of Darwin on the tropic bending of
canary grass and oat coleoptiles toward a unidirectional
light source provided the first indications of a transported
growth substance that mediates this adaptive response. A
few decades later, the Dutch botanist Went was the first
to isolate this growth substance, which was identified as
the signaling molecule indole-3-acetic acid (IAA) and
named auxin after the Greek word for ‘to grow’. The
asymmetric lateral distribution of auxin during tropic
responses was found to be mediated by its polar cell-
to-cell transport. This polar auxin transport (PAT) is
crucial for many developmental processes, creating auxin
gradients and maxima that regulate cell division,
elongation, and differentiation, and in this way are instru-
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
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mental for plant architecture and for its adaptation to
environmental changes [1].
The drivers of the auxin transport pathwayMolecular genetic research in Arabidopsis has identified
PIN-FORMED (PIN) transmembrane proteins as auxin
efflux carriers that determine the direction of PAT
through their asymmetric subcellular localization
[1,2�,3�]. The Arabidopsis thaliana genome has eight
PIN genes. The function of five of the encoded proteins
has been analyzed in detail, whereas that of PIN5, PIN6,
and PIN8 remains to be uncovered [1]. Besides PIN
proteins, the P-glycoprotein (PGP)-type ABC (ATP-
binding cassette) transporters, PGP1, and PGP19, have
been shown to act as auxin efflux carriers [2�,4]. Arabi-
dopsis PGP1 and PGP19 co-localize with PIN1 and PIN2
in root and shoot cells and enhance auxin transport
activity by physically interacting with these PINs [5].
Their predominantly apolar subcellular localization
suggests that PGPs are not major determinants in the
directionality of PAT [5]. PGP-type ABC transporters are
found from unicellular algae to angiosperms, and are
likely to be the ancient auxin efflux carriers, whereas
polar PIN proteins occur only later in evolution in multi-
cellular land plants for which directional auxin transport
has become a crucial aspect of development [6��]. In
addition to auxin efflux carriers, the AUXIN RESIST-
ANT1 (AUX1) permease-like protein and three homolo-
gous Like-AUX1 (LAX) proteins were identified in
Arabidopsis as auxin influx carriers [7,8]. AUX/LAX are
important enhancers of auxin transport and seem to
contribute to the directionality of PAT through feedback
on PIN polarity [9]. As PIN proteins are clearly the crucial
drivers of PAT, we will focus this review on the signaling
pathways that determine their asymmetric subcellular
localization and thereby modulate developmental pro-
grams in response to environmental stimuli.
General mechanisms in PIN polarity dynamicsPIN polar localization is highly dynamic, is regulated by
vesicle trafficking, protein degradation, and phosphoryl-
ation, and is responsive to both external and internal
signals, among which auxin [10–14]. Cyclic trafficking
of PIN-loaded vesicles was first demonstrated by using
the fungal antibiotic Brefeldin A (BFA) [10]. BFA blocks
exocytosis by targeting the large guanine nucleotide
exchange factors for the ARF-type G proteins (ARF-
GEFs) involved in endosomal recycling (Figure 1). In
Arabidopsis root cells, PIN1 basal polar recycling requires
the BFA-sensitive ARF-GEF GNOM, while apical recy-
cling of PIN2 and AUX1 is mediated by BFA-resistant
ARF-GEFs [10,11��,15]. Moreover, the basal retargeting
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
Current Opinion in Plant Biology 2008, 11:1–8
2 Cell Signalling and Gene Regulation
COPLBI-562; NO OF PAGES 8
Figure 1
Model for the role of the PINOID (PID) kinase as key regulator of the dynamic polar subcellular localization of PIN auxin efflux carriers. PIN proteins are
polarly localized auxin efflux carriers [1]. For a rapid regulation of their localization, they cycle between endosomal membrane compartments and the
plasma membrane, via exocytosis and clathrin- and sterol-dependent endocytosis [10,16,18]. This trafficking requires the action of ARF-GEF proteins.
In the basal PIN cycling pathway, the BFA-sensitive ARF-GEF GNOM is involved in exocytosis [10], whereas the BFA-resistant GNOM-LIKE1 (GNL1) is
thought to be required for endocytosis [17]. Other BFA-resistant ARF-GEF proteins are likely to be involved in the apical cycling route of PIN-loaded
vesicles. Movement of PIN-cargoes between the basal and apical cycling pathways is occurring via transcytosis [11��]. The PINOID (PID) protein
serine/threonine kinase is a membrane-associated protein that labels PIN cargoes for trafficking to the apical plasma membrane by phosphorylating
the hydrophilic loop of PIN proteins [22��]. Whether PID acts at the apical or basal plasma membrane or in endosomal compartments is not yet known.
PID activity is negatively or positively regulated through its calcium-dependent interaction with the calcium-binding proteins TOUCH3 (TCH3) or
PINOID-BINDING PROTEIN1 (PBP1), respectively [32]. Elevated auxin concentrations are well known to increase cytosolic calcium [27–30] and, apart
from the positive effect of auxin on PID expression [21], this could provide a second feedback loop by which auxin directs its own transport by
regulating the subcellular localization of PIN proteins.
of PIN1 during embryogenesis and lateral root formation
involves ARF-GEF-dependent transcytosis (Figure 1)
[11��]. This trafficking of PIN-loaded vesicles is actin
dependent [10] and PIN endocytosis is clathrin depend-
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
Current Opinion in Plant Biology 2008, 11:1–8
ent [16] and thought to be mediated by the BFA-resistant
GNOM-like 1 (GNL1) [17]. The membrane sterol com-
position is an important factor for polar endocytic recy-
cling of membrane proteins, and the polar localization of
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
www.sciencedirect.com
Kinases orienting auxin transport Robert and Offringa 3
COPLBI-562; NO OF PAGES 8
PIN1, PIN2, PIN3, and AUX1 is altered by mutations or
drug treatments affecting sterol composition [15,18,19].
In addition to protein targeting as a control for the
direction of PAT, protein degradation and turnover were
shown to feedback on the plasma membrane localized
PIN protein abundance in response to environmental
cues such as light and gravity. PIN2 is ubiquitinated
and degraded at the upper side of the root upon gravi-
stimulation [12] and in dark grown roots several PINs and
AUX1 are targeted to the vacuole for degradation [20].
AGC kinases as regulators of auxin transportpolarityThe first, and yet only identified molecular determinant
in the polar distribution of PINs is the PINOID (PID)
serine/threonine protein kinase [13]. Above threshold
levels of PID activity were found to induce movement
of PIN proteins from the basal (root tip facing) to the
apical (shoot tip facing) side of Arabidopsis root cells,
whereas in embryos and inflorescence tip epidermis cells,
loss-of-function pid mutations cause an apical-to-basal
shift [13]. In both cases, alteration of PIN polarity changes
the direction of PAT (Figure 1) and explains the pheno-
types of PID loss- and gain-of-function mutants. These
results indicate that PID-dependent phosphorylation is
instructing for PIN apical localization. Interestingly, PIDis an auxin responsive gene [21], suggesting that PID is
part of a feedback loop by which auxin controls the
direction of its own transport [14]. Recently it has been
demonstrated that PID directly phosphorylates PIN
proteins in their large central hydrophilic loop
(Figure 1) and that PP2A phosphatases counteract the
activity of this kinase [22��]. The kinase and the phos-
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
Figure 2
Developmental processes involving AGC kinase dependent PIN relocalizatio
PIN1::PIN1:GFP expression (light blue, plasma membrane) marks the incipie
nuclear) reported auxin maxima that persist in more mature primordia (P1–P
dark-grown wild-type Arabidopis (b) or phot1 mutant (C) seedlings following
Galvan-Ampudia); (d and e), Gravitropic growth of wild-type Arabidopsis se
hook-like structure at the root tip of 35S:PID seedlings, indicative for root m
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phatases co-localize with PINs at the plasma membrane,
and probably also in intracellular compartments [22��].Similar to PID overexpression (Figure 2e), loss of PP2Afunction results in root agravitropism and root meristem
collapse, which can be partially rescued by pid loss-of-
function. In correlation with these seedling phenotypes,
PIN localization in pp2a mutants is apical in Arabidopsis
roots. Although these data show that direct phosphoryl-
ation is an important signal in PIN polar targeting [22��],the actual mechanism behind the PIN polarity establish-
ment is still unclear. Key questions to address are (1)
where in the cell does PID dependent PIN phosphoryl-
ation occur, (2) how does the phosphorylation status of the
PINs signal their incorporation in the basal (GNOM
dependent and GNL1 dependent) or apical cycling route,
(3) are other PID phosphorylation targets involved or is
PIN phosphorylation sufficient, and (4) if PID-dependent
phosphorylation determines apical PIN localization, what
signals instruct lateral or basal PIN localization?
An answer to this last research question may come from
studying the PID-like kinases. PID belongs to the AGC
family of serine/threonine kinases, which includes and is
named after the well studied animal kinases (PKA, –G
and –C) that are involved in receptor mediated growth
factor signal transduction. In plants, the typical PKA,
PKG, and PKC are not present, but instead the plant-
specific subfamily of the AGC kinases, AGCVIII, has
been identified [6��]. In Arabidopsis, this family com-
prises PID, which groups to the AGC3 clade together
with WAG1 and WAG2, and the phototropins PHOT1
and PHOT2, blue light receptors that trigger phototropic
growth [6��,23]. Functional analysis of WAG1 and WAG2
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
n. (a) Phyllotactic patterning in the inflorescence apex [44]. Elevated
nt primordium I1 and precedes the DR5rev::3xVENUS-N7 (orange,
4, image kindly provided by Marcus Heisler); (b and c), A four-day-old
4 h stimulation with unidirectional light (images kindly provided by Carlos
edlings (d) and agravitropic growth of 35S::PID seedlings (e). Note the
eristem collapse [21].
Current Opinion in Plant Biology 2008, 11:1–8
4 Cell Signalling and Gene Regulation
COPLBI-562; NO OF PAGES 8
indicated that these kinases play a role in root growth.
The enhanced sensitivity of root growth of wag1 wag2loss-of-function seedlings to the PAT inhibitor NPA [23],
and the fact that the WAG kinases, like PID, are plasma
membrane-associated together suggest that the WAG
kinases are also involved in the regulation of PIN polar
targeting [6��].
Regulators of the PAT regulatorsPDK1
A common feature of animal AGC kinases is their acti-
vation by the 3-phosphoinositide dependent protein
kinase1 (PDK1). In animal cells, PDK1 recruits the kinases
by binding to their hydrophobic C-terminal PDK1 Inter-
acting Fragment (PIF) domain, and activates or enhances
the activity of the kinase by phosphorylation [24]. The
majority of the Arabidopsis AGCVIII kinases, including
PID, but excluding WAG1, WAG2, PHOT1, or PHOT2,
have a PIF domain and are in vitro substrates of PDK1
[25,26]. However, the in planta significance of their PDK1-
dependent phosphorylation is still unclear. PID, for
example, is an auto-activating kinase, and it may therefore
not need PDK1 during standard developmental programs,
but only to induce changes in PIN polarity in response to
specific stress conditions such as wounding [14].
Calcium signaling
Calcium has been reported as modulator of auxin responses
and PAT. A rapid increase in the cytosolic calcium con-
centration was detected within minutes after auxin appli-
cation using calcium fluorescent dyes or ion-sensitive
electrodes [27–29]. It was also found that PAT is sup-
pressed by application of the calcium chelator EDTA and
restored after application of a calcium solution [30]. Inter-
estingly, the PID kinase interacts with two calcium-bind-
ing proteins, TOUCH3 (TCH3) and PINOID BINDING
PROTEIN1 (PBP1), in a calcium-dependent manner, a
finding that provided the first molecular link between
calcium signaling and PAT [31]. TCH3 is a calmodulin-
like protein with six EF-hand domains encoded by a touch-
responsive gene [32]. PBP1 is a small protein with a single
EF-hand [33]. On the basis of in vitro phosphorylation
assays, neither of the calcium binding proteins is phospho-
target of PID, and instead, TCH3 represses, whereas PBP1
enhances PID kinase activity [31]. Recent results indicate
that this calcium-dependent regulation by TCH3 and
PBP1 is conserved for all four AGC3 kinases [Robert,
Galvan-Ampudia, and Offringa, unpublished].
For speculations on the in vivo significance of calcium in
regulating the AGC3 kinases, a first hint comes from the
phototropin signaling pathway. Phototropins consist of a
C-terminal kinase domain and an N-terminal photo-
receptor domain containing two LOV domains that bind
the chromophore flavin mononucleotide [34]. In dark,
PHOT is membrane-associated with both LOV domains
repressing the activity of the kinase domain. Light stimu-
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
Current Opinion in Plant Biology 2008, 11:1–8
lation results in the release of the LOV-dependent repres-
sion on the kinase domain [34], and an early event in the
transduction cascade is PHOT dependent calcium
release from the apoplast through plasma membrane
channels [35]. Although the role of calcium in the photo-
tropic response remains to be elucidated, an interesting
possibility could be that PHOT signaling initiates relo-
calization of PINs, by modulating the activity of the
AGC3 kinases through their calcium-induced interaction
with TCH3 and PBP1 (Figure 1).
BTB proteins
Recent results suggest the involvement of Broad-Com-
plex, Tramtrack, Bric-a-Brac (BTB) scaffold proteins in
the PID signaling pathway [36�,37�]. Scaffold proteins are
characterized by the presence of multiple protein–protein
interaction domains and are important for assembling
transcription and signaling complexes. BTB domain
proteins have been first described in Drosophila melano-gaster as scaffolds in transcription complexes, are found in
many eukaryotes including mammals and plants, and are
involved in various cellular processes [38,39]. The Arabi-
dopsis genome encodes 80 BTB proteins that have been
classified in 10 subfamilies on the basis of homology and
the presence of other domains [39].
Independent mutant screens identified the BTB protein
MACCHI-BOU4/ENHANCER OF PINOID/NAKED
PINS IN YUC MUTANTS1 (MAB4/ENP/NPY1) of
the NPH3-like family as a component in the PID path-
way, either as a PID interactor, an upstream regulator of
PID transcription, a downstream phospho-target of PID,
or in a parallel signaling pathway. mab4/npy1 loss-of-
function phenotypes suggest auxin transport defects
[36�,37�], and genetic analysis of mab4 indicates that
MAB4/ENP/NPY1 is involved in organogenesis synergis-
tically with PID by controlling PIN1 localization in
embryo and inflorescence meristems [36�].
Interestingly, two other BTB domain proteins of the
NPH3-like family, NON-PHOTOTROPIC HYPOCO-
TYL3 (NPH3) and ROOT PHOTOTROPISM2
(RPT2), interact with the phototropins [40,41]. Genetic
analyses suggest that both BTB proteins are required for
the phototropic response, with nph3 resembling phot1 and
rpt2 phenocopying phot2, indicating that they are down-
stream components of the PHOT1 and the PHOT2
signaling pathways, respectively. They are both associ-
ated with the plasma membrane where they may be part
of a PHOT complex in darkness [34,40,41]. Recent data
indicate that NPH3 is phosphorylated in the dark and that
its dephosphorylation in the light is dependent on
PHOT1 [42]. The two BTB proteins are proposed to
act as modular scaffold proteins bringing together a
dynamic signaling complex comprising PHOT1, or
PHOT2, and early actors of the phototropic response
such as phosphatases and kinases [40]. The possible
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
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Kinases orienting auxin transport Robert and Offringa 5
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interaction of NPH3 with CUL3 suggests that NPH3-
dependent recruitment of proteins for degradation by the
26S proteasome may be a crucial part of the phototropic
response [42].
Auxin dependent developmental processesregulated by plant AGC kinasesFrom the previous it becomes clear that the AGC3
kinases and phototropins are key switches that through
regulation of their expression and activity integrate many
biotic and abiotic signals to orient plant development by
directing PAT. Below we will use specific developmental
processes to exemplify how we think these kinases direct
PAT dependent patterning and growth (Table 1).
Organogenesis
Auxin plays a crucial role in the positioning and emer-
gence of new organs, such as lateral roots, leaves, and
flowers. In fact, auxin maxima precede the occurrence,
and thus indicate the positions of future organ primordia,
and the auxin efflux carrier PIN1 and the auxin influx
carrier AUX1 act in concert to generate such maxima
[1,9,43]. In the shoot apex, PIN1 is active in the epider-
mis, where its frequent polarity changes coincide with
new organ initiation (Figure 2a). Most of the current
mathematical models on the patterning of aerial organs
(phyllotaxis) assume that PIN1 polarity is self-directing to
cells with the highest auxin concentration, thereby orga-
nizing auxin maxima [44]. An important role for PID in
controlling PIN1 polarity during phyllotactic patterning is
evident, but it is not easy to envisage how the simple PID
dependent basal-to-apical switch in PIN1 polarity can
regulate this complex process [13]. Moreover, in contrast
to pin1 apices, the pin-like apex of the pid mutant does
still show phyllotactic patterning when auxin-containing
lanoline paste is applied [43]. An interesting possibility is
that the other AGC3 kinases may act partially redundant
with PID in elaborating PIN1 dynamics.
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
Table 1
Comparison of the developmental signaling pathways and their influ
phototropism, and gravitropism
Organogenesis
AGC kinases PINOID [13,43] PHO
Tran
Calcium ? Incre
calci
upon
Calcium BPs TCH3?, PBP1 [unpublished] ?
BTB proteins MAB4/ENP/NPY1 [36�,37�] NPH
PIN polarity changes PIN1 [13] PIN3
Direction of auxin transport Upward toward the shoot
tip [43]
Later
of th
Position of resulting
auxin maximum
At the future primordium
[43]
In th
side
Growth response Organ initiation [43] Hypo
the li
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One of the earlier phyllotaxis models implied mechanical
buckling between organ primordia as a positioning mech-
anism [45]. In view of the current auxin-based models,
however, a prominent role for mechanical force in phyl-
lotaxis is questionable [44]. Still, organ formation has
been found to be responsive to touch [46], and we there-
fore speculate on the involvement of mechanical stress
increased intracellular calcium in regulating PAT by
modulating the activity of the AGC3 kinases through
their interaction with the calcium-binding proteins
TCH3 and PBP1 [31]. Together with the BTB domain
scaffold protein MAB4/ENP/NPY1 [36�,37�], they inte-
grate auxin- and mechanico-signaling to fine tune the
PAT driven phyllotactic program in response to external
signals.
Phototropism
Phototropism is the blue light phototropin receptor trig-
gered differential cellular expansion between the lit side
and the shaded side resulting in bending of a plant organ
toward or away from a unidirectional light stimulus
(Figures 2b and c) [40]. To corroborate Darwin’s initial
hypothesis, DR5 promoter–reporter gene fusions and
auxin measurements have been used to show that this
growth response in hypocotyls or coleoptiles of etiolated
seedlings is mediated by lateral movement of auxin from
the lit to the shaded side [47–50]. Arabidopsis pin3 loss-of-
function mutants are defective in phototropic responses
and PIN3 proteins show lateral localization in the hypo-
cotyl [47]. The current model involves phototropin-
induced changes in PIN3 localization, which induces
lateral auxin transport to the dark side of the hypocotyl.
One interesting possibility is that the PHOT1-induced
calcium peaks modulate the activity of PID or the other
AGC3 kinases via their interaction with TCH3 and PBP1.
On the contrary, the expression of WAG kinases is rapidly
downregulated by light [23], which might provide an
alternative or parallel mechanism of downregulating
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
ence on auxin distribution, and transport during organogenesis,
Phototropism Gravitropism
T1, PHOT2 [40] AGC3?
sduction: AGC3? [42]
ased cytosolic
um concentration
light stimulation [35]
Calcium transport upon
gravi-stimulus [54,55]
?
3, RPT2 [40,41] ?
[47] PIN3 [47]
al toward the shaded side
e hypocotyl [47,48]
Lateral toward the lower
side of the root tip [51,52]
e epidermis at the shaded
of the hypocotyl [47,49]
In the lateral root cap and epidermis
at the lower side of the root [51,52]
cotyl bending toward
ght source [47–49]
Root tip bending toward
the gravity vector [43,51,53]
Current Opinion in Plant Biology 2008, 11:1–8
6 Cell Signalling and Gene Regulation
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AGC3 kinase activity and makes WAG1 and WAG2
candidate mediators of the phototropic growth responses.
As described above, the BTB protein NPH3 is a crucial
component in PHOT1 signaling. NPH3 is phosphory-
lated in the dark and dephosphorylated in a blue light
PHOT1-dependent manner [40,42,50]. Again, the AGC3
kinases may play a role in keeping NPH3 in the phos-
phorylated state that interacts with PHOT1, while repres-
sion of kinase activity followed by dephosphorylation of
NPH3 may lead to its release to the cytosol, where its
interaction with CUL3 initiates changes in PAT. NPH3
may be involved in transcriptional repression of WAG1 or
WAG2, or in mediating direct turnover of PIN3 at the lit
side.
Gravitropism
Another growth response orienting plant development is
gravitropism, the differential growth induced by changes
in the gravity vector (Figure 2d). Gravity is sensed by
plastid-derived-starch-filled organelles, which are found
in specialized cells in the columella root cap and in the
stem endodermis. Gravitropic growth of the root is well
studied, and dynamic auxin movement to the lower side
of gravi-stimulated root tips was observed using auxin
responsive DR5 promoter–reporter constructs [1,47,51].
The auxin efflux carriers PIN2 and PIN3 and the auxin
influx carrier AUX1 are the main players directing PAT
during root gravitropism. The PIN3 auxin efflux trans-
porter randomly localizes at the plasma membrane in the
root columella initials but rapidly relocates to the bottom
side of the cells upon changes in the gravity vector [47],
thereby initiating lateral movement of auxin to the lower
side of the root. PIN2 driven and AUX1 dependent
basipetal PAT via lateral root cap and the epidermis cells
subsequently establishes the auxin gradient that leads to
differential elongation between the upper and the lower
sides and results in the root reorientation toward the
gravity vector [1,51,52]. The PIN2 internalization and
degradation at the apical side [12] and its auxin-enhanced
plasma membrane localization at the basal side [12,53] act
synergistically to exaggerate this response. Curiously,
although PID overexpression induced PIN apicalization
leads to agravitropic root growth (Figure 2e); no AGC
kinase, or linked calcium binding or BTB protein has yet
been shown to be involved in root gravitropism response.
Moreover, the polarity of PIN3 does not seem to be
sensitive to PID overexpression [13]. However, in view
of the role of the WAG kinases in root waving [23] and the
role of calcium in PAT-directed gravitropism [54,55], it is
likely that the AGC3 kinases and their calcium binding
proteins play a regulatory function in gravitropic root
growth.
ConclusionsThe above indicates that specific plant AGC kinases are
key switches in the modulation of PAT to orient plant
development. The interaction of AGC3 kinases with
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Current Opinion in Plant Biology 2008, 11:1–8
calcium binding proteins clarifies the observed role of
calcium signaling in the regulation of PAT. Furthermore,
BTB proteins act as scaffolds of protein complexes in the
phototropin and the PID pathways. Further research is
needed to establish the connection between environmen-
tal signals, calcium signaling, and kinase dependent PIN
polarity, and the function of BTB proteins in the AGC
kinase signaling pathways. An elegant model suggests
that the AGC3 kinases could be the plant compass that
integrates different signals to direct PAT and thereby
orients plant development [6��]. It would be of extreme
interest to validate this hypothesis and to establish if and
how phosphorylation by these kinases differentiates PIN
polar targeting. ARF-GEFs, sterols, and BTB proteins are
all potential polarity marks that could assist in sorting
cargoes to specific cycling pathways in the cell on the
basis of their phosphorylation status.
AcknowledgementsWe would like to thank Marcus Heisler and Carlos Galvan-Ampudia forproviding images for Figure 2.
References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:
� of special interest
�� of outstanding interest
1. Tanaka H, Dhonukshe P, Brewer PB, Friml J: Spatiotemporalasymmetric auxin distribution: a means to coordinate plantdevelopment. Cell Mol Life Sci 2006, 63:2738-2754.
2.�
Petrasek J, Mravec J, Bouchard R, Blakeslee J, Abas M,Seifertova D, Wisniewska J, Tadele Z, Kubes M, Covanova M et al.:PIN proteins perform a rate-limiting function in cellular auxinefflux. Science 2006, 312:914-918.
3.�
Wisniewska J, Xu J, Seifertova D, Brewer PB, Ruzicka K, Blilou I,Rouquie D, Benkova E, Scheres B, Friml J: Polar PIN localizationdirects auxin flow in plants. Science 2006, 312:883-1883.
These two papers elegantly demonstrate that PIN proteins are auxinefflux carriers and that the subcellular localization of the PIN proteinsdoes matter in order to direct proper auxin flow in plant tissues.
4. Geisler M, Blakeslee JJ, Bouchard R, Lee OR, Vincenzetti V,Bandyopadhyay A, Titapiwatanakun B, Peer WA, Bailly A,Richards EL et al.: Cellular efflux of auxin catalyzed by theArabidopsis MDR/PGP transporter AtPGP1. Plant J 2005,44:179-194.
5. Blakeslee JJ, Bandyopadhyay A, Lee OR, Mravec J,Titapiwatanakun B, Sauer M, Makam SN, Cheng Y, Bouchard R,Adamec J et al.: Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis. Plant Cell 2007,19:131-147.
6.��
Galvan-Ampudia CS, Offringa R: Plant evolution: AGC kinasestell the auxin tale. Trends Plant Sci 2007, 12:541-547.
In this opinion paper the authors redefine the classification of the plant-specific AGC kinases, put forward the hypothesis that the four AGC3kinases, including PID, WAG1, and WAG2, constitute a compass thatorients plant development by directing PIN polar targeting, and providean interesting view point on the origin of this plant compass in evolution.
7. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,Walker AR, Schulz B, Feldmann KA: Arabidopsis AUX1 gene: apermease-like regulator of root gravitropism. Science 1996,273:948-950.
8. Yang Y, Hammes UZ, Taylor CG, Schachtman DP, Nielsen E:High-affinity auxin transport by the AUX1 influx carrier protein.Curr Biol 2006, 16:1123-1127.
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
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Kinases orienting auxin transport Robert and Offringa 7
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9. Bainbridge K, Guyomarc’h S, Bayer E, Swarup R, Bennett M,Mandel T, Kuhlemeier C: Auxin influx carriers stabilizephyllotactic patterning. Genes Dev 2008, 22:810-823.
10. Geldner N, Friml J, Stierhof YD, Jurgens G, Palme K: Auxintransport inhibitors block PIN1 cycling and vesicle trafficking.Nature 2001, 413:425-428.
11.��
Kleine-Vehn J, Dhonukshe P, Sauer M, Brewer PB, Wisniewska J,Paciorek T, Benkova E, Friml J: ARF GEF-dependenttranscytosis and polar delivery of pin auxin carriers inArabidopsis. Curr Biol 2008, 18:526-531.
In this paper, the authors describe for the first time the mechanism oftranscytosis in plant cells. This mechanism of vesicle transport betweentwo sides of a cell is illustrated from important PIN polarity switchesduring plant development such as embryogenesis and lateral rootinitiation.
12. Abas L, Benjamins R, Malenica N, Paciorek T, Wisniewska J,Moulinier-Anzola JC, Sieberer T, Friml J, Luschnig C: Intracellulartrafficking and proteolysis of the Arabidopsis auxin-effluxfacilitator PIN2 are involved in root gravitropism. Nat Cell Biol2006, 8:249-256.
13. Friml J, Yang X, Michniewicz M, Weijers D, Quint A, Tietz O,Benjamins R, Ouwerkerk PB, Ljung K, Sandberg G et al.:A PINOID-dependent binary switch in apical-basal PINpolar targeting directs auxin efflux. Science 2004,306:862-865.
14. Sauer M, Balla J, Luschnig C, Wisniewska J, Reinohl V, Friml J,Benkova E: Canalization of auxin flow by Aux/IAA-ARF-dependent feedback regulation of PIN polarity. Genes Dev2006, 20:2902-2911.
15. Kleine-Vehn J, Dhonukshe P, Swarup R, Bennett M, Friml J:Subcellular trafficking of the Arabidopsis auxin influx carrierAUX1 uses a novel pathway distinct from PIN1. Plant Cell 2006,18:3171-3181.
16. Dhonukshe P, Aniento F, Hwang I, Robinson DG, Mravec J,Stierhof YD, Friml J: Clathrin-mediated constitutiveendocytosis of PIN auxin efflux carriers in Arabidopsis. CurrBiol 2007, 17:520-527.
17. Teh OK, Moore I: An ARF-GEF acting at the Golgi and inselective endocytosis in polarized plant cells. Nature 2007,448:493-496.
18. Men S, Boutte Y, Ikeda Y, Li X, Palme K, Stierhof YD,Hartmann MA, Moritz T, Grebe M: Sterol-dependentendocytosis mediates post-cytokinetic acquisition of PIN2auxin efflux carrier polarity. Nat Cell Biol 2008, 10:237-244.
19. Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K,Scheres B: Cell polarity and PIN protein positioning inArabidopsis require STEROL METHYLTRANSFERASE1function. Plant Cell 2003, 15:612-625.
20. Laxmi A, Pan J, Morsy M, Chen R: Light plays an essential role inintracellular distribution of auxin efflux carrier PIN2 inArabidopsis thaliana. PLoS ONE 2008, 3:e1510.
21. Benjamins R, Quint A, Weijers D, Hooykaas P, Offringa R: ThePINOID protein kinase regulates organ development inArabidopsis by enhancing polar auxin transport. Development2001, 128:4057-4067.
22.��
Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A,Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F et al.:Antagonistic regulation of PIN phosphorylation by PP2A andPINOID directs auxin flux. Cell 2007, 130:1044-1056.
This work unraveled the mechanism by which the PINOID kinase isapically targeting the PIN proteins and discovered the antagonisticcomponents of PINOID in this signaling pathway. Not only the pheno-types of loss- and gain-of-function mutants in PINOID and PP2A, but alsothe activity of the two enzymes are shown to be complementary.
23. Santner AA, Watson JC: The WAG1 and WAG2 protein kinasesnegatively regulate root waving in Arabidopsis. Plant J 2006,45:752-764.
24. Mora A, Komander D, van Aaltem DM, Alessi DR: PDK1, themaster regulator of AGC kinase signal transduction. Semin CellDev Biol 2004, 15:161-170.
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
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25. Bogre L, Okresz L, Henriques R, Anthony RG: Growth signallingpathways in Arabidopsis and the AGC protein kinases. TrendsPlant Sci 2003, 8:424-431.
26. Zegzouti H, Anthony RG, Jahchan N, Bogre L, Christensen SK:Phosphorylation and activation of PINOID by the phospholipidsignaling kinase 3-phosphoinositide-dependent proteinkinase 1 (PDK1) in Arabidopsis. Proc Natl Acad Sci U S A 2006,103:6404-6409.
27. Felle H: Auxin causes oscillations of cytosolic free calcium andpH in Zea mays coleoptiles. Planta 1988, V174:495-499.
28. Gehring CA, Irving HR, Parish RW: Effects of auxin and abscisicacid on cytosolic calcium and pH in plant cells. Proc Natl AcadSci U S A 1990, 87:9645-9649.
29. Shishova M, Lindberg S: Auxin induces an increase of Ca2+
concentration in the cytosol of wheat leaf protoplasts. J PlantPhysiol 2004, 161:937-945.
30. Dela Fuente RK, Leopold AC: A role for calcium in auxintransport. Plant Physiol 1973, 51:845-847.
31. Benjamins R, Galvan-Ampudia CS, Hooykaas PJ, Offringa R:PINOID-mediated signaling involves calcium-bindingproteins. Plant Physiol 2003, 132:1623-1630.
32. Sistrunk ML, Antosiewicz DM, Purugganan MM, Braam J:Arabidopsis TCH3 encodes a novel Ca2+ binding protein andshows environmentally induced and tissue-specificregulation. Plant Cell 1994, 6:1553-1565.
33. Reddy VS, Day IS, Thomas T, Reddy AS: KIC, a novel Ca2+
binding protein with one EF-hand motif, interacts with amicrotubule motor protein and regulates trichomemorphogenesis. Plant Cell 2004, 16:185-200.
34. Celaya RB, Liscum E: Phototropins and associated signaling:providing the power of movement in higher plants. PhotochemPhotobiol 2005, 81:73-80.
35. Harada A, Shimazaki K: Phototropins and blue light-dependentcalcium signaling in higher plants. Photochem Photobiol 2007,83:102-111.
36.�
Furutani M, Kajiwara T, Kato T, Treml BS, Stockum C, Torres-Ruiz RA, Tasaka M: The gene MACCHI-BOU 4/ENHANCER OFPINOID encodes an NPH3-like protein and reveals similaritiesbetween organogenesis and phototropism at the molecularlevel. Development 2007, 134:3849-3859.
These two papers describe the identification of an NPH3-like protein bymutant screens for alteration of organogenesis. NPH3 proteins are alsoinvolved in the phototropins signaling pathway, which allows to hypothe-size on an interesting parallel between the phototropism and organogen-esis signaling pathways.
37.�
Cheng Y, Qin G, Dai X, Zhao Y: NPY1, a BTB-NPH3-like protein,plays a critical role in auxin-regulated organogenesisin Arabidopsis. Proc Natl Acad Sci U S A 2007,104:18825-18829.
These two papers describe the identification of an NPH3-like protein bymutant screens for alteration of organogenesis. NPH3 proteins are alsoinvolved in thephototropinssignalingpathway,which allows tohypothesizeon an interesting parallel between the phototropism and organogenesissignaling pathways.
38. Stogios PJ, Downs GS, Jauhal JJ, Nandra SK, Prive GG:Sequence and structural analysis of BTB domain proteins.Genome Biol 2005, 6:R82.
39. Gingerich DJ, Gagne JM, Salter DW, Hellmann H, Estelle M, Ma L,Vierstra RD: Cullins 3a and 3b assemble with members of theBroad Complex/Tramtrack/Bric-a-Brac (BTB) protein familyto form essential ubiquitin-protein ligases (E3s) inArabidopsis. J Biol Chem 2005, 280:18810-18821.
40. Liscum E: Phototropim: mechanisms and outcomes. In TheArabidopsis Book. Edited by Meyerowitz EM, Somerville CR.American Society of Plant Biologists; 2002doi: 10.1199/tab.0042.
41. Inada S, Ohgishi M, Mayama T, Okada K, Sakai T: RPT2 is a signaltransducer involved in phototropic response and stomatalopening by association with phototropin 1 in Arabidopsisthaliana. Plant Cell 2004, 16:887-896.
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
Current Opinion in Plant Biology 2008, 11:1–8
8 Cell Signalling and Gene Regulation
COPLBI-562; NO OF PAGES 8
42. Pedmale UV, Liscum E: Regulation of phototropic signaling inArabidopsis via phosphorylation state changes in thephototropin 1-interacting protein NPH3. J Biol Chem 2007,282:19992-20001.
43. Reinhardt D, Pesce ER, Stieger P, Mandel T, Baltensperger K,Bennett M, Traas J, Friml J, Kuhlemeier C: Regulation ofphyllotaxis by polar auxin transport. Nature 2003, 426:255-260.
44. Heisler M, Jonsson H: Modelling meristem development inplants. Curr Opin Plant Biol 2007, 7:92-97.
45. Green PB, Steele CS, Rennich SC: Phyllotactic patterns: abiophysical mechanism for their origin. Ann Bot 1996,77:515-527.
46. Braam J: In touch: plant responses to mechanical stimuli. NewPhytol 2005, 165:373-389.
47. Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K: Lateralrelocation of auxin efflux regulator PIN3 mediates tropism inArabidopsis. Nature 2002, 415:806-809.
48. Esmon CA, Tinsley AG, Ljung K, Sandberg G, Hearne LB,Liscum E: A gradient of auxin and auxin-dependenttranscription precedes tropic growth responses. Proc NatlAcad Sci U S A 2006, 103:236-241.
49. Briggs WR: Mediation of phototropic responses of corncoleoptiles by lateral transport of auxin. Plant Physiol 1963,38:237-247.
Please cite this article in press as: Robert HS, Offringa R, Regulation of auxin transport polarit
Current Opinion in Plant Biology 2008, 11:1–8
50. Haga K, Takano M, Neumann R, Iino M: The Rice COLEOPTILEPHOTOTROPISM1 gene encoding an ortholog ofArabidopsis NPH3 is required for phototropism ofcoleoptiles and lateral translocation of auxin. Plant Cell 2005,17:103-115.
51. Ottenschlager I, Wolff P, Wolverton C, Bhalerao RP, Sandberg G,Ishikawa H, Evans M, Palme K: Gravity-regulateddifferential auxin transport from columella to lateralroot cap cells. Proc Natl Acad Sci U S A 2003,100:2987-2991.
52. Swarup R, Kramer EM, Perry P, Knox K, Leyser HM, Haseloff J,Beemster GT, Bhalerao R, Bennett MJ: Root gravitropismrequires lateral root cap and epidermal cells for transportand response to a mobile auxin signal. Nat Cell Biol 2005,7:1057-1065.
53. Paciorek T, Zazımalova E, Ruthardt N, Petrasek J, Stierhof Y,Kleine-Vehn J, Morris D, Emans N, Jurgens G, Geldner N, Friml J:Auxin inhibits endocytosis and promotes its own efflux fromcells. Nature 2005, 435:1251-1256.
54. Lee JS, Evans ML: Polar transport of auxin acrossgravistimulated roots of maize and its enhancement bycalcium. Plant Physiol 1985, 77:824-827.
55. Plieth C, Trewavas AJ: Reorientation of seedlings in the earth’sgravitational field induces cytosolic calcium transients. PlantPhysiol 2002, 129:786-796.
y by AGC kinases, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.06.004
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