1 Centre for Molecular Biology of Plants, University Tübingen, Auf der
Morgenstelle 3, 72076 Tübingen, Germany
2 Department of Plant Systems Biology, Flanders Interuniversity Institute for
Biotechnology, Gent University, Technologiepark 927, B-9052 Gent,
Belgium
3 Department of Biotechnology, Institute of General and Molecular Biology,
87-100 Toru, Poland
4 Institute for Applied Genetics and Cell Biology, University of Applied Life
Sciences and Natural Resources, Muthgasse 18, A-1190 Vienna, Austria
* Author for correspondence (e-mail: jiri.friml{at}zmbp.uni-tuebingen.de)
Accepted 4 August 2005
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SUMMARY |
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Key words: Arabidopsis, Auxin transport, PINs, Functional redundancy
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Introduction |
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Here we identify and describe synergistic interactions within the auxin transport network, which correlate with specific ectopic expression and proper polar targeting of PIN proteins in certain cells. This phenomenon involves feedback between auxin distribution and PIN gene expression as well as PIN protein stability. The identified complex regulations provide a mechanistic basis for compensatory properties of a functionally redundant auxin distribution network.
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Materials and methods |
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Growth conditions and drug treatments
Arabidopsis seedlings were grown in a 16 hours light/8 hours dark
cycle at 18-25°C on 0.5 x MS with sucrose. Short-time exogenous drug
application was performed by incubation of 4-5-day-old seedlings in liquid 0.5
x MS with or without 1% sucrose supplemented with indole-3-acetic acid
(IAA); 2,4-Dichlorophenoxyacetic acid (2,4-D); N-1-naphthylphthalamic acid
(NPA) or 1-naphthalene acetic acid (NAA) for 24 hours. Long-time treatment was
done by growing seedlings for 4 days on 0.5 x MS with 1% sucrose and
NAA. The sirtinol treatment was done by growing the seedlings for 5 days on
0.5 x MS with 1% sucrose plus 20 µmol/l Sirtinol.
Quantitative RT-PCR and Northern blot analyses
RNA was extracted using the RNeasy kit (QIAGEN) from root samples. Poly(dT)
cDNA was prepared out of 1 µg total RNA using Superscript III Reverse
Transcriptase (Invitrogen, Belgium) as recommended by Invitrogen.
Quantifications were performed on a Bio-Rad Icycler apparatus with the qPCR
Core Kit for SYBR green I (Eurogentec) upon recommendations of the
manufacturer. PCR was carried out in 96-well optical reaction plates heated
for 10 minutes to 95°C to activate hot start Taq DNA polymerase, followed
by 40 cycles of denaturation for 60 seconds at 95°C and
annealing-extension for 60 seconds at 58°C. Target quantifications were
performed with specific primer pairs designed using Beacon Designer 4.0
(Premier Biosoft International, Palo Alto, CA). Expression levels were
normalized to ACTIN2 expression levels. All RT-PCR experiments were
at least performed in triplicates and the presented values represent means.
The statistical significance was evaluated by t-test. Northern
analysis of PIN2 expression was performed with Col-O seedlings (6
DAG) grown in liquid 0.5 x MS under continuous illumination. Prior to
the experiment, seedlings were transferred into the dark for 16 hours. NAA (10
µmol/l) was added and samples were harvested at indicated time points.
Total RNA (10 µg) was loaded per lane. As a loading control, UBQ5
was used. For the quantification of PIN2::GUS activity, the GUS activity was
determined as described (Sieberer et al.,
2000). PIN2::GUS seedlings (6 DAG) were pre-adapted in
the dark for 16 hours, treated with 10 µmol/l NAA and subsequently
processed at indicated time points. Protein concentrations were normalized
with Bradford reagent (Biorad).
Expression profiling experiments
Growth conditions were as described
(Himanen et al., 2004). For
the timecourse experiments, plants were grown on 10 µmol/l NPA for 72 hours
before they were transferred to 10 µmol/l NAA containing medium. For the
RNA preparation only the differentiated segments were used. The root apical
meristems were cut off and the shoots were removed by cutting below the
root/shoot junction. RNA was isolated using RNeasy kit according to the
manufacturer's instructions. A more detailed description of the microarray,
including the data evaluation, is given elsewhere
(Vanneste et al., 2005
).
In-situ expression and localization analysis
Histochemical staining for GUS activity and immunolocalization were
performed as described (Friml et al.,
2003a). For PIN2::GUS, stainings were performed with 10-fold lower
concentration of the X-GLUC substrate. The following antibodies and dilutions
were used: anti-PIN1 (Benková et
al., 2003
) (1:500), anti-PIN2
(Paciorek et al., 2005
)
(1:400) and anti-PIN4 (Friml et al.,
2002a
) (1:200), anti-HA (mouse) (Babco, 1:1000); and FITC (1:200)
and CY3-conjugated anti-rabbit (1:500) or anti-mouse (1:500) secondary
antibodies (Dianova). For GFP visualization, samples were fixed for 1 hour
with 4% paraformaldehyde, mounted in 5% glycerol and inspected. Microscopy was
done on a Zeiss Axiophot equipped with an Axiocam HR CCD camera. For confocal
laser scanning microscopy, a Leica TCS SP2 was used. Images were processed in
Adobe Photoshop and assembled in Adobe Illustrator.
Phenotype analysis
For embryo phenotype analysis, for each condition and stage, at least 40
embryos were analysed as described (Friml
et al., 2003b). Root phenotypes were examined in 4-day-old
seedlings. Root length was measured from hypocotyl junction to root apex, and
root meristem size from the position in which epidermis cells rapidly elongate
to quiescent centre as described (Blilou et
al., 2005
). Microscopy inspection of roots and embryos was done on
a Zeiss Axiophot equipped with an Axiocam HR CCD camera using differential
interference contrast optics.
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Results |
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PIN functional redundancy in root development involves cross-regulation of PIN gene expression
In the root meristem, five PIN genes are known to be expressed
(Fig. 2A). The PIN1
expression pattern is somewhat variable, but under our experimental conditions
PIN1 could be found predominantly at the basal (lower) side of stele and
endodermis cells with occasional weak expression in the quiescent centre and
up to the four youngest epidermis and cortex daughter cells
(Friml et al., 2002a)
(Fig. 2D). PIN2 is
expressed in a non-overlapping pattern in the lateral root cap and older
epidermis and cortex cells with apical (upper) polarity in the epidermis and
predominantly basal polarity in the cortex
(Müller et al., 1998
,
Friml et al., 2003a
)
(Fig. 2G). PIN4 is
expressed in the central root meristem with a polar subcellular localization
pointing predominantly towards the columella initials
(Friml et al., 2002a
)
(Fig. 2J). By contrast, PIN3
(Friml et al., 2002b
) and PIN7
(Blilou et al., 2005
) are
localized in largely overlapping patterns in columella and stele of the
elongation zone. However, with the exception of PIN2, which when mutated
causes agravitropic root growth, removal of any of the other PINs causes no,
or rather subtle, root phenotypes
(Sabatini et al., 1999
;
Friml et al., 2002a
;
Friml et al., 2003b
). By
contrast, pin1,2 double mutants displayed strong root growth defects
reflected in significantly shorter roots and the formation of a smaller root
meristem, when compared with either single mutant
(Fig. 2B,C) or any other double
mutant combination (Blilou et al.,
2005
). These strong synergistic interactions between PIN1 and PIN2
may indicate a functional cross-regulation similar to that observed with PIN4
and PIN7 in the embryos. Indeed, the analysis of expression and abundance of
PIN1 and PIN2 in the respective mutants reveals that PIN1 became
ectopically induced in the PIN2 expression domain in cortex and
epidermis cells of pin2 (Fig.
2E). Reciprocally, in pin1 mutants, PIN2 was
ectopically expressed in the endodermis and weakly in the stele
(Fig. 2H) along with ectopic
upregulation of the PIN4 expression in the stele
(Fig. 2K). Remarkably,
ectopically expressed PIN proteins exhibited the polar localization of the PIN
protein that had been replaced. PIN2 and PIN4 were basally localized, when
upregulated in endodermis or stele of pin1
(Fig. 2I,L), whereas PIN1
showed apical localization in epidermis and basal localization in cortex cells
when upregulated in roots of the pin2 mutant
(Fig. 2F). These findings
demonstrate that the functional redundancy of PIN proteins involves
cross-regulation of PIN gene expression and polar targeting in a cell-specific
manner, which potentially explains the observed synergistic interactions.
Accordingly, ectopic upregulation of PINs, as observed in pin
mutants, could be sufficient to compensate for the function of missing PIN
genes.
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Auxin-dependent signalling controls PIN gene expression in a tissue-specific manner
Next we addressed potential mechanisms underlying the observed
cross-regulation of PIN gene expression. As NPA treatment and various
pin mutants change the pattern of auxin distribution in roots and
embryos (Luschnig et al.,
1998; Sabatini et al.,
1999
; Friml et al.,
2002a
; Friml et al.,
2003b
), we tested whether auxin itself can directly influence PIN
gene expression. Treatments with different biologically active auxins such as
IAA, NAA and 2,4-D led to an increase in GUS activity in PIN4::GUS
(Fig. 3I-K) and
PIN2::GUS (Fig. 3M,N and not shown) roots. Importantly, both NAA and 2,4-D, which differ in their
transport properties (Delbarre et al.,
1996
), induced PIN gene expression in a similar way, indicating
that auxin influences PIN gene expression without the need of the active auxin
transport. This was further confirmed by analysis of the effects of sirtinol
a compound that is not a substrate of the auxin transport system but
is converted to a substance with auxin effects
(Zhao et al., 2003
;
Dai et al., 2005
). The effect
of sirtinol seemed to be somewhat delayed when compared to auxin effects, but
prolonged treatments had the same impact on the induction of PIN gene
expression as auxins, as shown, for example, by the upregulation of
PIN4::GUS and PIN1:GFP (Fig.
3L,P).
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In summary, these results show that the expression of PINs is directly or indirectly controlled by auxin in a tissue-specific manner, which provides a plausible mechanism for the observed cross-regulations in PIN functional redundancy.
The auxin effect on PIN gene expression is time- and concentration-dependent
Analyses of GUS activity in PIN1::GUS, PIN3::GUS, PIN4::GUS and
PIN7::GUS roots revealed that the auxin effect on the activity of PIN
promoters is time- and concentration-dependent
(Fig. 5A-D). Staining
conditions here were chosen to maximize the dynamic range of staining
intensities in order to better resolve the differences in GUS expression
levels after auxin treatments rather than to obtain optimal overall staining
patterns. Thus, for example, untreated PIN7::GUS seedlings, when
optimally stained, also showed GUS signal in the stele
(Fig. 5D inset), which is in
accordance with earlier observations of PIN7 expression
(Blilou et al., 2005).
Interestingly, independently of the time and concentration of the auxin
treatment, the upregulation remained largely confined to the same tissues,
further confirming the cell-type-specific effect on PIN gene expression.
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Auxin regulates PIN gene expression through an Aux/IAA-dependent pathway
We then addressed the molecular mechanism by which auxin regulates PIN gene
expression. Even when the protein synthesis was inhibited by cycloheximide,
auxin induced the expression of PIN proteins (not shown), demonstrating that
the auxin-dependent PIN upregulation does not require de novo synthesis of any
factors and thus PINs are primary response genes. Significantly, a
treatment with cycloheximide alone was sufficient to induce expression of
PIN1, PIN2, PIN3 and PIN4 to roughly maximum levels
(Fig. 6C), implying that PIN
gene expression is controlled by an unstable transcriptional repressor. The
auxin effect on gene expression is known to involve a rapid, auxin-dependent
degradation of the Aux/IAA transcriptional repressors
(Gray et al., 2001). Indeed,
in the solitary-root-1 (slr-1) mutant, which carries the
stabilized version of the IAA14 repressor
(Fukaki et al., 2002
),
auxin-dependent upregulation of PIN gene expression is severely compromised
(Fig. 6C), suggesting that
auxin utilizes Aux/IAA-dependent signalling to regulate PIN gene expression.
In addition, we used transgenic plants harbouring a stabilized version of
IAA17 (AXR3) under the control of a heatshock promoter (HS::axr3-1)
(Knox et al., 2003
). The
induction of axr3 expression by 2 hours of 37°C treatment concomitantly
abolished the auxin-dependent upregulation of PIN1, PIN2, PIN3, PIN4
and PIN7 expression (Fig.
6D), directly linking the regulation of PIN gene expression to the
Aux/IAA signal transduction pathway. These conclusions gained additional
support from global expression analysis following auxin-dependent induction of
lateral root formation (Vanneste et al.,
2005
). Microarray-based analysis was performed at different time
points after auxin application to seedlings that were grown under inhibited
auxin transport (by NPA). Only the differentiated part of the primary root was
analysed to minimize the influence of different tissue- and
developmental-stage-specific factors
(Vanneste et al., 2005
). Under
these conditions, the expression of PIN1, PIN3 and PIN7 was
rapidly and strongly induced by auxin, along with a number of well-known
primary auxin response genes as well as PINOID and related genes
(Fig. 6G). Expression of other
PIN genes was also analysed (e.g. PIN genes were also on the ATH1 Affymetrix
chip) but were not induced in this experiment (data not shown). Importantly,
the observed auxin-dependent induction of PIN gene expression was completely
abolished when the expression profiling experiment was performed in the
slr-1 mutant (Fig.
6G). In summary, these experiments demonstrate that
tissue-specific PIN gene expression is regulated by auxin through
AUX/IAA-dependent signalling.
Auxin-dependent post-transcriptional downregulation of PIN proteins
Our results suggest that auxin is able to modulate PIN levels by regulating
PIN gene expression in a highly specific way. Additional levels of regulations
might occur due to effects on PIN protein stability, as at least PIN2
degradation was shown to be regulated by auxin levels
(Sieberer et al., 2000). To
address the post-transcriptional effects of auxin on the abundance of PIN
proteins, we utilized GUS and GFP translation fusions with PIN1, PIN2, PIN4
and PIN7. Comparisons of the auxin effects on PIN7::GUS and
PIN7::PIN7:GUS transgenic plants clearly showed a time- and
concentration-dependent transcriptional upregulation of PIN7 promoter
activity (Fig. 5D), but a
downregulation of PIN7:GUS levels (Fig.
5E). Similarly, PIN7:GFP (Fig.
7A) and PIN2:GFP (Fig.
7B) abundance decreased at higher auxin concentrations (higher
than 100 nmol/l 2,4-D). However, at lower concentrations, the PIN2 and PIN7
protein amount increased (best at 10 nmol/l), suggesting that both the
transcriptional and the post-transcriptional auxin effects on PIN expression
overlap. In support of this notion, the transcriptional upregulation of
PIN2 expression in stele, which occurs in PIN2::GUS
seedlings following auxin treatment (Fig.
3N), cannot be observed in PIN2::PIN2:GFP seedlings
(Fig. 7B). In
PIN1::PIN1:GFP roots, the optimal 2,4-D concentration for the
PIN1:GFP upregulation in epidermal cells was 100 nmol/l. At higher
concentrations, the PIN1:GFP level decreased also in its stele expression
domain (Fig. 7C), albeit to a
lesser extent than in the case of PIN2 and PIN7 reporter proteins. However,
there was no visible decrease in the PIN4:GFP amount following auxin treatment
(not shown). These results show that higher auxin concentrations, besides
modulating PIN gene expression, post-transcriptionally downregulate the
abundance of specific PIN proteins. This provides an additional level of
regulation for modulating of different PIN protein amounts in different
cells.
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Discussion |
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Model for feedback regulations and compensatory properties in auxin distribution
Plant development is characterized by its flexibility and adaptability,
which allow the optimal adjustment of plant shape according to the
environment. The auxin distribution network is supposed to enable the
integration of multiple environmental and developmental signals to allow the
flexible changes in auxin accumulation patterns that underlie the adaptive
nature of plant development. The regulation of PIN polar targeting,
degradation and differential regulation of expression are potential upstream
control points for mediating the dynamic auxin gradients. For example, the
PIN3 polarity can be rapidly modulated by environmental signals such as
gravity, which through asymmetric auxin distribution ultimately leads to
gravitropic bending (Friml et al.,
2002a). Also, developmental signals can mediate dynamic changes in
PIN polarity and thus mediate apical-basal axis specification in embryos
(Friml et al., 2003b
), trigger
specific patterns of organ positioning
(Reinhardt et al., 2003
) or
perpetuate organ primordium development
(Benková et al.,
2003
).
Under conditions of an ever-changing environment and constant stimulation,
a dynamic system such as the PIN-dependent auxin transport network requires a
mechanism(s), which would at some point stabilize and perpetuate its
readjustments. For this purpose, biological systems typically accommodate
feedback regulatory loops. Long-standing physiological models, such as the
canalization hypothesis, proposed that auxin itself can modulate its own
transport and its polarity (Sachs,
1988) and thus mediate regenerative properties of plant
development, especially the de novo formation of vascular strands
(Sachs, 2000
;
Berleth and Sachs, 2001
). The
canalization hypothesis assumes, besides positive feedback on transport
activity, a directional polarization of auxin flow relative to the position of
the auxin source. Our data show that auxin itself, together with
cell-type-specific factors, can positively control PIN transcription, which
involves the activity of Aux/IAA transcriptional repressors. The complementary
evidence for the influence of auxin distribution on PIN gene expression came
from the expression profiling experiments in poplar
(Schrader et al., 2003
) and
from analysis of flavonoid mutants, where both the auxin transport and the
distribution of PIN proteins are affected
(Peer et al., 2004
). However,
the effect of auxin on PIN polarity or on polarity of auxin flow has not been
demonstrated so far. Our data show that ectopically expressed PIN proteins in
various pin mutants always adopt the correct polar localization,
suggesting a tight cell-type-based control, apparently requiring direct or
indirect regulation by auxin. Such a functional link is also provided by the
recent analysis of regulators of PIN polarity, such as the Ser/Thr protein
kinases of the PINOID type (Friml et al.,
2004
). It has been reported previously that PINOID is a
primary auxin response gene (Benjamins et
al., 2001
). Also our expression profiling data show that
PINOID and homologous genes are upregulated along with the PIN genes
in the same tissues. It is thus conceivable that auxin mediates changes of
cellular PIN polarity via control of PINOID expression. In such a
scenario, both cellular PIN levels and PIN localization can be influenced by
auxin itself. Such feedback regulations may contribute to the compensatory
properties of the auxin distribution network. In the simplest model, the
defect in auxin flow caused, for example, by a mutation in a specific PIN
protein, would lead to auxin accumulation within affected cells. This in turn
would lead to the upregulation of expression and polar retargeting of other
PIN family member(s), which, in this manner, could functionally compensate.
This unique, so far undescribed, type of regulatory redundancy explains
observed genetic redundancy and provides a possible mechanism for the
stabilization of changes in auxin distribution. The fine interplay between the
modulating external signals and the stabilizing internal feedback in the
PIN-based auxin transport network might thus contribute to both the flexible
and robust nature of plant development.
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ACKNOWLEDGMENTS |
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