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Intracellular trafficking and proteolysis of the
Arabidopsis auxin-efflux facilitator PIN2 are
involved in root gravitropism
Lindy Abas1,5, René Benjamins1,5, Nenad Malenica1,5, Tomasz Paciorek2, Justyna Wiřniewska2,3,
Jeanette C. Moulinier–Anzola1, Tobias Sieberer1, Jiří Friml2,4 and Christian Luschnig1,6
Root gravitropism describes the orientation of root growth along the gravity vector and is mediated by differential cell elongation
in the root meristem. This response requires the coordinated, asymmetric distribution of the phytohormone auxin within the root
meristem, and depends on the concerted activities of PIN proteins and AUX1 — members of the auxin transport pathway. Here,
we show that intracellular trafficking and proteasome activity combine to control PIN2 degradation during root gravitropism.
Following gravi-stimulation, proteasome-dependent variations in PIN2 localization and degradation at the upper and lower
sides of the root result in asymmetric distribution of PIN2. Ubiquitination of PIN2 occurs in a proteasome-dependent manner,
indicating that the proteasome is involved in the control of PIN2 turnover. Stabilization of PIN2 affects its abundance and
distribution, and leads to defects in auxin distribution and gravitropic responses. We describe the effects of auxin on PIN2
localization and protein levels, indicating that redistribution of auxin during the gravitropic response may be involved in the
regulation of PIN2 protein.
Numerous plant-growth responses have been linked to the polarized
transport of auxin throughout the entire plant body1–3. Arabidopsis PIN
and AUX1 proteins are candidates that may facilitate this polarized auxin
transport4–7. In gravity-responding roots, PIN2 (also known as EIR1 and
AGR) seems to function in the distribution of auxin as pin2 mutants fail to
establish a lateral auxin gradient after gravi-stimulation and exhibit pronounced deficiencies in root gravitropism4–6,8. The expression and subcellular localization of PIN2 are consistent with a function in polarized auxin
transport, that is, the localization of PIN2 at the apical (upper) side of lateral
root cap and epidermis cells seems to direct the auxin flow from the root
tip into the elongation zone. Following gravi-stimulation, more auxin is
directed into the elongation zone at the lower side of the root when compared with the upper side. The resultant auxin gradient causes inhibition of
growth on the lower side of the root and thus, the root bends5,6,8,9.
Recently some of the molecular mechanisms that control subcellular
PIN localization and expression have been revealed. Inhibition of the
intracellular cycling of PINs by brefeldin A treatment (BFA, an inhibitor
of vesicular budding) was found to interfere with the subcellular localization of several PINs and correlated with deficiencies in gravitropic
root bending10. Ectopic expression of the PINOID serine–threonine
kinase causes similar effects that have been linked to deficiencies in the
establishment of auxin gradients11. Proteasome activity and auxin-mediated control of endocytosis also affect PIN abundance and intracellular
distribution12,13 — highlighting the importance of post-transcriptional
mechanisms in the control of PIN proteins.
To investigate the post-transcriptional mechanisms involved in the gravitropic responses of roots, reporter constructs that express functional PIN2
from its endogenous as well as heterologous promoters were generated.
We found that gravi-stimulation resulted in asymmetric PIN2 distribution,
with more protein degraded at the upper side of the gravi-stimulated root.
This occurred regardless of whether PIN2 was expressed under the control
of its endogenous promoter or a heterologous promoter, indicating that
the asymmetric distribution of PIN2 may be caused by post-transcriptional mechanisms. Moreover, we found that the intracellular localization,
abundance and ubiquitination of PIN2 were perturbed by inhibitors of
the proteasome or of intracellular cycling. Analysis of a mutant pin2 allele
showed that PIN2 localization and degradation were perturbed in this
mutant and this correlated with deficiencies in gravitropic root growth.
Further experiments performed with exogenously added auxin indicated
that auxin itself could act as a modulator of PIN2 abundance. We propose
that intracellular trafficking, and the proteasome, regulate steady-state
levels of PIN2 and thereby auxin distribution in gravi-stimulated roots.
1
Institute for Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences – BOKU, Muthgasse 18, A-1190 Wien, Austria. 2Center
for Molecular Biology of Plants, University Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany. 3Department of Biotechnology, Institute of General
and Molecular Biology, 87–100 Torun, Poland. 4Masaryk University, Department of Functional Genomics and Proteomics, Laboratory of Molecular Plant Physiology,
Kamenice 5, CZ-625 00, Brno, Czech Republic. 5These authors contributed equally to this work.
6
Correspondence should be addressed to C.L. (e-mail: [email protected])
Received 7 November 2005; accepted 17 January 2006; published online 19 February 2006; DOI: 10.1038/ncb1369
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a
c
b
d
transverse side of epidermal and lateral root-cap cells, and to the lower
sides of cortical cells (Fig. 1b, e). Similarly, in all eir1–4 35S:PIN2–GFP
lines analysed, and in DEX-inducible eir1–4 TA:PIN2–GFP lines, PIN2–
GFP was found to be restricted to the upper transverse cell walls of epidermal and lateral root-cap cells, with only weak signals in other parts of
the root meristem (Fig. 1c, d, f). Similar results were obtained with eir1–4
35S:PIN2 plants (Fig. 1a). In addition, prominent PIN2:GFP signals were
found in the columella cells of eir1–4 35S:PIN2–GFP and of eir1–4 TA:
PIN2–GFP plants, where they are not found in wild-type plants (Fig. 1c,
f ). However, the apparent restoration of eir1–4 root gravitropism by 35S:
PIN2–GFP expression indicates that this ectopic PIN2 accumulation did
not interfere with root bending, and further confirms the importance of
lateral root-cap and epidermis cells for root gravitropism15.
To investigate auxin distribution within the roots of gravi-stimulated seedlings, we used the auxin reporter construct DR5rev–GFP (the
expression of which correlates with endogenous auxin distribution)16,17.
eir1–4 35S:PIN2 showed an asymmetric DR5rev–GFP signal in the roots
of gravi-stimulated seedlings (with a strong GFP signal at the lower
side of the gravi-stimulated root tip), indicating auxin accumulation
in these cells. (see Supplementary Information, Fig. S1e). This pattern resembled that observed in gravi-stimulated wild-type roots,
whereas this pattern was not detected in gravi-stimulated eir1–4 (see
Supplementary Information, Fig. S1e).
Irc
e
e
f
Figure 1 Analysis of PIN2 expression and subcellular localization in primary
root meristems. (a) Immunostaining of eir1–4 35S:PIN2 (green) revealed
a polar signal in lateral root cap and epidermis cells (arrows) No additional
prominent signal was detectable. (b) PIN2 showed polar localization in the
lateral root-cap, epidermis and cortex cells of wild-type roots. (c) PIN2–GFP
in DEX-treated eir1–4 TA:PIN2–GFP roots. A prominent signal was detected
in columella, lateral root-cap and epidermis cells. (d) Epidermis (e) and
lateral root-cap (lrc) cells of DEX-treated eir1–4 TA:PIN2–GFP roots. There
was a polar localization of the GFP signal in these cells. (e, f) Comparison of
eir1–4 PIN2:PIN2–GFP (e) and eir1–4 35S:PIN2–GFP roots (f). Expression
of eir1–4 35S:PIN2–GFP was pronounced in lateral root-cap and epidermis
cells, but, in contrast with the wild-type protein, also accumulated in
columella root-cap cells. PIN2 signals are indicated in green. Propidium
iodide staining of the cell walls is indicated in red. Scale bars represent
20 µm in a–c, 10 µm in d and 50 µm in e and f.
RESULTS
PIN2 expressed from heterologous promoters rescues pin2 and
shows a subcellular localization similar to endogenous PIN2
Previous work has shown that expression of PIN2 by the heterologous,
constitutive CaMV 35S-promoter is sufficient to rescue the agravitropism of eir1-1 (a pin2 allele)12. Similar results were obtained with eir1–4
TA:PIN2 plants that express the PIN2 cDNA under control of a dexamethasone (DEX)-inducible promoter (TA)14, confirming that PIN2 is
capable of rescuing the root gravitropism of pin2 mutants regardless of
the promoter (see Supplementary Information, Fig. S1a–c). However,
the gravitropic response is slower than that of wild-type plants (see
Supplementary Information, Fig. S1d).
As PIN2 overexpression constructs restore gravitropism in pin2
mutants, PIN2 localization in the transgenic lines should be similar to
that of the wild-type protein. In wild-type plants and in plants expressing the PIN2:PIN2–GFP construct , PIN2 was restricted to the upper
250
PIN2 protein turnover is affected by the proteasome and by
endosomal cycling
As proteasome activity has previously been associated with the control of PIN2 protein turnover12, the impact of proteasome inhibitors on
PIN2 was assessed. Treatment of wild-type or eir1–4 35S:PIN2 seedlings
with the proteasome inhibitors MG132 and clasto-lactacystin-β-lactone
resulted in increased PIN2 levels as seen in western blots (Fig. 2b). This
demonstrated that both endogenous and overexpressed PIN2 are stabilized when the proteasome is inhibited. No changes in PIN2 transcript
levels were detected when eir1–4 35S:PIN2 seedlings were treated with
MG132, but in wild-type seedlings there was a decrease in endogenous
PIN2 transcript levels — indicating that the stabilizing effect of proteasome inhibitors on PIN2 steady-state protein levels is mediated by a
post-transcriptional mechanism (Fig. 2b).
To further address the involvement of proteasome activity in the control of PIN2, we tested for PIN2 ubiquitination. Ubiquitination is characteristic of proteins that are recognized by the proteasome18. Proteasome
activity has also been implicated in the targeting of ubiquitinated membrane proteins to the lysosome or vacuole19. A high-molecular weight
signal was recognized by both anti-ubiquitin and anti-PIN2 in PIN2-specific immunoprecipitates, indicating that a fraction of PIN2 is covalently
modified by ubiquitin (Fig. 2c). Moreover, proteasome inhibition caused
a pronounced increase in the amounts of ubiquitinated PIN2 (Fig. 2c).
Degradation of eukaryotic membrane proteins seems to be regulated
through controlling their intracellular transport and sorting them to a
proteolytic compartment20. In this context, it is significant that the control
of PIN protein function has been associated with its constitutive cycling
between the plasma membrane and endosomes, as demonstrated by treatment with BFA10,21,22. Whether or not PIN2 cycling is related to its turnover
was tested by analysing the effect of BFA on eir1–4 35S:PIN2 seedlings. BFA
treatment caused a dramatic increase in PIN2 protein levels while PIN2
transcript levels remained unaffected (Fig. 2d). To exclude the possibility
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DMSO MG132
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36 UBQ5
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eir1−4
35S : PIN2
8
DMSO BFA
PIN2
PIN2
UBQ5
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Coomassie
1
2
3
4
5
Figure 2 Control of proteasome activity and vesicular transport affects the
steady-state levels of PIN2. (a) Total membrane protein fractions of eir1–4
(lane 1) and eir1–4 35S:PIN2 (lane 2) were probed with an affinity-purified
antibody for PIN2. A specific signal with an Mr(K) of 60–70 (arrowhead)
was detected in eir1–4 35S:PIN2. A non-specific band (asterisk) was seen
in some blots. (b) PIN2 protein levels increased when eir1–4 35S:PIN2
seedlings were treated with 50 µM MG132 or 5 µM of clasto-lactacystin-βlactone (LAC) for 150 min. A similar increase in PIN2 levels was observed
in wild-type (Col–O) seedlings treated with MG132 for 150 min. Controls
were treated with DMSO. No signal was detected in eir1–4 extracts. Lower
panels indicate Coomassie-staining of the protein samples. No differences
in 35S:PIN2 transcript levels (left) were detected after MG132-treatment
for 150 min, whereas the transcript levels of wild-type PIN2 even decreased
upon proteasome inhibition (UBQ5 was used as a loading control). The
PIN2-specific signal in RNA isolated from 35S:PIN2 seedlings was
markedly more intense than the signal found in wild-type preparations.
(c) Immunoprecipitation was performed using an affinity-purified antibody
for PIN2 and blots were probed with ubiquitin or PIN2 affinity–purified
antibodies. The high-molecular-weight ubiquitin-specific signal increased
in MG132-treated eir1–4 35S:PIN2 membrane fractions compared with
DMSO-treated controls. Lower panel: the same blot was probed for PIN2
and showed an increase in both PIN2 levels (arrowhead) and in the high
molecular weight PIN2-specific fraction when plants were treated with
MG132. DMSO or MG132-treated eir1–4 membrane fractions were used
as controls. No protein extract was added to the sample in the third lane.
(d) Time-course of eir1–4 35S:PIN2 seedlings treated with 10 µM BFA.
PIN2 levels increased over 8 h, whereas no increase was observed when
seedlings were treated with DMSO. Eir1–4 treated with BFA were used as a
control. Coomassie-staining of the protein samples is indicated in the lower
panel. No differences in PIN2 transcript levels were detected in eir1–4
35S-PIN2 seedlings after 8 h BFA treatment (right panel; UBQ5 was used
as loading control).
that BFA inhibited the proteasome (as this would then affect PIN2 levels)
it was confirmed that there was no general increase in the total amount of
ubiquitinated proteins after treatment with BFA (data not shown).
It was found that both BFA and MG132 interfered with the asymmetric distribution of PIN2 in the upper and lower side of gravi-stimulated
eir1–4 35S:PIN2–GFP roots, and that they caused an increase in PIN2:
GFP signals (Fig. 3g–i). These findings agree with the increases in PIN2
protein seen by western-blot analysis (Fig. 2b) and correlate with the
inhibition of root gravitropism seen in eir1–4 35S:PIN2 roots treated
with BFA or MG132 (see Supplementary Information, Fig. S2). Thus,
eir1–4 35S:PIN2 mediates normal gravitropism only under conditions
where vesicular transport or proteasome activity are not inhibited and
the establishment of a PIN2 gradient is possible.
Cell-specific proteolytic turnover of PIN2 responds to gravistimulation
To investigate the role of the proteolytic turnover of PIN2 in root
gravitropism, the expression and distribution of PIN2 were examined
after gravi-stimulation. Whether or not these processes were affected
by treatment with MG132 or BFA was also analysed. In gravi-stimulated wild-type roots, asymmetric PIN2 distribution can be detected
by comparing the upper and the lower side of the root. Within 1–2 h of
gravi-stimulation, PIN2-specific signals were weaker at the upper than
the lower side of vertically positioned roots. This difference was most
pronounced in epidermal cells (Fig. 3a, b). After 6 h of gravi-stimulation, this asymmetric PIN2 distribution was no longer visible (Fig. 3c).
The distribution of PIN2:GFP in gravi-stimulated eir1–4 35S:PIN2–GFP
roots showed a similar pattern, but was delayed; the most pronounced
asymmetry was seen after 2–4 h of gravi-stimulation (Fig. 3d–f). Thus,
both endogenous and overexpressed PIN2 resulted in asymmetric levels
of PIN2 in the upper versus the lower side of the root tip.
Intracellular relocation of PIN2 in gravi-stimulated roots
depends on proteasome activity
As it is assumed that PINs function in a specific subcellular location, the localization of PIN2 was analysed using a functional
haemagglutinin(HA)-tagged PIN2 expressed under control of the PIN2
promoter in eir1-1 seedlings after gravi-stimulation. Intracellular accumulation of HA-specific signals was observed in the upper side of gravistimulated roots, together with a gradual disappearance of the signal in
elongating epidermis cells (Fig. 4a, b). In contrast, PIN2:HA-specific signals in the lower side of gravi-stimulated roots remained restricted to the
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a
d
g
b
e
h
c
f
i
Figure 3 Gravi-stimulation mediates the establishment of a PIN2 gradient
that is sensitive to inhibition of vesicular transport and depends on
proteasome activity. (a–c) PIN2 immunolocalization in wild-type seedlings
gravi-stimulated for 0 (a), 90 (b) and 360 (c) min. After 90 min, PIN2
levels in the upper side of the root were less pronounced than those in the
lower side (arrowheads). After 360 min no pronounced differences in the
abundance of PIN2 signals were detectable. (d–f) eir1–4 35S:PIN2–GFP
roots gravi-stimulated for 0 (d), 240 (e) and 360 (f) min. After 240 min
of gravi-stimulation, PIN2:GFP signals were pronounced only in the
lower side of the root (arrowheads). The delayed kinetics (compared with
those in a–c), correspond to those shown in Supplementary Information,
Fig. S1d. (g–i) eir1–4 35S:PIN2–GFP roots gravi-stimulated for 240 min
were pretreated with DMSO (g), 20 µM BFA (h) and 50 µM MG132 (i).
Drug treatments resulted in increased PIN2:GFP signals, specifically in
the outermost cell files, and interfered with the formation of a PIN2:GFP
gradient. Scale bars represent 50 µm.
proximity of the cell wall (Fig. 4a). In marked contrast, no intracellular
relocation of PIN2:HA was observed in gravi-stimulated roots that were
pre-treated with MG132 (Fig. 4c, d), indicating a proteasomal involvement in the intracellular distribution of PIN2 after gravi-stimulation.
the apparent accumulation of auxin in the elongation zone of wav6–52
seedlings indicates that PIN2wav6–52 still mediates auxin transport into
these cells. Stabilization and mis-localization of PIN2 thus results in a
striking alteration in auxin distribution.
Stabilised PIN2 is defective in root gravitropism
Our data indicate that interference with PIN2 turnover and intracellular
trafficking blocks gravitropic root bending. This is further supported
by the analysis of wav6–52, a severe pin2 allele with a glycine-to-glutamate substitution at position 541 of the predicted PIN2 protein that
fails to respond to gravity (agravitropic)23. Analysis of the expression of
the PIN2wav6–52 protein in western blots revealed a protein of a comparable size to that of wild type PIN2 (Fig. 5a). However, the amount of
PIN2wav6–52 protein was higher than wild-type PIN2, although wav6–52
transcript levels were similar to wild type (Fig. 5a). Thus, the point
mutation in PIN2wav6–52 seems to stabilize the protein. In situ analysis
showed that PIN2wav6–52 still localizes to the plasma membrane but also
accumulates in intracellular vesicular structures (Fig. 5b, c). Noticeably,
gravi-stimulation of wav6–52 roots did not cause significant changes in
the overall abundance and subcellular distribution of PIN2wav6–52. Instead,
the mutant protein was detected at similar intensities in the upper and
lower sides of the root (Fig. 5d, e). Testing of the expression pattern of the
auxin-responsive reporter DR5rev–GFP in wav6–52 revealed a signal in
columella and in lateral root-cap cells that extended into the root elongation zone, whereas DR5rev–GFP expression in wild-type seedlings is only
abundant in columella root-cap cells (Fig. 5f, h). DR5rev–GFP expression
in wav6–52 contrasted with that seen in the eir1–4 knock-out allele,
where a strong DR5rev–GFP signal was visible in root columella and in
lateral root-cap cells proximal to the root tip (Fig. 5f, g). Thus, although
the absence of PIN2 seems to cause auxin retention in the root cap7,15,
Auxin affects PIN2 protein levels
The auxin gradient that is established during gravi-stimulation may
have implications for the stability and localization of PIN2 itself12,13.
Previous work has shown that extended treatment with auxin reduced
PIN2 reporter protein abundance, indicating that auxin may promote the
degradation of PIN212. Short-term auxin treatment has also been shown
to result in the retention of PIN2 at the plasma membrane13. However,
PIN2 protein levels were not quantified. Quantitative analysis of PIN2
in the membrane fraction of auxin-treated eir1–4 35S:PIN2 seedlings
revealed a decrease in PIN2 protein levels after treatment with 10 µM
naphthylene-1-acetic acid (NAA) although transcript levels were unaffected (Fig. 6a). Similarly, lower amounts of PIN2 were observed after
auxin treatment of wild-type seedlings, when compared with untreated
controls (Fig. 6b). Depending on the growth conditions, timing of the
auxin-mediated decrease in PIN2 steady-state levels varied, indicating
that additional parameters impinge on the turnover of PIN2 (L. A. & C. L.,
unpublished observations). To investigate the role of auxin in the control
of PIN2 distribution in gravi-stimulated roots, PIN2:HA localization was
analysed in eir1−1 PIN2:PIN2:HA roots treated with the auxin-analogue
2,4-dichlorophenoxy acetic acid (2,4-D). After 90 min of gravi-stimulation, the control roots internalized PIN2:HA in the upper side of the
root, whereas in the presence of 2,4-d, PIN2:HA signals were restricted
to the proximity of the cell walls (Fig. 6c, e). After 3 h of 2,4-d treatment,
however, PIN2:HA gave a diffuse signal in the upper and the lower
side of the root (Fig. 6d, f). Thus, whereas short-term auxin treatment
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a
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c
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Figure 4 Intracellular distribution of PIN2:HA after gravi-stimulation
in eir1−1 PIN2:PIN2:HA. (a, b) Gravi-stimulation for 150 min caused
intracellular accumulation of HA-specific signals (b, arrowhead) in the
upper side of the root. This response correlated with the disappearance
of the plasma membrane-localized signal in epidermis cells (b, arrow). In
contrast, the signal remained at the plasma membrane in the lower side of
the root (a). (c, d) Gravi-stimulation of seedlings that were pre-treated with
50 µM MG132 for 150 min restricted the HA-specific signal to the plasma
membrane in the upper and lower side of the root. Scale bars represent
50 µm in a and c and 10 µm in b and d.
interferes with the accumulation of intracellular PIN2:HA in the upper
side of gravi-stimulated roots, extended auxin incubation delocalizes
PIN2 distribution, which may lead to degradation of the protein.
DISCUSSION
Here, we show that the subcellular localization and abundance of PIN2
are crucial for its role in mediating the gravitropic response, and that
PIN2 is regulated by a mechanism that involves proteasome activity
during PIN2-mediated growth responses. We demonstrate that a PIN
protein can be ubiquitinated and present quantitative analysis indicating
that interference with the control of PIN2 levels affects its function.
PIN proteins cycle continuously between the plasma membrane and
endosomal compartments. This process has been implicated in the
control of the subcellular localization of PIN proteins, which in turn
determines the direction of auxin transport21. Our findings — demonstrating that inhibition of vesicle transport results in accumulation
of PIN2 — indicate that intracellular cycling of PIN2 also determines
steady-state levels of the protein. The importance of PIN2 turnover for
root gravitropism is also highlighted by analysis of the wav6–52 allele.
The agravitropic root phenotype of this allele seems to result from stabilized PIN2wav6–52, which interferes with the distribution of auxin within
the root meristem. The findings that both the amount and distribution
of PIN2 are jointly affected in the wav6–52 seedlings correlates with the
data from BFA and MG132-treated plants, where both parameters are
also jointly affected.
Proteolytic turnover of eukaryotic plasma-membrane proteins
requires a mechanism that regulates protein levels through intracellular sorting and targeting24. A key function in the sorting process of
endocytosed membrane proteins has been attributed to ubiquitination25–27. Furthermore, proteasome activity has been implicated in the
targeting of membrane proteins to the vacuole and/or the lysosome,
although the mechanisms remain unknown19,20,28,29. Our findings predict the involvement of the proteasome in the turnover of membrane
proteins in plants. We found that MG132 treatment results in the accumulation of ubiquitinated PIN2 and interferes with the internalization
of PIN2 in gravi-stimulated roots. Thus, it is possible that ubiquitination of PIN2 serves as a signal to influence sorting and degradation of
PIN2 in a proteasome-dependent manner. This signalling may control
the proportion of PIN2 that is recycled back to the membrane relative to the proportion that is internalized and targeted for proteolytic
degradation and would thus determine the steady-state level of this
protein (Fig. 7a).
Our findings can be integrated into existing models for the control of
root gravitropism: A gravity stimulus would lead to the lateral relocation
of the PIN3 auxin-transport facilitator to the lower side of columella
root cap cells. This would facilitate the establishment a highly localized
auxin gradient with the maximum auxin levels located in the lower side
of the root cap (Fig. 7b and see ref. 22). The putative auxin-uptake carrier AUX1, and PIN2, would then control auxin transport to the root
elongation zone15. In this respect, auxin-mediated retention of PIN2 at
the plasma membrane in the lower side of the root13, versus internalization and degradation of PIN2 in the upper side, may promote differential
auxin-flux rates, thereby maintaining the lateral auxin gradient initially
established by PIN3 and AUX1.
Auxin was previously shown to effect the endocytosis of PIN213, however, we also found that auxin can promote the degradation of PIN2.
This effect may be part of a homeostatic mechanism that controls PIN2
levels at later stages of root gravitropism. In such a scenario, after gravistimulation elevated levels of auxin would build up in the lower side of
the root and could be amplified further through an auxin reflux loop. It
has been proposed that this loop proceeds through root epidermis and
cortex cells30. Once auxin threshold levels have been reached, auxininduced PIN2 degradation would reduce auxin transport in the lower
side of the root and thus prevent further bending of the root.
It has previously been shown that treatment with auxin (and BFA) for
2 h seemed to interfere with the endocytosis of PIN213, however, quantitative western experiments presented in our study indicate a net decrease
in total PIN2 levels after 2 h of NAA-treatment. Although these discrepancies may be due to different experimental conditions, our analyses of
PIN2 distribution in auxin-treated gravi-stimulated roots show that the
effects of auxin on PIN2 are time-dependent. The finding that shortterm auxin treatment interfered with the accumulation of intracellular
PIN2-specific signals in the upper side of gravi-stimulated roots could be
interpreted as an inhibitory effect of auxin on the endocytosis of PIN2.
In contrast, longer periods of auxin incubation caused an enrichment of
intracellular PIN2-specific signals that may reflect enhanced turnover
of the protein. These data suggest that auxin first interferes with PIN2
internalization and then causes internalization and degradation of the
protein.
The data presented here show the importance of post-transcriptional
control of PIN2 for this auxin efflux facilitator to function correctly. Our
findings agree with previous studies, indicating that post-transcriptional
control can override transcriptional regulation of PIN2 (refs 12, 31, 32).
It is clear however, that transcriptional and post-transcriptional mechanisms must combine for the complex changes in PIN2 abundance seen
during the gravitropic response to occur33. Future work should address
how these mechanisms integrate to regulate PIN2 function.
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a
Ler
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wav6-52
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PIN2
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c
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Figure 5 Analysis of wav6-52 roots. (a) Western blot of wav6–52 and Ler
(corresponding wild type) membrane protein extracts probed with antiPIN2. PIN2 protein levels were increased in the wav6–52 mutant allele
(left panel). Coomassie-staining of the protein samples is indicated in the
lower panel. A northern blot of PIN2 and UBQ5 (loading control, right
panels) revealed no differences in PIN2 transcript levels.
(b, c) Immunolocalization showing the subcellular localization of PIN2
in wav6–52 (b) and wild-type plants (c). Unlike the control, which
showed polar PIN2 localization at the plasma membrane, wav6–52 also
accumulated PIN2 ectopically, in intracellular vesicles (b).
(d, e) PIN2 distribution in wav6–52 (d) and wild-type (e) roots after
240 min of gravi-stimulation. Wild-type roots showed increased PIN2 levels
at transversal epidermis cell walls at the lower side of the root, whereas no
difference was detected in wav6–52 roots. (f–h) Comparison of DR5rev–GFP
expression in wav6–52 (f), eir1–4 (g) and wild-type (h) roots. Unlike
wild-type, wav6–52 roots showed strong GFP signals in lateral root-cap
cells extending into the root elongation zone (f, arrowheads), indicative
of ectopic accumulation of auxin. Eir1–4 showed strong DR5rev–GFP
expression in lateral root-cap cells in the proximity of the root tip (g,
arrowheads) Scale bars represent 10 µm in b and c and 50 µm in d–h.
METHODS
was performed with affinity-purified PIN2 serum and anti-HA (1:1000; Babco,
Richmond, CA)38,39. Fluorescein isothiocyanate (FITC)- and CY3-conjugated
anti-rabbit (1:500) or anti-mouse (1:500) IgG (Dianova, Hamburg, Germany)
were used as secondary antibodies.
Growth conditions and plant material. Plants were grown on soil in continuous light, or on plant nutrient agar (PNA)34 supplemented with 1% (w/v)
sucrose under 16 h light then 8 h dark conditions. Eir1–4 was obtained from the
Arabidopsis Information Resource (TAIR; SALK_547613; ref. 35). Plants were
transformed using the floral dip protocol36.
Growth assays. Drugs were prepared in DMSO. Gravitropic root reorientation of
plants was assayed on horizontally positioned, dark-incubated seedlings (5–7 days
after germination; DAG) at the indicated time points. To assay distribution of the
reporter proteins after gravi-stimulation, plants were transferred to microscope
slides that were covered with a thin layer of PNA. After gravi-stimulation, expression of the reporter proteins was analysed on a Leica TCS SP2 CLSM.
Nucleic-acid manipulations and constructs. Standard experimental procedures
that have previously been described were used37. 35S:PIN2 has been described previously12. For the generation of 35S:PIN2–GFP, PCR-amplified GFP was introduced
after base pair 1442 of the PIN2 coding sequence and the cassette was placed behind
a tandem 35S-promoter, the endogenous PIN2 promoter12, or into pTA700214 (kindly
provided by N. Chua, Rockefeller University, NY). The PIN2:PIN2-HA construct
was generated by fusion of the PIN2 promoter and the PIN2 cDNA with the HA
epitope tag at the carboxy-terminus.
Antibodies and immunolocalization. A PIN2-specific polyclonal antibody
was raised against amino acids 189–477 of the predicted PIN2 open reading
frame, which was expressed in vector pGEX-4T-2. Immunohistochemistry
254
Membrane protein extraction and analysis. Up to 2 g of seedlings (5–6 DAG)
were homogenized. and resuspended in extraction buffer (50 mM Tris at pH 6.8,
5% (v/v) glycerol, 1.5% (w/v) insoluble poly-vinylpolypyrrolidone, 150 mM KCl,
5 mM Na EDTA, 5 mM NaEGTA, 1 mM 1,4-dithioerythritol (DTE), 50 mM NaF,
20 mM β-glycerophosphate, 0.5% (w/v) casein40 and protease inhibitors: 1 mM
benzamidine, I mM PMSF, 3.5 µg ml–1 E64, 1 µg ml–1 pepstatin, 1 µg ml–1 leupeptin, 1 µg ml–1 aprotinin and 1 Roche complete mini protease inhibitor tablet per
10 ml). Samples were clarified by centrifugation (3,800g for 20 min), filtered and
then centrifuged (100,000g for 90 min). Pellets were resuspended in 50 mM Tris
at pH 7.5, 20% glycerol, 2 mM EGTA, 2 mM EDTA, 50–500 µM DTE, 10 µg ml–1
solubilized casein and protease inhibitors as above. Equal amounts of protein
were separated by 10% SDS–Urea PAGE, probed with affinity-purified anti-PIN2,
followed by HRP-conjugated goat anti-rabbit IgG (1:100,000; Pierce, Rockford,
IL). For immunoprecipitations membrane protein (up to 100 µg) was solubilized
in RIPA buffer with protease inhibitors and incubated overnight with affinitypurified rabbit anti-PIN2. Immuno-complexes were pulled down with Protein A
Sepharose 6MB. Blots were probed with a mouse monoclonal antibody to ubiquitin (clone P4D1, 1:100; Santa Cruz, Santa Cruz, CA), followed by HRP-conjugated
goat anti-mouse IgG (1:20,000; Dianova, Hamburg, Germany). Stripped blots
were reprobed with affinity-purified mouse anti-PIN2 and secondary antibody
as above (1:40,000).
NATURE CELL BIOLOGY VOLUME 8 | NUMBER 3 | MARCH 2006
©2006 Nature Publishing Group
A RT I C L E S
a
a
eir1-4
35S : PIN2
C
eir1-4
35S : PIN2
NAA
C
PIN2
PIN2
Coomassie
UBQ5
MG132
NAA
BFA
b
Col−O
Col−O
C NAA
C
PIN2
PIN2
Coomassie
UBQ5
c
e
d
f
NAA
b
Figure 6 Auxin promotes degradation of PIN2. (a) PIN2 abundance in total
membrane protein fractions that were isolated from eir1–4 35S:PIN2 treated
with 10 µM NAA for 2 h (left; C, no auxin added). Coomassie-staining is shown
below. PIN2 transcript levels in eir1–4 35S:PIN2 after treatment with 10 µM
NAA for 2 h (right). UBQ5 was used as a loading control. (b) PIN2 protein
levels in auxin-treated wild-type roots (left; C: no auxin added). Two hours of
treatment with 10 µM NAA resulted in decreased PIN2 levels when compared
with the control. Coomassie-staining is shown below. PIN2 transcript levels
increased after NAA-treatment for 2 h (right). (c–f) Subcellular distribution
and abundance of PIN2:HA in gravi-stimulated eir1–1 PIN2:PIN2:HA roots
treated with 10 µM 2,4-D. After 90 min (c), epidermis cells in the upper side
of untreated control roots showed a pronounced intracellular PIN2:HA signal,
whereas signals in the lower side remained in the proximity of transverse
cell walls. A similar distribution was seen after 180 min (d) of gravistimulation. Treatment with 2,4-D for 90 min (e) prevented the accumulation
of intracellular PIN2:HA signals in epidermis cells at the upper side of root
(arrowheads). After 180 min (f) of 2,4- D treatment, internalized PIN2:HAspecific signals became visible in the upper and the lower side of the root
(arrowheads). Scale bar represents 50 µm.
Figure 7 A diagram illustrating the role of PIN proteins in the regulation of
root gravitropism. (a) Continuous cycling of PIN2 (red circles) between the
plasma membrane and an intracellular compartment (green circle) controls
subcellular distribution and steady-state levels of the auxin transport
facilitator. A fraction of ubiquitinated (blue circle) endocytosed protein is not
recycled to the plasma membrane but is degraded. Inhibition of proteasome
activity primarily interferes with intracellular distribution of PIN2 and
thereby gives rise to an accumulation of the protein. (b) Variations in PIN
localization and abundance direct asymmetric auxin flow in gravi-stimulated
roots. In vertically oriented roots, PIN3 (green) is equally distributed at the
plasma membrane of columella cells, giving rise to a uniform distribution of
auxin (blue arrows). PIN2 (red) ensures further auxin transport into the rootelongation zone. Under these conditions, roots will grow in the direction of
the gravity vector. After gravi-stimulation, PIN3 relocates transiently to the
lower side of columella cells, redirecting the auxin stream to the lower side
of the gravi-stimulated root tip. Asymmetric, auxin-controlled distribution
of PIN2 then mediates differential auxin transport resulting in downward
bending of the root. On completion of root reorientation, auxin-mediated
degradation of PIN2 may reset PIN2 distribution to default levels.
Note: Supplementary Information is available on the Nature Cell Biology website.
ACKNOWLEDGEMENTS
This work has been supported by grants from the Austrian Science Fund (FWF,
P16311 and P15441) to C.L., by Volkswagenstiftung to J.F. and J.W, the Deutsche
Forschungsgemeinschaft (SFB 446) to T.P., the Foundation for Polish Science to
J.W., by the Institute of Applied Genetics and Cell Biology (MSM0021622415) and
by the ‘Hochschuljubiläumsfonds der Stadt Wien’. R.B. was a recipient of a TALENT
fellowship from the Netherlands Organisation for Scientific Research. We would
like to thank M. Bennett, L. Mach, E. Benková, M. Hauser, O. Mittelsten Scheid and
K. Stockhammer for advice and valuable comments on the manuscript. L.A. thanks
M. Guppy for good discussions. C.L. is indebted to J. Glössl for his ongoing and
substantial support.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
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A RT I C L E S
Published online at http://www.nature.com/naturecellbiology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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