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First published online 11 September 2008
doi: 10.1242/dev.021071
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3,4ek3,4
pa3,4
ímalová3
í Friml1,2,*
1 Department of Plant Systems Biology, VIB, and Department of Molecular
Genetics, Ghent University, 9052 Gent, Belgium.
2 Center for Plant Molecular Biology (ZMBP), University of Tübingen,
D-72076 Tübingen, Germany.
3 Institute for Experimental Botany, Academy of Sciences of the Czech Republic,
Rozvojová 263, 165 02 Praha 6, Czech Republic.
4 Department of Plant Physiology, Faculty of Science, Charles University,
Vini
ná 5, 128 44 Praha 2, Czech Republic.
* Author for correspondence (e-mail: jiri.friml{at}psb.ugent.be)
Accepted 20 August 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: PGP, PIN, Auxin transport, Embryogenesis, Organogenesis, Tropisms
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Because the auxin molecule is charged inside cells and,
thus, membrane impermeable, its intercellular transport relies on
carrier-mediated cellular influx and efflux (reviewed by
Kerr and Bennett, 2007;
Vieten et al., 2007). Genetic
approaches in Arabidopsis thaliana have identified two groups of
proteins that are involved in auxin export from cells: PIN-FORMED (PIN)
proteins and several ABC transporter-like phosphoglycoproteins (PGPs)
(reviewed by Bandyopadhyay et al.,
2007; Vieten et al.,
2007).
The PIN protein family consists of plant-specific integral plasma membrane
proteins that have been identified based on mutants defective in organogenesis
(pin-formed1 or pin1)
(Okada et al., 1991;
Gälweiler et al., 1998)
and tropism (pin2/agr1/eir1)
(Luschnig et al., 1998). The
Arabidopsis genome encodes eight PIN-related sequences, most of which
have been already characterized at cellular and developmental levels (reviewed
by Vieten et al., 2007;
Zaímalová et al.,
2007). PIN proteins are expressed in different parts of the plant
and are almost universally required for all aspects of auxin-related plant
development, including embryogenesis
(Friml et al., 2003),
organogenesis (Benková et al.,
2003; Reinhardt et al.,
2003), root meristem patterning and activity
(Friml et al., 2002a;
Blilou et al., 2005), tissue
differentiation and regeneration
(Scarpella et al., 2006;
Xu et al., 2006;
Sauer et al., 2006a), and
tropisms (Luschnig et al.,
1998; Friml et al.,
2002b). Most phenotypic aberrations in pin
loss-of-function alleles can be phenocopied by external application of auxin
efflux inhibitors, such as 1-naphthylphthalamic acid (NPA)
(Tanaka et al., 2006). When
expressed in plant and non-plant cultured cells, PIN proteins perform a
rate-limiting function in cellular auxin efflux
(Petráek et al.,
2006). Importantly, PIN proteins show distinct polar subcellular
localization that determines auxin flux direction, as predicted by classical
models of directional auxin transport
(Wi
niewska et al.,
2006). The dynamic regulation of the intracellular movement of
PINs, their polar targeting and their protein stability provides a means to
regulate directional throughput of auxin flow
(Friml et al., 2004;
Paciorek et al., 2005;
Abas et al., 2006;
Michniewicz et al., 2007).
Moreover, the PIN-dependent auxin distribution network involves redundancy and
auxin-mediated crossregulation of PIN expression and PIN targeting
(Sauer et al., 2006a;
Blilou et al., 2005;
Vieten et al., 2005). A
crucial role for PIN-dependent auxin efflux in generation of morphogenetic
asymmetric auxin distribution has recently been suggested by mathematical
modelling (Grieneisen et al.,
2007).
Plant orthologues of the mammalian multidrug-resistance proteins
(Martinoia et al., 2002;
Verrier et al., 2008) PGP1
(ABCB1) and PGP19 (MDR1/ABCB19), similarly to PIN proteins, have been shown to
perform cellular auxin efflux in both plant and heterologous systems;
accordingly, basipetal auxin transport is decreased in pgp1 and
pgp19 mutants (Noh et al.,
2001; Geisler et al.,
2005; Petráek
et al., 2006). In addition, these proteins bind auxin efflux
inhibitors, such as NPA (Murphy et al.,
2002). Phenotypic defects caused by loss of PGP function are most
pronounced in vegetative organs and include dwarfism, curly wrinkled leaves,
twisted stems and reduced apical dominance, supporting their role in
auxin-based development (reviewed by
Bandyopadhyay et al., 2007).
However, expression, localization and roles of PGPs in patterning processes
are less characterized compared with PINs. The cellular localization of PGPs
is mainly apolar, but instances of asymmetric cellular distribution have been
reported (Geisler et al.,
2005; Blakeslee et al.,
2007; Wu et al.,
2007).
Important questions in auxin research relate to the roles of these two
types of auxin efflux proteins in auxin transport. Do they represent
independent mechanisms? What would be the functional requirements for two
distinct transport systems? Do they cooperate and how? The only partial
colocalization of PINs and PGPs at the plasma membrane and the difference in
the corresponding mutant phenotypes favours a scenario in which PGPs and PINs
have independent functions. Nevertheless, recent biochemical studies have
demonstrated an interaction between PIN and PGP proteins that is functionally
relevant in heterologous systems, because it influences the rate of efflux,
its substrate specificity and its sensitivity to inhibitors
(Blakeslee et al., 2007;
Bandyopadhyay et al., 2007).
However, the relevance of the interaction between PINs and PGPs in planta and,
eventually, in asymmetric auxin distribution, remains unclear.
Here, we present evidence that PINs and PGPs define independent auxin transport mechanisms that cooperate to mediate auxin distribution-mediated development during embryogenesis, organogenesis and root gravitropism. Our data suggest a model for how non-polar auxin efflux mediated by PGPs is linked with vectorial transport driven predominantly by PINs.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), pin1pgp1pgp19
(Blakeslee et al., 2007),
pin1-1 (Okada et al.,
1991), pin1-3xDR5rev::GFP (R i ka
et al., 2007), rcn1-1 (Garbers et
al., 1996), eir1-3
(Luschnig et al., 1998),
eir1pgp1pgp19 (Blakeslee et al.,
2007); and transgenic lines DR5::GUS
(Sabatini et al., 1999),
DR5rev::GFP (Friml et al.,
2003), XVE-PIN1
(Petráek et al.,
2006), pPGP1::PGP1-myc and pPGP19::PGP19-HA
(Blakeslee et al., 2007).
pPGP1::PGP1-myc was generated as previously described for
pPGP19::PGP19-HA (Blakeslee et
al., 2007). For pPGP19::PGP19-GFP and
pPGP1::PGP1-GFP, tag sequences in pPGP19::PGP19-HA and
pPGP1::PGP1-myc, respectively
(Blakeslee et al., 2007), were
replaced by an enhanced green fluorescent protein (GFP) sequence to create
C-terminal fusion constructs and was transformed to pgp19 or
pgp1 mutants. The pGVG-PGP1-myc and pGVG-PGP19-HA plasmids
were constructed by cloning the whole genomic coding region of PGP1
and PGP19 genes fused with the respective tag by primer extension PCR
to pTA7002 (Aoyama and Chua,
1997), and transformed to the UAS::GUS
(Weijers et al., 2003) line.
Fifteen independent transgenic lines were analysed for each construct.
Growth conditions
Arabidopsis plants were grown in a growth chamber under long-day
conditions (16 hours light/8 hours dark) at 18-23°C. Seeds were sterilized
with chlorine gas or ethanol, and stratified for 2 days at 4°C. Seedlings
were grown vertically on half Murashige and Skoog medium with 1% sucrose and
supplemented with 5 µmol/l NPA, 4 µmol/l β-estradiol (EST) or 5
µmol/l dexamethasone (DEX). Drugs were purchased from Sigma-Aldrich (St
Louis, MO, USA).
BY-2 cell lines
The transgenic lines GVG-PIN4, GVG-PIN6, GVG-PIN7, GVG-PGP19-HA
and pPIN1::PIN1-GFP
(Petráek et al.,
2006; Benková et al.,
2003) of Nicotiana tabacum Bright Yellow-2 (BY-2) cells
(Nagata et al., 1992) were
grown as described (Petráek
et al., 2006). Expression of PIN7 and PGP19 was induced by the
addition of 1 µM DEX at the beginning of subcultivation. For the NPA
effect, 10 µM NPA was added together with DEX. For microscopy, an Eclipse
E600 microscope (Nikon, Tokyo, Japan) and a colour digital camera 1310C (DVC,
Austin, TX, USA) were used. Reciprocal plots of cell size distribution
represent individual cell lengths and diameters measured by LUCIA image
analysis software (Laboratory Imaging, Prague, Czech Republic). At least 170
cells in total were measured on five optical fields for each variant.
Immunotechniques and microscopy
Arabidopsis embryos and roots were stained immunologically as
described (Sauer et al.,
2006b). The antibodies used were anti-PIN1
(Paciorek et al., 2005)
(1:1000), anti-c-myc (1:500) from rabbit and anti-GFP (1:500) from
mouse (Roche Diagnostics, Brussels, Belgium) (1:500). Fluorescein
isothiocyanate isomer I (FITC) or Cy3-conjugated anti-rabbit or anti-mouse
secondary antibodies were purchased from Dianova (Hamburg, Germany) and
diluted 1:600. The microscopic analyses were carried out on a SP2 confocal
microscope (Leica-Microsystems, Wetzlar, Germany). GFP samples were scanned
without fixation.
Phenotype analyses
Plates with grown seedlings (5 or 10 days old) were scanned on a flatbed
scanner and measured with ImageJ software
(http://rsb.info.nih.gov/ij/).
The vertical growth index (VGI) was calculated as described
(Grabov et al., 2005). The
hypocotyl twisting index was determined as the relation between hypocotyl
length and the distance from the root base to the apical hook. For embryo and
root tip morphology analyses, we used chloral hydrate clearing
(Friml et al., 2003) and
microscopy was carried out on an Axiophot microscope (Zeiss, Jena, Germany)
equipped with a digital camera. Lateral roots were analysed and GUS staining
was performed as described (Benková
et al., 2003).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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ek et al.,
2006). After induction of GVG-PGP19-HA or
GVG-PIN7 constructs, cells accumulated less auxin because of the
increased auxin efflux
(Petráek et al.,
2006). To study how increased auxin efflux influences cellular
behaviour in BY-2 cells, we examined the growth and morphology of DEX-treated
and untreated cells in these lines. After induction of PIN7 or PGP19-HA
expression, identical phenotypical changes occurred: cells ceased to divide,
started to elongate, and formed and accumulated starch granules
(Fig. 1A,B,D,E). A similar set
of morphological changes was observed in induced GVG-PIN4 and
GVG-PIN6 lines, and in a line constitutively expressing PIN1-GFP that
also showed increased auxin efflux (see Fig. S1A-D,H,I in the supplementary
material). Importantly, this cellular behaviour could be mimicked by
cultivation of cells in medium with a decreased amount of or no auxin (see
Fig. S1E-G in the supplementary material). These experiments imply that the
enhanced efflux after induction of either PIN7 or PGP19-HA expression leads to
depletion of auxin from cells, resulting in a change in the developmental
program reflected by the switch from cell division to cell elongation.
All phenotypic changes induced by overexpression of PIN7 in the
GVG-PIN7 line were completely reversed by application of the auxin
efflux inhibitor NPA (Fig.
1C,G; see Fig. S1J,K in the supplementary material). By contrast,
after induction of PGP19-HA expression in the GVG-PGP19-HA line, NPA
treatment was ineffective in rescuing auxin starvation phenotypes
(Fig. 1F,G; see Fig. S1J,K in
the supplementary material). These observations are in line with previously
reported differences in sensitivities of PGP19- and PIN7-mediated auxin efflux
to NPA (Petráek et al.,
2006). These data collectively suggest that, although PGP and PIN
proteins play similar cellular roles in mediating auxin efflux and inducing
the switch from cell division to cell elongation, they define two distinct
auxin efflux mechanisms that differ in sensitivity to auxin efflux
inhibitors.
Effect of PIN- and PGP-inducible overexpression in Arabidopsis seedlings
To study what effects overexpression of PINs and PGPs may have at the
multicellular level, we analysed transgenic lines overexpressing PIN1, PGP1 or
PGP19. After induction of PIN1 expression in the estradiol-inducible
XVE-PIN1 line
(Petráek et al.,
2006) (Fig. 2A,B),
seedlings lost the gravitropic response and had retarded root growth
(Fig. 2F,R). This effect was
stronger at increased concentrations of oestradiol, supporting the
rate-limiting function of PIN proteins
(Fig. 2R). Immunolocalization
confirmed that ectopically expressed PIN1 was localized predominantly on the
basal side of root epidermal cells (Fig.
2A,B), consistent with previous reports
(Winiewska et al.,
2006). In hypocotyls of dark-grown seedlings, we observed a
previously uncharacterized phenotype. Unlike the straight-growing hypocotyls
of non-induced plants, induced XVE-PIN1 plants had a twisted growth
along the vertical axis. Twisting was usually more pronounced close to the
hypocotyl base (Fig. 2I,J). To
test for possible changes in auxin distribution after induction of PIN1
expression, we crossed the XVE-PIN1 line with the DR5::GUS
reporter line, a widely used auxin response reporter
(Hagen and Guilfoyle, 2002).
DR5::GUS staining of the XVE-PIN1 line after estradiol
treatment suggested a stronger auxin accumulation in the root tip, which
rationalizes the root agravitropic phenotype
(Fig. 2N,O). In dark-grown
seedlings, weak uniform GUS staining along the hypocotyl axis was seen in
non-induced controls. This pattern changed after induction of PIN1 expression,
and DR5 signal became stronger, with random maxima along the hypocotyl
(Fig. 2L,M) reflecting
differential cell elongation and hypocotyl twisting. Similar to the situation
in BY-2 cells, PIN1-inducible overexpression phenotypes (root elongation and
hypocotyl twisting) could be partially rescued by exogenous application of NPA
(Fig. 2K,S). Notably, similar
phenotypic aberrations and NPA treatment-based rescue were also detected at
different levels of induced PIN expression (data not shown) and in other
PIN1-overexpressing lines, such as DEX-inducible GVG-PIN1 and
constitutive 35S::PIN1 (data not shown). This confirmed that the
phenotypic changes and changes in patterns of DR5 activity observed are due to
the overexpression of PIN1 protein.
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)
introduced into GVG-PGP19-HA plants revealed a reduced DR5
activity in the root tip, contrasting with the increased DR5 signal
after PIN1 overexpression (Fig.
2P,Q). Furthermore the sensitivity to NPA was not visibly altered
in these lines (Fig. 2T). Although other post-transcriptional events may influence the outcome of these overexpression experiments, observations that overexpression of PIN1 when compared with that of PGP1 and PGP19 leads to qualitatively different phenotypes suggest that, unlike in cultured cells, overexpression of PINs and PGPs in planta have different effects on auxin distribution and seedling development. This hypothesis is consistent with a scenario in which PIN and PGP efflux machineries are distinct and might have both overlapping and distinct functions in auxin transport-dependent development.
PGP proteins are expressed and synergistically interact with PIN1 protein during embryogenesis
To study whether distinct PIN- and PGP-dependent transport mechanisms have
common developmental roles, we studied role of PIN- and PGP-dependent
transport during different auxin transport-mediated processes. Many data on
the developmental roles of various members of the PIN family are available
(Tanaka et al., 2006), but
comparable information for PGP1 and PGP19 is still largely lacking.
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;
Weijers et al., 2005). Various
PIN proteins are expressed at different stages of embryogenesis in distinct
cells, and PIN gene mutants (single and double), as well as mutants
with defective PIN localization, are defective in embryo patterning
(Steinmann et al., 1999;
Friml et al., 2003;
Friml et al., 2004;
Weijers et al., 2005;
Michniewicz et al., 2007).
However, in pgp mutants, no embryo patterning defects have been
reported and the function of PGPs in embryogenesis has not been studied so
far. To investigate expression and cellular localization of PGP1 and PGP19
during embryogenesis, we used the functional tagged constructs
pPGP1::PGP1-myc (Blakeslee et al.,
2007) and pPGP19::PGP19-GFP (see Fig. S3 in the
supplementary material). PGP1-myc was expressed from the earliest
embryo stages onwards in all pro-embryo and suspensor cells. Cellular
localization was mainly apolar, but more intense signals were observed between
freshly divided cells (Fig.
3A,B). PGP19-GFP was also expressed at early stages, predominantly
in the basal cell lineage that forms the suspensor until approximately the
octant stage (Fig. 3C). At
dermatogen (16-cell) stage, PGP19-GFP expression extended from suspensor to
the lower tier cells of the pro-embryo
(Fig. 3D). At mid-globular and
later stages, PGP19-GFP expression was gradually confined to the outer layers,
including protoderm and cells surrounding vascular precursor cells with no
expression between initiating cotyledon primordia
(Fig. 3E). At later stages,
PGP19-GFP expression persisted in cells surrounding the forming vasculature
(pericycle, endodermis and protoderm) (Fig.
3H). As for PGP1, no apparent polar localization was observed.
Interestingly, the PGP19-GFP expression pattern at globular and later stages
was roughly complementary to that of PIN1
(Fig. 3E-J). PGP19-expressing
cells appeared to separate the inner basipetal and outer acropetal auxin
streams, which are defined by PIN1 expression and localization
(Friml et al., 2003;
Vieten et al., 2005)
(Fig. 3F,I).
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5% of seedlings had a defective cotyledon
formation, including tricots and fused cotyledons
(Fig. 4B,G), as reported
previously (Okada et al.,
1991). In the progeny of pgp1pgp19pin1+/- plants, almost
25% of seedlings had fused cotyledons and some were cup-shaped, a feature that
is extremely rarely seen in pin1 seedlings
(Fig. 4D,E,G). Corresponding
mutant phenotypes were also seen during embryogenesis at heart and later
stages (Fig. 4A-D). As reported
(Blakeslee et al., 2007), later
post-embryonic development in pin1pgp1pgp19 mutants was also very
strongly affected (Fig.
4F). These results reveal a previously unknown role of PGPs during embryogenesis. PGP1 and PGP19 are not strictly required for embryo development, but they act synergistically with PIN1 protein, mainly during cotyledon formation.
PGP genetically interacts with RCN1 in embryogenesis and root development
To test further the functional interaction between PGP- and PIN-dependent
auxin transport systems in embryogenesis and patterning, we generated
rcn1pgp1pgp19 mutant that, besides lacking PGP-mediated efflux, is
defective in RCN1 (Root Curling on NPA). This gene encodes a
subunit of protein phosphatase 2A (PP2A) that has been shown to be involved in
various developmental and signalling processes
(Garbers et al., 1996;
Kwak et al., 2002;
Larsen and Cancel, 2003).
Importantly, PP2A phosphatase, together with PINOID kinase, regulates PIN
polar targeting and, thus, directionality of PIN-dependent auxin transport
(Friml et al., 2004;
Michniewicz et al., 2007).
Some features of the rcn1 mutant are similar to those of the
pgp1pgp19 mutant, such as wavy root growth, but they do not include
embryonic patterning defects (Garbers et
al., 1996) (Fig.
4G). Strikingly, rcn1pgp1pgp19 triple mutants exhibited
strong embryonic and post-embryonic auxin-related phenotypes. Some seedlings
of the rcn1pgp1pgp19 mutant were defective in apical-basal patterning
(11/97) or cotyledon formation (3/97) (Fig.
5E,F). During embryogenesis, rcn1pgp1pgp1 exhibited
aberrant hypophysis divisions (16/20) at the globular stage
(Fig. 5A-D). This spectrum of
developmental aberrations is typical for mutants with strong defects in auxin
transport (for example, pin1pin3pin4pin7)
(Friml et al., 2003) or auxin
signalling (monopteros and bodenlos)
(Hardtke and Berleth, 1998;
Hamann et al., 2002). In
post-embryonic development, roots of rcn1pgp1pgp19 seedlings were
reduced in length and showed enhanced defects in the gravitropic response and
differentiation of columella cells when compared with rcn1 single or
pgp1pgp19 double mutants (Fig.
5H-J).
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Diverse functions of PGP1 and PGP19 in lateral root organogenesis
Next, we studied the role of PGP-dependent auxin efflux and its possible
interaction with PIN-dependent mechanisms in lateral root initiation and
emergence, other processes that involve PIN-dependent auxin transport.
Pharmacological or genetic modulation of local transport-dependent auxin
distribution inhibits lateral root initiation and its morphogenesis
(Benková et al., 2003;
Casimiro et al., 2003). The
role of PGP1 and PGP19 in lateral root development has already been proposed
(Lin and Wang, 2005;
Wu et al., 2007), but their
precise function and interaction with PINs in initiation and emergence remains
unknown.
We determined expression and localization patterns of PGP1 and PGP19 during
early post-embryonic development by using pPGP1::PGP1-GFP and
pPGP19::PGP19-GFP constructs. pPGP1::PGP1-GFP and
pPGP19::PGP19-GFP complemented most aspects of the corresponding
pgp1 and pgp19 mutant phenotypes, such as hypocotyl
elongation defect (see Fig. S3A-E in the supplementary material). Similarly to
expression during embryogenesis, PGP1-GFP did not exhibit tissue-specific
expression pattern and was detected in all cells of hypocotyls and roots
(Fig. 6A,C,D), except root-tip
columella cells. The expression of PGP19-GFP was also found in hypocotyls and
main roots, but, in contrast to PGP1-GFP, exhibited a more tissue-specific
pattern, with strongest expression in endodermal and pericycle tissues
(Fig. 6B,E,F), in agreement
with published data on PGP19-HA (Blakeslee
et al., 2007) and MDR1(PGP19)-GFP
(Wu et al., 2007). In
addition, PGP19-GFP expression was detected also in root tip epidermal cells,
which is not supported by the published pattern of MDR1(PGP19)-GFP
(Wu et al., 2007). Both
proteins are also expressed during all stages of lateral root development and
persisted after lateral root emergence as shown previously
(Geisler et al., 2005;
Wu et al., 2007). PGP1-GFP
expression was observed from the first stage of lateral root primordium
organogenesis on (Fig. 6G).
During developmental stage I, PGP1-GFP was localized to anticlinal membranes
of short initials, and, later, when lateral root primordia are formed, apolar
membrane localization was detected in all cells of primordia. Expression and
membrane localization of PGP19-GFP at developmental stage I fully overlapped
with PGP1, but at later developmental stages PGP19-GFP expression was more
restricted to endodermis and pericycle. In emerged lateral roots, PGP19-GFP
was detected also in cortical and epidermal cells, similar to its expression
in primary root tips (Fig.
6H).
Next, we investigated the consequence of PGP1 and PGP19 loss in lateral
root initiation and emergence, and their functional interaction with PIN1 in
this process. Both pgp single mutants initiated fewer lateral roots
(Fig. 6I), whereas,
interestingly, the combination of pgp1 and pgp19 mutations
almost completely rescued the effect of single mutations
(Fig. 6I). This genetic
complementation of pgp1 and pgp19 mutations was also
observed during lateral root emergence
(Fig. 6J). Both single mutants
had a reduced progression rate through consecutive stages of lateral root
development, but this defect was largely recovered in pgp1pgp19
double mutants (Fig. 6J).
Addition of pin1 to the pgp1pgp19 mutant surprisingly led to
rescue of pin1 phenotype, which is characterized by delayed lateral
root primordium development (Benková
et al., 2003), and even slightly increased lateral root initiation
above wild-type level (Fig.
6I,J).
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Antagonistic and synergistic effects of PIN- and PGP-dependent transport on the spatial pattern of auxin responses
To gain further insights into how PGP- and PIN-mediated transport
mechanisms together regulate auxin-dependent plant development, we analysed
changes in auxin distribution, as visualized indirectly by the
DR5rev::GFP auxin response reporter
(Friml et al., 2003). During
embryogenesis, spatial distribution of DR5rev::GFP signal did not
dramatically change in pgp1pgp19 mutants, but auxin response maxima
in cotyledon primordia and at the root pole were enhanced
(Fig. 7A,B). Conversely,
pin1 mutant embryos had a reduced DR5 signal at the root
pole and cotyledon primordia (Fig.
7A,C). When pin1 and pgp1pgp19 mutations were
combined, the spatial pattern of DR5 activity distribution in embryos
was strongly distorted. The DR5 activity maxima were less well
defined and DR5 signal was more diffuse
(Fig. 7D). These results
clearly show that both PGP- and PIN1-mediated transport systems are required
together for the spatial pattern of auxin distribution and formation of
well-defined auxin maxima during embryogenesis. This observation also fully
explains the observed synergistic genetic interaction between pin1
and pgp1pgp19 mutations (Fig.
4A-E,G).
In post-embryonic roots, the situation is somewhat different: maintenance
of auxin maxima in the quiescent centre/columella region is crucial for
controlling root meristem activity
(Sabatini et al., 1999;
Friml et al., 2002a;
Grieneisen et al., 2007). We
detected quantitative changes in DR5 activity similar to those during
embryogenesis, including and increase in DR5 activity in
pgp1pgp19 mutants, but a reduction in pin1 mutant roots
(Fig. 7E-G). However, when
pgp1pgp19 and pin1 mutations were combined, the spatial
pattern of DR5 activity was not impaired and the level of activity
was roughly restored to that of the wild type
(Fig. 7H). This apparent
difference between the requirements of PGP and PIN transport systems for
spatial patterning of the auxin response in embryos and seedling roots is
probably due to pronounced functional redundancy of PIN proteins for auxin
delivery to the central root meristem during post-embryonic development
(Blilou et al., 2005;
Vieten et al., 2005).
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), where
auxin redistribution to the lower side of gravistimulated root is more
pronounced in pgp1pgp19 (see Fig. S4A,B in the supplementary
material), but inhibited in pin2 mutants
(Luschnig et al., 1998).
Contrasting functions of PIN and PGP are also supported by the synergistic
effects of PIN1 gain-of-function and PGP loss-of-function alleles. For
example, defects in root elongation and hypocotyl twisting were more
pronounced after XVE-PIN1 induction in pgp1pgp19 mutant than
they were after PIN1 induction in wild type
(Fig. 8A). In summary, these data show that even when PGP- and PIN-dependent auxin transport mechanisms have opposite or even antagonistic effects on auxin distribution and development during several developmental processes, both systems are complementary and are required together to maintain a dynamic spatial pattern of auxin distribution and subsequent development.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Vieten et al., 2007). PGP and
PIN proteins from Arabidopsis are both involved in auxin efflux
(Geisler et al., 2005;
Petráek et al.,
2006) and can physically and functionally interact in mediating
this process (Blakeslee et al.,
2007). However, the developmental relevance of the PGP and PIN
interactions is unclear. Here, we have systematically studied the distinct and
common roles of both auxin efflux systems in auxin distribution-dependent
development and provide new insights into understanding the purpose of these
two independent auxin efflux mechanisms for regulating plant development.
PGPs and PINs define two distinct auxin efflux systems
Since the identification of both PINs and PGPs as being involved in the
same process of cellular auxin efflux
(Geisler et al., 2005;
Petráek et al.,
2006), an important issue is whether they represent independent
transport systems or act together as necessary parts of one transport system.
Our results strongly support the scenario in which PGPs and PINs characterise
two distinct auxin efflux mechanisms. The earlier observations of the largely
non-overlapping phenotypes observed in pin and pgp
loss-of-function mutants (Vieten et al.,
2007) indicated a different role for these protein families that
could be explained by the distinct expression patterns of the family members.
More significantly, when both proteins are overexpressed under comparable
general promoters in the present study, the resulting phenotypes, although
similar in cultured cells, appear to be distinct in planta, and show different
sensitivities to the auxin efflux inhibitor NPA. Phenotypes resulting from PIN
overexpression can be reversed by NPA, but similar sensitivities to NPA are
not possible to demonstrate in PGP overexpression lines. The different effects
of PIN and PGP overexpression, together with the different responses of these
transporters to inhibitors, clearly favours the scenario that PIN and PGP
protein families define two distinct auxin efflux machineries.
However, the identity of the molecular mechanism that underlies the effect
of auxin efflux inhibitors such as NPA still has to be clarified. Previous
reports have clearly shown that NPA binds PGP proteins
(Noh et al., 2001;
Murphy et al., 2002;
Rojas-Pierce et al., 2007),
that PGP-mediated auxin efflux activity is inhibited by NPA in heterologous
systems (Petráek et al.,
2006; Blakeslee et al.,
2007) and that NPA inhibits PGP19 action in phototropism
(Nagashima et al., 2008). It
is possible that NPA and other auxin efflux inhibitors have multiple binding
and regulatory sites with different affinities. Moreover, auxin efflux
inhibitors might have multiple effects, including modification of actin-based
subcellular dynamics (Dhonukshe et al.,
2008) or of the PGP-PIN interaction
(Blakeslee et al., 2007).
PGP- and PIN-mediated transport are required together for embryogenesis and organogenesis
Another important argument for distinct roles of the PGP- and PIN-dependent
transport systems are the divergent phenotypes in pin
loss-of-function mutants when compared with pgp1 and pgp19
mutants. PGP proteins play an important role in determining the plant
architecture during vegetative growth (Noh
et al., 2001; Lin and Wang,
2005; Wu et al.,
2007). For example, agriculturally interesting dwarf mutations in
maize and sorghum results from loss of PGP activity and from a reduction in
auxin transport (Multani et al.,
2003). However, pgp mutants do not show clear patterning
defects, whereas pin single and multiple mutants show defects in
embryogenesis, organogenesis, tissue differentiation and meristem activity
(Tanaka et al., 2006;
Vieten et al., 2007). We found
that an important role of PGP1 and PGP19 in patterning processes can be
unmasked if the PIN-dependent transport is compromised. During embryogenesis,
the pgp1pgp19 mutation enhances greatly the effect of the
pin1 mutation on the formation of embryonic leaves. Furthermore,
analysis of mutant combinations of pgp1pgp19 and rcn1 [a
gene encoding a regulatory subunit of PP2A phosphatase that does not influence
the PIN function directly, but through the phosphorylation-dependent the PIN
polar subcellular targeting (Michniewicz
et al., 2007)] revealed very strong embryo and seedling patterning
phenotypes that are not observed in any of the single combinations. The
phenotypes are reminiscent of those found in mutants that are strongly
defective in auxin transport, such as gnom/emb30 (Steinman
et al., 1999) and pin1pin3pin4pin7
(Friml et al., 2003) mutants.
These results, together with expression and localization patterns of PGP1 and
PGP19 during embryogenesis, reveal a previously unknown role of the
PGP-dependent auxin transport in development and support the notion that the
common functions of these two transport mechanisms contribute to patterning
processes.
Concerted action of PGP- and PIN-mediated transports is required for auxin distribution
Auxin transport executes its effect on plant development largely by
generating asymmetric auxin distribution, which is often indirectly monitored
using the auxin response. The effects of gain- and loss-of-function mutations
in PGPs and PIN proteins on the quantity of local auxin response and resulting
development are, at times, seemingly opposite. For example, in root tip, PIN1
overexpression, but not pgp1pgp19 loss of function, enhances auxin
accumulation in central root meristem. Conversely, pin1 loss of
function and PGP19 overexpression both produce the opposite effect: a decrease
in auxin response in the root-tip region. Similarly, during embryogenesis, the
pin1 mutation decreases auxin response maxima, but pgp1pgp19
increases them. These results show that PGP- and PIN-dependent transport
systems play, in some instances, opposing roles in mediating auxin
accumulation in specific cells.
The opposite actions of PGP and PIN proteins can also be observed at the
developmental level. Some features of PIN1 overexpression, such as twisting
hypocotyls, can also be seen in the pgp1pgp19 mutant
(Noh et al., 2001) or in the
mutant of the apparent activator of the PGP function - immunophilin-like
protein TWISTED/DWARF (TWD) (Geisler et
al., 2003). Furthermore, hypergravitropic root growth in
pgp1pgp19 seedlings (Lin and
Wang, 2005) (see Fig. S4 in the supplementary material) contrasts
with agravitropic growth of pin2 or pin3 seedlings.
Significant also are the additive effects of pgp1pgp19 mutations on
the PIN1 overexpression-induced phenotypes. Interestingly, despite these
opposite roles for PGPs and PINs in mediating the quantitative distribution of
the auxin response, both transport systems are required together for
generating proper spatial patterns of auxin distribution. This effect was
observed for auxin distribution in pin1pgp1pgp19 mutant embryos
(Fig. 7H) or in
pin2pgp1pgp19 seedlings during the gravitropic response
(Blakeslee et al., 2007)
(Fig. 5J). Thus, both the PGP-
and PIN-dependent auxin transport mechanisms play distinct, sometimes
opposite, roles in auxin distribution, but both transport systems cooperate to
generate and maintain the spatial pattern of auxin distribution that is
necessary for patterning and tropisms.
Model of the PGP and PIN interaction in local auxin distribution in meristematic tissues
Based on previous and novel findings, we propose a model for the functional
interaction between PGP- and PIN-dependent auxin transport mechanisms in
embryos and root meristems (Fig.
8C). We take into account the prevalent non-polar cellular
localization of PGP1 and PGP19 (Fig.
3, Fig. 6A-H,
Fig. 8B) as well as the related
loss-of function and overexpression phenotypes. In cells where PINs and PGPs
are co-expressed, two types of interactions might take place. A direct
interaction between PIN and PGP, which takes place at PIN polar membrane
domains, contributes to the specificity and modulation of auxin efflux rate
(Blakeslee et al., 2007). The
proportion of PGPs that do not colocalize with PINs act multilaterally in
auxin efflux and, thus, regulate the effective cellular auxin concentration
available for PIN-mediated transport. This combined action of PIN and PGP
action determines how much auxin flows through auxin channels. Observation of
a higher cellular auxin concentration in pgp mutants
(Bouchard et al., 2006) that
might enhance PIN-mediated transport directly supports this scenario. However,
establishment of such a specific auxin concentration by enhanced PGP19
expression in cells, where there is no direct PIN1-PGP19 colocalization (such
as basal protoderm and endodermal initials in embryos), additionally focuses
auxin flow. It is likely that for long-distance transport, e.g. in stems,
another mode of PGP and PIN interaction applies, as suggested by strong auxin
transport defects in pgp mutant stems
(Noh et al., 2001;
Geisler et al., 2005).
It is important to note that different internal or external cues, such as
light, can influence the extent and mode of PIN-PGP interactions, for instance
at the level of functional pairing of PINs and PGPs
(Blakeslee et al., 2007) or by
producing distinct effects on either PIN or PGP functions. Moreover, the
activity of previously uncharacterized PGPs may also significantly contribute
to auxin transport. In summary, our model could provide an explanation of the
existence of two auxin transport mechanisms that ensure precise and proper
formation of spatial and temporal auxin distribution in plants.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3345/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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J. Pan, S. Fujioka, J. Peng, J. Chen, G. Li, and R. Chen The E3 Ubiquitin Ligase SCFTIR1/AFB and Membrane Sterols Play Key Roles in Auxin Regulation of Endocytosis, Recycling, and Plasma Membrane Accumulation of the Auxin Efflux Transporter PIN2 in Arabidopsis thaliana PLANT CELL, February 1, 2009; 21(2): 568 - 580. [Abstract] [Full Text] [PDF]
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