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First published online 3 October 2007
doi: 10.1242/dev.009654
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1 Graduate School of Biological Sciences, Nara Institute of Science and
Technology, Nara 630-0101, Japan.
2 Lehrstuhl für Genetik, Technische Universität München,
Wissenschaftszentrum Weihenstephan, Am Hochanger 8, 85350 Freising,
Germany.
Author for correspondence (e-mail:
m-tasaka{at}bs.naist.jp)
Accepted 10 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Polar auxin transport, Organ development, PIN1, PID, NPH3, Arabidopsis thaliana
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Local accumulation of auxin is induced by a directed intercellular transport
system from the site of its biosynthesis, referred to as polar auxin
transport. In this process, auxin efflux carriers play a key role. They are
localized at the plasma membrane with a uniform polarity between cells. This
is supposed to permit directional auxin transport through organs and tissues.
Recently, genetic analyses and studies in heterologous expression systems
indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins
function in mediating auxin efflux from cells. In Arabidopsis,
mutations of PIN family genes cause phenotypes consistent with impaired polar
auxin transport, as pin-formed inflorescences in pin1, reduce tropic
response in pin2 and pin3, and disrupt the apical-basal axis
in pin7 embryos (Okada et al.,
1991; Gälweiler et al.,
1998; Luschnig et al.,
1998; Chen et al.,
1998; Muller et al.,
1998; Friml et al.,
2002; Friml et al.,
2003). Mutations of PGP genes in Arabidopsis, maize and
sorghum result in reductions of growth and polar auxin transport
(Noh et al., 2001;
Noh et al., 2003;
Geisler et al., 2003;
Geisler et al., 2005;
Multani et al., 2003;
Lin and Wang, 2005). When
expressed in yeast and mammalian cell lines, both PIN and PGP proteins
activate the efflux of indole-3-acetic acid, the natural and main auxin, and
an artificial auxin (Petrásek et
al., 2006; Geisler et al.,
2005). PIN-mediated auxin efflux appears to be necessary to
establish and maintain the proper auxin distribution, as there are obvious
defects of development in pin mutants that have not been reported in
pgp mutants.
In the SAM, PIN1 localizes to the plasma membrane of the epidermis and the
vasculature of organ primordia. In the meristem L1 layer, PIN1 localization is
preferentially oriented toward the center of incipient organs
(Reinhardt et al., 2003). In
cotyledon development, PIN1 localization is restricted to the apical side of
the plasma membrane in the protodermal cells, as well as in the SAM
(Steinmann et al., 1999;
Benková et al., 2003;
Treml et al., 2005). These
data indicate that auxin is transported from the site of biosynthesis via the
meristem L1 layer into the SAM or the protodermal cell layer in the embryo.
Auxin is redistributed and accumulated at the sites where it will promote the
initiation of organs. In organogenesis, the mechanism of the regulation of
PIN1 polarity and auxin accumulation at the site where a primordium will
initiate has remained an important question.
Previous studies have shown that auxin itself modulates the subcellular
localization of PIN proteins. PIN proteins constitutively cycle between the
plasma membrane and endosomes (Geldner et
al., 2001; Geldner et al.,
2003). Auxin treatment blocks PIN endocytosis, and promotes PIN
accumulation and activity at the plasma membrane in Arabidopsis roots
(Paciorek et al., 2005). In
pea epicotyls, local auxin accumulation leads to rearrangements in polar
localization of PIN proteins (Sauer et
al., 2006). These data provide a mechanism for the feedback
regulation of auxin transport. By genetic studies, several factors were
identified as regulators of PIN localization. In Arabidopsis,
mutations in GNOM/EMB30, BIG/TIR3/DOC1,
PID, and ENHANCER OF PINOID (ENP) genes disrupt the
localization of PIN protein. The GNOM/EMB30 gene encodes a
GDP-GTP exchange factor for small G-proteins of the ARF class, involved in
coat recruitment and cargo-selective vesicle trafficking
(Shevell et al., 1994). The
gnom/emb30 mutation leads to a disorganized PIN localization
(Steinmann et al., 1999). The
BIG/TIR3/DOC1 gene encodes a calossin-like protein.
In the mutants, treatment with polar auxin transport inhibitors causes
mislocalization of PIN1 protein to an unidentified intracellular compartment
(Gil et al., 2001). The
PID gene encodes a Ser/Thr kinase, involved in polar auxin transport
(Christensen et al., 2000;
Benjamins et al., 2001). In
pid inflorescences, PIN1 polarity is completely reversed in the
meristem L1 layer. Inversely, overexpression of PID induces a reverse
redistribution of PIN proteins. These results suggest that PID
controls PIN polarity (Friml et al.,
2004). ENP is also suggested to control PIN1 polarity in
concert with PID, as the enp mutation, when combined with
the pid mutation, causes a reverse localization of PIN1 protein in
the apex of the embryo resulting in a loss of cotyledon primordia
(Treml et al., 2005). In
addition, blue light-photostimulation has been shown to delocalize PIN1
protein in hypocotyls on the side distal to the light source
(Blakeslee et al., 2004).
Apparently, this phenomenon is mediated by PHOTOTROPIN 1/NON-PHOTOTROPIC
HYPOCOTYL 1 (PHOT1/NPH1), which belongs to the same kinase family as PID,
since PIN1 delocalization was not observed in blue light-treated
phot1/nph1 mutant hypocotyls.
In this paper, we report the identification of the macchi-bou 4
(mab4) mutant, which is defective in organogenesis, as a
pinoid (pid) enhancer mutant. Whereas mab4 and
pid single mutants display minor cotyledon phenotypes, pid
mab4 double mutants completely lack cotyledons. We found that
MAB4 was identical to ENP, which is suggested to control the
PIN1 polarity (Treml et al.,
2005). MAB4/ENP encodes a novel protein, which
belongs to NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family supposed to function as a
signal transducer in phototropic response and regulate lateral translocation
of auxin. MAB4/ENP is expressed in the protodermal cell
layer of the embryo and in the meristem L1 layer at the site of organ
initiation. In mab4-1 embryos, PIN1:GFP (green fluorescent protein)
abundance in the plasma membrane of the protodermal cells was severely
reduced. Moreover, in cultured Arabidopsis cells, MAB4/ENP, existed
in a fraction of endosomes and unidentified intracellular compartments,
partially colocalized with PID. These results demonstrate that
MAB4/ENP functions as a regulator of polar auxin transport
in organogenesis in concert with PID and imply similarities in
molecular mechanisms between organogenesis and phototropism.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
pin1-3 [Lansberg erecta (Ler)]
(Bennett et al., 1995),
pid-15 (Ler) (Treml et
al., 2005), pid-2 (Ler)
(Christensen et al., 2000),
pid-3 (Col) (Bennett et al.,
1995). mab4-1 was isolated from the M2 population of Col
that had been mutagenized by fast neutron exposure (Lehle Seeds, Round Rock,
TX, USA). enp was isolated from the M2 population of Ler
mutagenized by ethyl methanesulfonate
(Treml et al., 2005).
mab4-2 carries a T-DNA insertion at the 5' untranslated region.
This allele was obtained from the Arabidopsis Biological Resource
Center (SALK_104491) (Alonso et al.,
2003) and was backcrossed three times to wild-type Col before any
analysis and construction of pid mab4 double mutants. Plants were
grown on soil as previously described
(Fukaki et al., 1996), and
siliques were collected for analyses of embryo phenotypes and in situ
hybridization. Stages of embryogenesis were as defined previously
(Jürgens and Mayer,
1994). For analysis of seedling phenotypes, seeds were surface
sterilized and germinated on Murashige and Skoog plates, as previously
described (Aida et al.,
1997).
Allelism tests
To examine allelism in MAB4 and ENP, pid-3/+
mab4-1/mab4-1 plants were crossed to pid-15/+
enp/enp. The F1 progeny segregated cotyledon-lacking
seedlings (2 out of 11 seedlings). Genotypes of the PID locus in the
F1 seedlings were confirmed by using PCR primers that detected the
pid-3 and pid-15 mutations.
Mapping and cloning of the MAB4/ENP gene
The mab4-1 mutant was crossed with pid-2 (Ler)
for mapping of the MAB4/ENP locus. Using the 
300
cotyledonless seedlings in F2 progeny, the MAB4 locus was mapped
between the T15N24 CAPS marker and nga1139, a well-known SSLP marker on
chromosome 4. Moreover, the MAB4 locus was mapped to the 315-kb
region between the F3L17 CAPS marker (one recombinant) and the F10M6 CAPS
marker (six recombinants) on chromosome 4 after F2 or F3 analysis of 1400
F2 plants. The primers used for amplification were: T15N24_F
(5'-GATCTGCCCATCATGAGATC-3') and T15N24_R
(5'-CTTGTTCGGTTTCTCGTTGC-3') for T15N24 CAPS marker; F3L17_F
(5'-CTTGGTACCGAAGCCCCGAC-3') and F3L17_R
(5'-GACTGGCGTGATTGACGAAG-3') for F3L17 CAPS markers; F10M6_F
(5'-GGTCTAAAGATCGGCAAAGC-3') and F10M6_R
(5'-TCACCGTTTACGGATTTACG-3') for F10M6 CAPS markers. The PCR
products were digested with EcoRI, HindIII and
DraI, respectively.
The 9.4-kb DNA fragment that included the 6.1-kb upstream region of the
At4g31820 gene and the 0.5-kb downstream region was cloned into the
binary vector pBIN19. The construct was transformed into Agrobacterium
tumefaciens strain MP90 and transformed into the mab4-1 plants
by the floral dip method (Clough and Bent,
1998).
Microscopy
Scanning electron microscopy images were obtained as previously described
(Ishida et al., 2000).
Fluorescence was imaged by confocal laser-scanning microscopy (FV1000;
Olympus, Tokyo, Japan). For confocal microscopy, dissected embryos were
mounted in 7% glucose.
In situ hybridization
In situ hybridization was performed as previously described
(Takada et al., 2001).
Hybridization was performed at 45°C. Templates for transcription of a
MAB4/ENP antisense probe were derived from a PCR-amplified
1098 bp fragment corresponding to a region that spanned amino acids
45-410.
Subcellular localization of MAB4/ENP
MAB4/ENP and PID cDNAs were amplified by RT-PCR
from the Col wild type. The fragment was subcloned under the control of the
cauliflower mosaic virus 35S promoter and the Nos terminator. GFP (S65T) was
translationally fused to both ends of the MAB4/ENP protein and the N terminus
of PID protein with a triple glycine linker. Monomeric red fluorescent protein
(mRFP) (Campbell et al., 2002)
was also fused in-frame to the N terminus of MAB4/ENP with a triple glycine
linker. 35S::ARA6-mRFP (Ueda et al.,
2004), 35S::mRFP-ARA7 (Ueda et
al., 2004), 35S::Venus-SYP31
(Nagai et al., 2002;
Uemura et al., 2004) and
35S::Venus-SYP41 (Uemura et al.,
2004) were used as intracellular markers of late endosome, early
endosome, cis-Golgi and trans-Golgi network (TGN), respectively.
Co-introduction and double transient expression of XFP-tagged MAB4/ENP,
GFP-PID, PIN1:GFP-2 (Wisniewska et al.,
2006) and intracellular markers in the protoplasts of cultured
Arabidopsis cells were performed as previously described
(Takeuchi et al., 2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Benjamins et al.,
2001; Furutani et al.,
2004). However, its contribution to cotyledon formation is partial
as pid-3 mutants displayed milder defects of cotyledon development
than pin1-201 pid-3 double mutants, which lack cotyledons
(Fig. 1D)
(Furutani et al., 2004). This
indicates that two pathways, dependent on PIN1 and PID,
might function in cotyledon formation. To identify factors in the pathways, a
screening for pid enhancers was carried out. The screening focused on
the mutant loci displaying severe defects of cotyledon development when
combined with the pid mutation. We identified a pid
enhancer, named macchi-bou 4 (mab4) in a screen of fast
neutron-mutagenized lines. Seedlings of mab4-1 single mutants
exhibited aberrant cotyledon number and cotyledon fusion at a very low
frequency (Fig. 1C;
Table 1). The mab4-1
mutation also caused defects in postembryonic organogenesis. Whereas lateral
organ formation was normal in the inflorescence meristem in mab4-1
mutants (Fig. 1G,I,M,N),
aberrant initiation of floral organs such as sepals, petals and stamens, and
fusion of them within the same whole or sometimes different whorls were
observed in the mutant flowers (Fig.
1M,N,Q; Table 2).
In addition, they displayed short valves and decreased valve numbers
(Fig. 1O,P;
Table 2). When combined with
the pid-3 mutation, the mab4-1 mutation caused severe
defects of organogenesis. The seedlings of double mutants completely lacked
cotyledons (Fig. 1E). Like
pin1-201 pid-3 double mutants, the pid-3 mab4-1 mutants
could not produce any organs in flower meristems
(Fig. 1J,K), whereas
pid-3 single mutants were able to form floral organs, albeit to a
lesser extent than the wild type (Fig.
1H). Recently, it was reported that a combination of mutations in
the PID and ENP genes also caused severe defects of
organogenesis (Fig. 1F,L)
(Treml et al., 2005). These
data demonstrated the possibility that MAB4 is identical to
ENP. A series of crosses revealed that the mab4-1 mutation
could not complement the enp mutation, indicating that mab4
is allelic to enp. These results showed that
MAB4/ENP is involved in organogenesis synergistically with
PID.
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) (Fig.
2A). mab4-2 mutants displayed the same mild defects in
cotyledon development and floral organ development and to the same extent as
the mab4-1 allele, whereas the enp allele was less potent
(Tables 1,
2). When mab4-2 was
coupled with a pid-3 mutation, cotyledons and lateral organs were
completely absent, nearly identical to pid-3 mab4-1 and pid-15
enp (data not shown). These results showed that
mab4/enp phenotypes are caused by a loss of function of the
At4g31820 gene and that both mab4-1 and mab4-2
represent stronger alleles than enp.
The structure of MAB4/ENP
MAB4/ENP encodes a member of the NPH3-family proteins,
composed of 571 amino acid residues with a molecular mass of 66.4 kDa
(Fig. 2B). The NPH3 family
consists of 31 members in Arabidopsis
(Fig. 2C)
(Kimura and Kagawa, 2006).
Among NPH3-family genes, five genes (MAB4/ENP, At5g67440,
At4g37590, At2g14820, At2g23050) show high similarity to one another and
are less similar to Arabidopsis NONPHOTOTROPIC HYPOCOTYL 3
(NPH3), ROOT PHOTOTROPISM 2 (RPT2), which had been
identified previously in Arabidopsis, as their mutations display
defects of phototropic response (Fig.
2B,C) (Motchoulski and Liscum,
1999; Sakai et al.,
2000). The rice genome consists of at least 26 members of the NPH3
family and two members (Os06g0184500, Os09g0420900) exhibit high similarity to
MAB4/ENP and are less similar to COLEOPTILE PHOTOTROPISM 1 (CPT1), the rice
NPH3 ortholog, the mutation of which caused a lack of phototropic response in
the coleoptile (Fig. 2C)
(Haga et al., 2005).
Whereas NPH3, RPT2 and CPT1 contain a BTB/POZ (broad complex, tramtrack,
and bric à brac/pox virus and zinc finger) domain at the N-terminal
region and a coiled-coil domain at the C terminus, MAB4/ENP contains a BTB/POZ
domain at the N-terminal region, but no distinct coiled-coil domain. The
BTB/POZ domain has been found in a large number of proteins and is known as a
protein-protein interaction motif (Stogios
et al., 2005). In addition, in mab4-1, three amino acid
residues, G L and Y, from 408 to 410 were deleted
(Fig. 2B). Of the three amino
acids, tyrosine is a highly conserved amino acid among the NPH3-family
proteins. In case of NPH3, the strong nph3-2 allele carries a
deletion of this conserved tyrosine residue
(Motchoulski and Liscum,
1999). These data indicate that the tyrosine is important for the
molecular function of MAB4/ENP and for that of other family members.
The MAB4/ENP gene has an auxin responsive element
(AuxRE), TGTCTC (Ballas et al.,
1993; Ulmasov et al.,
1999), at positions -228 to -223 from the initiation codon and a
WUSCHEL (WUS)-binding site, TTAATGG
(Lohmann et al., 2001), at
position -201 to -195.
MAB4/ENP mRNA expression
In order to investigate the temporal and spatial expression pattern of the
MAB4/ENP gene in wild-type development, in situ
hybridization analyses were performed. In wild-type embryos,
MAB4/ENP expression was detected uniformly in the embryo at
the 8-cell stage (Fig. 3A).
Differential expression of the MAB4/ENP gene in the embryo
began at the 32-cell stage (Fig.
3B). Detection of MAB4/ENP mRNA continued in the
protodermal cells but decreased in the inner cells of the embryo. At this
stage, the hypophysis is formed at the junction between the embryo proper and
the suspensor, continuous with the protodermal cells surrounding the inner
cells. However, MAB4/ENP was not expressed in the
hypophysis. The differential localization of MAB4/ENP mRNA
became more obvious in globular stage embryos
(Fig. 3C). As cotyledon
primordia develop, MAB4/ENP mRNA in the protodermal cell
layer was gradually restricted to the tip of cotyledon primordia and radicles
(Fig. 3D,E). In addition,
MAB4/ENP was strongly expressed in several inner cells at
the tips of the cotyledon primordia (Fig.
3E). From the late-heart stage, MAB4/ENP began
to be expressed in the presumptive shoot apical meristem (SAM), and was
strongest in the protodermal layer (Fig.
3E,F).
In the postembryonic stage, MAB4/ENP mRNA was detectable in the organ primordia and the SAM. MAB4/ENP was strongly expressed in the lateral regions of young leaf primordia and weakly in vascular tissue (Fig. 3G). In inflorescence meristems, the MAB4/ENP mRNA signal was detected in the meristem L1 layer at the site of flower initiation (Fig. 3H). As flower primordia initiated, MAB4/ENP expression was induced in the inner cell layers (Fig. 3H,I). In young flower primordia, MAB4/ENP expression was elevated at the site of sepal induction, and later where the inner floral whorl organs developed (Fig. 3J,K). During the course of ovule development, MAB4/ENP mRNA was found at the tips of the nucellus and the outer integuments and expression was weaker in the inner integuments (Fig. 3L).
MAB4/ENP expression in pin1 pid embryos
Expression analysis of MAB4/ENP reveals that
MAB4/ENP expression is induced at the site of incipient
organ initiation and that in developing organs it is restricted to the tips of
organ primordia, where auxin maxima are established. To determine whether the
MAB4/ENP expression pattern depends on auxin distribution,
we examined the expression of MAB4/ENP in pin1-3
pid-2 embryos developing in siliques of pin1-3/+
pid-2/pid-2 plants. In these embryos an auxin maximum is not
established in the apex. Until the heart stage, we could not detect any
defects of MAB4/ENP expression (data not shown). At the
heart stage, when the double mutants were confirmed by distinct phenotypes
that cotyledon primordia lacked, MAB4/ENP mRNA was usually
found in the protodermal cell layer of mutant embryos
(Fig. 4A). Later,
MAB4/ENP was expressed in the protodermal cell layer of the
presumptive SAM (Fig. 4D), as
in wild-type embryos. However, MAB4/ENP transcripts were not
detectable in inner cells of the double-mutant embryos, whereas in wild type
MAB4/ENP is normally expressed in inner cells of tips of
cotyledon primordia (compare Fig.
3E and Fig. 4B).
Even if rudimentary primordia were occasionally developed in the apex of the
double mutants, we could not detect MAB4/ENP transcripts in
the inner cells at the tips of these rudimentary primordia
(Fig. 4C,D). These results
suggest that PIN1 and PID are not essential for the
expression of MAB4/ENP in the protodermal layer. However,
the expression of MAB4/ENP transcripts in inner cells of
cotyledon primordia depend on the activity of PIN1 and PID,
possibly through the auxin distribution established by these genes in the apex
of embryos.
|
). We
analyzed the localization of PIN1 and auxin distribution in mutant embryos. We
used the stronger pid and mab4/enp alleles,
pid-3 and mab4-1, instead of the previously used alleles
pid-15 and enp (Treml et
al., 2005).
|
) in embryos of the pid-3 and mab4-1 single
mutant. In wild-type embryos, PIN1:GFP was expressed in the protodermal cells
of cotyledon primordia and provascular cells, and was localized on the apical
side of cells in the protodermal cell layer of the apex
(Fig. 5A,C).
DR5rev::GFP was found to be present in the tips of cotyledon
primordia (Fig. 5E,G). As
compared with wild-type embryos, we found the same defects of PIN1:GFP and
DR5rev::GFP expression in embryos homozygous for pid-3
similar to those previously reported for pid-15 embryos
(Treml et al., 2005).
pid-3 embryos frequently combined the apical and basal localization
of PIN1 in the protodermal cell layer of cotyledon primordia (data not shown).
The DR5rev::GFP signal was often missing in the apex of
pid-3 embryos (data not shown). Interestingly, although no defects in
PIN1 localization, per se, were detectable in provascular tissues of
mab4-1 embryos, the abundance of PIN1 signal was severely reduced in
the protodermal cells. The defects of PIN1 localization in mab4-1
embryos were detectable from the heart stage. At the heart stage, PIN1
expression was reduced especially on the abaxial side of cotyledon primordia
of mab4-1 mutants (Fig.
5B). At later stages, PIN1:GFP expression in the tips of cotyledon
primordia was severely reduced in mab4-1 embryos
(Fig. 5D) compared to the wild
type. This effect of the single mab4-1 mutation has not been reported
in the previous analysis of ENP. By contrast, no defects of
DR5rev::GFP expression could be found. In mab4-1 embryos,
expression of DR5rev::GFP was normally detectable in the tips of
cotyledon primordia from the heart stage, as in wild-type embryos
(Fig. 5E-H).
To examine the combined effects of pid and
mab4/enp mutations on polar auxin transport, we carried out
expression analyses of PIN1:GFP and DR5rev::GFP in pid-3
mab4-1 embryos. The same results were obtained as previously described in
pid-15 enp embryos (Treml et al.,
2005). In the apex of pid-3 mab4-1 embryos, PIN1 polarity
was shifted to the lateral and basal side of the plasma membrane in the
protodermal layer and no DR5rev::GFP expression was detectable (data
not shown). Taken together, these results demonstrate that
MAB4/ENP regulates PIN1 localization in the protodermal cell
layer and is involved in the establishment of auxin maxima in the apex of the
embryo in concert with PID.
Subcellular localization of PIN1, PID and MAB4/ENP
Synergistic interactions between MAB4/ENP and
PID, detected by the genetic analysis, and the control of PIN1
abundance on the plasma membrane by MAB4/ENP suggest a
distinct subcellular distribution of MAB4/ENP and PIN1 or PID. To test this
possibility, we confirmed the subcellular distribution of PIN1, PID and
MAB4/ENP. First, the subcellular localization of GFP-tagged PIN1 was analyzed
in transfected Arabidopsis protoplasts by confocal laser scanning
microscopy. GFP-tagged PIN1 was transiently expressed under the control of its
own promoter. The functionality of the construct was verified by
complementation of the pin1 mutant
(Wisniewska et al., 2006). The
fluorescence of GFP-tagged PIN1 protein was located in the plasma membrane and
distributed in a dot-like pattern throughout the cell
(Fig. 6A). Previously, it was
shown that PIN proteins cycle between the plasma membrane and endosomes
(Geldner et al., 2001;
Geldner et al., 2003). To
confirm whether PIN cycling is reflected in cultured Arabidopsis
cells, Venus- and mRFP-tagged subcellular marker genes under the control of
the CaMV 35S promoter were co-introduced with GFP-tagged PIN1, and their
subcellular locations were examined. As expected, the fluorescence of
GFP-tagged PIN1 overlapped, in part, with the late-endosomal marker ARA6-mRFP
(see Fig. S1A in the supplementary material) and the early-endosomal marker
mRFP-ARA7 (see Fig. S1B in the supplementary material), but not with the
cis-Golgi apparatus marker Venus-SYP31 (see Fig. S1C in the supplementary
material) or the trans-Golgi network (TGN) marker Venus-SYP41 (see Fig. S1D in
the supplementary material). These results indicate that PIN1 is localized in
the plasma membrane and endosomes in cultured Arabidopsis cells as
well as in whole plants. To examine the colocalization of MAB4/ENP with PIN1,
GFP-tagged PIN1 was co-introduced with mRFP-tagged MAB4/ENP under the control
of the CaMV 35S promoter and their subcellular locations were analyzed. The
fluorescence of mRFP-tagged MAB4/ENP protein was distributed in a dot-like
pattern throughout the cell. A fraction of the mRFP-MAB4/ENP fluorescence was
very close to some intracellular fluorescence from PIN1-GFP, but the two did
not merge (Fig. 6B). These
results showed that MAB4/ENP is not colocalized with PIN1 in cultured
Arabidopsis cells.
|
|
). To
examine the subcellular colocalization of MAB4/ENP with PID, GFP-tagged PID
was co-introduced with mRFP-tagged MAB4/ENP and their subcellular
localizations were analyzed. A fraction of mRFP-MAB4/ENP florescence was
colocalized with a fraction of the GFP-PID intracellular florescence
(Fig. 6D). These results
indicate that MAB4/ENP is in part colocalized with PID in the intracellular
compartments. To identify the intracellular compartment where MAB4/ENP and PID coexist, the subcellular localization of MAB4/ENP and PID was analyzed in detail by using subcellular marker genes. Venus- and mRFP-tagged subcellular marker genes were co-introduced with GFP-tagged MAB4/ENP and PID and their respective subcellular locations were examined. The fluorescence of GFP-tagged MAB4/ENP did not overlap with that of mRFP-ARA7 (Fig. 7B), Venus-SYP31 (Fig. 7C) or Venus-SYP41 (Fig. 7D). A fraction of MAB4/ENP-GFP fluorescence merged with that of the late-endosome marker ARA6-mRFP (Fig. 7A). At the same time, the fluorescence of GFP-tagged PID did not overlap with mRFP-ARA6 (see Fig. S2A in the supplementary material) and Venus-SYP31, but occasionally existed in very close proximity to Venus-SYP31 (see Fig. S2C in the supplementary material). A fraction of GFP-PID florescence merged with that of ARA7-mRFP (see Fig. S2B in the supplementary material) and SYP41 (see Fig. S2D in the supplementary material). These results indicate that MAB4/ENP is localized to a subpopulation of late endosomes as well as in unidentified organelles and that PID localizes to the plasma membrane and to a subpopulation of endosomes and the TGN. Furthermore, MAB4/ENP and PID may be colocalized in unidentified intracellular compartments.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Among 31
members of the Arabidopsis NPH3 family, NPH3 and RPT2 have been
identified as signal transducers involved in phototropism. The nph3
mutant has a defect in phototropism of hypocotyls and roots
(Liscum and Briggs, 1996). The
rpt2 mutant has defects in phototropism of roots, and blue
light-induced stomatal opening (Sakai et
al., 2000; Inada et al.,
2004). Mutation of rice CPT1, an ortholog of
NPH3, results in a lack of coleoptile phototropism and severely
reduced root phototropism (Haga et al.,
2005). In the cpt1 coleoptiles, asymmetrical auxin
distribution was not established in response to blue light, suggesting that
CPT1 regulates auxin transport in phototropism. These data imply that
a part of the NPH3 gene family might control auxin transport. Furthermore, the
difference in the subfamily, to which NPH3, RPT2 and
MAB4/ENP belong, might reflect their particular roles in
development. In the Arabidopsis genome, there are at least four MAB4/ENP-like genes (At2g23050, At4g37590, At5g67440, At2g14820). As mab4/enp single mutants displayed mild defects in organogenesis, it was postulated that these genes function redundantly with MAB4/ENP in organogenesis. T-DNA insertion lines disrupting these loci did not result in any phenotypes in organogenesis (T.K., B.T., R.A.T.-R. and M.T., unpublished). We are currently constructing multiple mutants between these loci and MAB4/ENP.
|
|
; Hadfi et al.,
1998; Nemhauser et al.,
2000), suggesting that MAB4/ENP is involved in
the action of auxin in organogenesis. In the protodermal cell layer of the
early embryo and the meristem L1 cell layers at the site of organ initiation,
where MAB4/ENP is expressed, PIN1 is localized to the plasma
membrane in a polar fashion. The layers are suggested to be an auxin route to
the tip of organ primordia, where auxin maxima are established. These data
strongly suggest a correlation between MAB4/ENP function and
auxin distribution. Indeed, in pid-15 enp embryos PIN1 polarity was
completely reversed in the protodermal cell layer and auxin maxima were not
established in the apex (Treml et al.,
2005). The mab4-1 mutation caused a reduction of PIN1
abundance in the plasma membrane of the protodermal cells of cotyledon
primordia, although DR5rev::GFP was normally expressed in the tips of
cotyledon primordia (Fig. 5).
We speculate that the normal expression of DR5rev::GFP in the mutant
is due to other members of the PIN family redundantly functioning to establish
auxin distribution. We also suggest that the DR5rev::GFP system could
not detect subtle defects of auxin distribution in the mutant.
In the apex of pin1 pid embryos, MAB4/ENP mRNA
was not detected in the inner cell layers, whereas it was normally expressed
in the protodermal cell layer (Fig.
4). It is possible that MAB4/ENP expression
depends upon the auxin distribution in the inner cells but not in the
protodermal cells. In this context, it is interesting to note that the
MAB4/ENP gene contains an auxin responsive sequence, AuxRE
(Fig. 2)
(Ballas et al., 1993;
Ulmasov et al., 1999).
MAB4/ENP might therefore be able to respond to auxin in the
inner cells. However, expression of the MAB4/ENP gene in the
protodermal cell layer during early embryogenesis is very similar to that of
Arabidopsis thaliana MERISTEM LAYER 1 (ATML1)
(Lu et al., 1996). Recently,
it was revealed that a small fragment containing known binding sites for
homeodomain transcription factors, the ATML1-binding L1 box
(Abe et al., 2001) and
WUS-binding site (Lohmann et al.,
2001), was responsible for the ATML1 expression pattern
(Takada and Jürgens,
2007). Although the MAB4/ENP gene has no L1 box,
it contains the WUS-binding site (Fig.
2). These data suggest a regulatory mechanism through the
WUS-binding site, resulting in a similar expression pattern to ATML1
in early embryogenesis.
MAB4/ENP regulates localization of PIN1 protein
MAB4/ENP could control the activity of PIN1 in
organogenesis. This idea is supported by the following important results. (1)
The mab4/enp mutation enhanced phenotypes of pid
mutants in the same way as does the pin1 mutation. (2) In
mab4-1 embryos, PIN1 abundance in the plasma membrane was severely
reduced. (3) In pid-15 enp embryos, PIN1 polarity was completely
reversed (Treml et al., 2005).
(4) The MAB4/ENP expression domain overlaps with PIN1
(Reinhardt et al., 2003;
Benková et al., 2003;
Heisler et al., 2005). What is
the action mechanism of MAB4/ENP for the activity of PIN1?
Considering that MAB4/ENP is localized in a subpopulation of endosomes and
unidentified intracellular compartments, we suggest that
MAB4/ENP is involved in trafficking of PIN1 protein to the
plasma membrane. In addition, in cultured Arabidopsis cells, MAB4/ENP
partially colocalizes with PID, but not with PIN1, suggesting that MAB4/ENP
does not modulate PIN1 trafficking in its vicinity, but functions distantly
together with PID. Recently, PID protein kinase has been shown to regulate
polar auxin transport through the control of PIN localization.
Loss-of-function mutants of PID display an apical-to-basal shift in
PIN1 polarity at the inflorescence apex. Conversely, ectopic expression of
PID induces a basal-to-apical shift of PIN polarity, suggesting that
PID functions as a switch that regulates PIN localization
(Friml et al., 2004). Lee and
Cho (Lee and Cho, 2006) also
showed that PID positively regulates auxin efflux through
acceleration of PIN trafficking to the plasma membrane using the
auxin-sensitive root hair cell system. Taking into consideration the
colocalization of PID with PIN1 in the plasma membrane (see Fig. S3 in the
supplementary material), we speculate that PID modulates PIN1 trafficking to
the plasma membrane and that MAB4/ENP mediates
PID-dependent modulation of PIN1 trafficking in the intracellular
compartment. In this study the subcellular localization analyses were
performed in cultured Arabidopsis cells that lack polarity, and
additional analyses are necessary in plant cells with polarity in order to
uncover the functional interaction between MAB4/ENP, PIN1 and PID.
An analogy between organogenesis and phototropism
Based on the structural similarities of the components involved in
organogenesis and phototropism, we conclude that both processes share common
molecular principles. MAB4/ENP is a member of the NPH3 family. PID, a key
regulator of polar auxin transport in organogenesis, is a member of the AGC
kinase family (Bögre et al.,
2003). PHOTOTROPIN 1/NON-PHOTOTROPIC HYPOCOTYL 1 (PHOT1/NPH1),
which functions as a blue light receptor
(Huala et al., 1997), also
belongs to the same AGC Vlll subfamily. PHOT1/NPH1 is a plasma
membrane-associated protein and forms a complex with NPH3 and RPT2 supposed to
function as the signalosome in phototropism
(Motchoulski and Liscum, 1999;
Inada et al., 2004). Finally,
both processes use PIN proteins to transport auxin such that it is
asymmetrically distributed to lateral domains.
In phototropism, asymmetric distribution of auxin is established in a
lateral direction on light stimulation
(Friml et al., 2002). In this
process, PIN-dependent polar auxin transport is suggested to play a
significant role. Mutations in the PIN3 gene alter differential
growth in response to light stimulation
(Friml et al., 2002).
Furthermore, upon blue light stimulation PIN1 proteins are asymmetrically
localized across Arabidopsis hypocotyls. This response, which is then
followed by tropic bending, is apparently controlled by PHOT1/NPH1 since the
phot1/nph1 mutation blocks the PIN1 delocalization in
light-treated plants (Blakeslee et al.,
2004). Likewise, mutation of the rice CPT1 gene, an
ortholog of NPH3, caused defects in lateral translocation of auxin
(Haga et al., 2005). These
data suggest that the PHOT1/NPH1-NPH3 signaling complex is involved in the
phototropic lateral redistribution of auxin through the control of PIN
localization.
The organogenesis also requires the asymmetric distribution of auxin. In
cotyledon development, two auxin maxima are established opposite to each other
at lateral positions in the apex of the embryo
(Friml et al., 2003;
Treml et al., 2005). At the
globular stage, when cotyledon primordia start to develop, PID
transcripts are detectable in two domains, each encompassing approximately
three-quarters of the embryo (Furutani et
al., 2004), while MAB4/ENP is expressed in the
protodermal cell layers in the embryo proper
(Fig. 3). As the cotyledon
primordia develop, PID transcripts accumulate mainly at the
boundaries of these primordia and slightly in the regions surrounding their
base (Furutani et al., 2004)
while the expression domain of MAB4/ENP is restricted to the
tips of the cotyledon primordia (Fig.
3). A comparable expression pattern of MAB4/ENP
and PID is visible at the site of floral anlagen and floral organ
initiation (Fig. 3)
(Christensen et al., 2000).
Taken together, the expression domains of the PID and
MAB4/ENP genes partially and temporarily overlap during
embryogenesis. Both genes coordinately control the polar localization of PIN1,
which in turn directs the correct formation of auxin maxima at the tips of
growing cotyledon primordia (this study)
(Treml et al., 2005).
The molecular interaction of PID, MAB4/ENP and PIN1 is less clear. We could show, that the fluorescent protein-tagged PID and MAB4/ENP are partially colocalized in some intracellular compartments when co-introduced in cultured Arabidopsis cells (Fig. 6). These data support the possibility of an interaction between PID and MAB4/ENP. However, when we used the yeast two-hybrid method to confirm a direct interaction between PID and MAB4/ENP, we could not detect an interaction between them (M.F. and M.T., unpublished). Nevertheless, it is possible that another factor could function as an adaptor protein between them to form the complex. Alternatively, the yeast two-hybrid system might be unsuitable for analyses of membrane-associated proteins.
Certainly, significant differences in the detailed molecular interactions
of both processes exist as well. This can be inferred, for instance, from the
structures of the participating molecules. PID has no LOV (light,
oxygen or voltage) domains required for the activation
of PHOT1/NPH1 (Briggs and Christie,
2002). MAB4/ENP has no apparent coiled-coil domains, which have
been shown to be important for the direct interaction between PHOT1/NPH1 and
NPH3 (Motchoulski and Liscum,
1999). It should also be considered that both processes take place
at different developmental stages and in different cells and tissues. Both
will also require quite different, as yet unidentified, molecules. However,
the use of similar key components demonstrates an astonishing economy in
plants to solve two disparate biological problems. In the end, one major
difference is the output of laterally increased auxin concentrations, as one
leads to a cotyledon primordium, whereas the other to a tropic bending towards
light.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/21/3849/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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