1 Graduate School of Biological Sciences, Nara Institute of Science and
Technology, Nara 630-0101, Japan
2 INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 Versailles
Cedex, France
Author for correspondence (e-mail:
m-aida{at}bs.naist.jp)
Accepted 20 July 2004
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SUMMARY |
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Key words: Embryogenesis, Cotyledon development, CUC, PIN1, PID, Auxin, Arabidopsis thaliana
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Introduction |
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Previous studies have indicated that auxin is involved in various
patterning processes, including apical patterning during embryogenesis. Auxin
displays asymmetric distribution that changes dynamically throughout early
embryogenesis and polar auxin transport is important for this distribution
(Sabatini et al., 1999;
Friml et al., 2002
;
Benková et al., 2003
;
Friml et al., 2003
). Treatment
of embryos with exogenous auxin or polar transport inhibitors causes variable
defects in the apical pattern formation, including abnormal positioning or
fusion of cotyledons (Liu et al.,
1993
; Hadfi et al.,
1998
; Friml et al.,
2003
). These results suggest that proper auxin distribution is
important for the symmetrical positioning of cotyledon primordia and the
establishment of cotyledon boundaries.
Genetic studies support the role of auxin in the above-mentioned processes.
In Arabidopsis, mutations in the PIN-FORMED1
(PIN1), MONOPTEROS (MP) and PINOID
(PID) genes disrupt the patterning of cotyledons
(Okada et al., 1991;
Berleth and Jürgens, 1993
;
Bennett et al., 1995
). These
mutants are also defective in lateral organ formation from the postembryonic
shoot meristem, indicating their significant role in lateral shoot organ
development. The PIN1 gene encodes a member of the putative auxin
efflux regulator proteins that promote polar auxin transport
(Gälweiler et al., 1998
)
and is suggested to promote organ formation by regulating auxin distribution
(Benková et al., 2003
;
Reinhardt et al., 2003
). The
MP gene encodes a transcriptional activator that binds in vitro to an
auxin-responsive cis-element and is suggested to promote primordium formation
by mediating auxin-induced transcriptional activation
(Ulmasov et al., 1997a
;
Hardtke and Berleth, 1998
).
The PID gene encodes a serine/threonine kinase, the transcription of
which is induced by exogenous auxin
(Christensen et al., 2000
;
Benjamins et al., 2001
).
Similar to the pin1 mutant, the pid mutant displays a
reduction of polar auxin transport in the stem
(Bennett et al., 1995
).
Moreover, plants overexpressing PID exhibit reduced root growth and
this phenotype is suppressed by treatment with polar auxin transport
inhibitors. These results suggest that PID functions as a positive
regulator of polar auxin transport
(Benjamins et al., 2001
).
The expression of CUP-SHAPED COTYLEDON1 (CUC1) and its
functionally redundant homolog, CUC2, has been analyzed in
pin1 and mp mutant embryos
(Aida et al., 2002). These
genes encode transcription factors of the NAC family, promote cotyledon
separation at the boundaries and cause cotyledon fusion when both of them are
mutated. Although these two genes are normally expressed in a stripe between
cotyledon primordia, CUC1 expression is expanded to the periphery of
the apical region and CUC2 expression is restricted to the center.
The effects of pin1 or mp mutations on CUC1 and
CUC2 expression are well correlated with the fusion phenotype of
pin1 or mp mutants as well as their double mutant
combinations with cuc1 or cuc2. These results suggest that
PIN1 and MP regulate boundary formation by regulating the
CUC1 and CUC2 genes. The SHOOT MERISTEMLESS
(STM) gene, a kn1-type homeobox gene required for SAM
formation and maintenance (Barton and
Poethig, 1993
; Clark et al.,
1996
; Endrizzi et al.,
1996
; Long et al.,
1996
), also participates in promoting cotyledon separation and its
contribution is particularly prominent in the pin1 mutant background
(Aida et al., 2002
).
To further investigate the molecular relationship between auxin and apical pattern formation in the Arabidopsis embryo, we examined the functions of PIN1 and PID genes in this process. We found that pin1 and pid mutations, when combined in the double mutant, lead to a striking seedling phenotype that is represented by a radially symmetric shape without any cotyledons. This phenotype is associated with the prolonged expansion of CUC1, CUC2 and STM expression domains to the periphery of the embryo apex, and contrasts with the mild and transient changes in CUC1 and CUC2 expression observed in the pin1 or pid single mutant. Triple and quadruple mutant analysis indicates that the ectopic expression of CUC1, CUC2 and STM genes in the pin1 pid double mutant is mainly responsible for the growth inhibition of cotyledon primordia. Our results thus demonstrate that the overlapping function of PIN1 and PID is largely responsible for the establishment of bilateral symmetry and cotyledon outgrowth, and that the latter process involves the negative regulation of boundary-specific downstream effectors, the CUC and STM genes.
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Materials and methods |
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Construction of double, triple and quadruple mutants
For the construction of the pin1-3 pid-2 double mutant, plants
heterozygous for pin1-3 were crossed with pid-2 homozygotes.
Among the F2 population, plants homozygous for pid-2 and heterozygous
for pin1-3 were selected by using PCR primers that detected the
mutations and self-fertilized. Among F3 populations, double mutants were
selected by PCR-based genotyping or the presence of the novel specific
phenotype (see Results). pin1-6 pid-7.1.2.6 and pin1-201
pid-3 double mutants also displayed the same phenotype. For the
construction of the pin1 pid cuc1, pin1 pid cuc2 or pin1 pid
stm triple mutants, plants heterozygous for pin1-3 and
homozygous for pid-2 were crossed with cuc1-1 homozygous,
cuc2 homozygous or stm-1 heterozygous plants, respectively.
Among the F2 populations, plants homozygous for cuc1-1 or
cuc2, or heterozygous for stm-1 were selected by PCR-based
genotyping. These plants were further selected for the heterozygous
pin1-3 and homozygous pid-2 mutation by PCR, and for the
pin-shaped inflorescence phenotype (Bennett
et al., 1995). Phenotypes of the triple mutants were examined in
the F3 populations and their genotypes were confirmed by PCR. The STM
and PIN1 loci were located on chromosome 1 and closely linked. The
occurrence frequency of the novel phenotype was 2.4% (n=127), which
was nearly identical to that of the pin1 stm double mutant, as
previously described (Aida et al.,
2002
). For the construction of the pin1 pid cuc1 cuc2
quadruple mutant, plants heterozygous for pin1-3 and homozygous for
pid-2 and cuc1-1 were crossed with plants heterozygous for
pin1-3 and homozygous for pid-2 and cuc2. Among the
F2 populations, plants heterozygous for both pin1-3 and
cuc1-1, and homozygous for cuc2 were selected. Seedling
phenotypes of the quadruple mutants were examined in the F3 populations.
Microscopy
For visualization of vasculature, seedlings were cleared as previously
described (Aida et al., 1997).
Scanning electron microscopy images were obtained as described previously
(Aida et al., 1999
).
In situ hybridization
In situ hybridization was performed as previously described
(Aida et al., 2002).
Hybridization was performed at 45°C. Templates for transcription of a
PID antisense probe were derived from a PCR-amplified 1122 bp
fragment corresponding to a region that spanned amino acids 44-417. Probes for
the following genes have been reported previously: ANT
(Long and Barton, 1998
),
CUC1 (Takada et al.,
2001
), CUC2 (Aida et
al., 1999
), FIL (Sawa
et al., 1999
) and STM
(Long et al., 1996
). As
controls, we confirmed the expression patterns of FIL, PID, CUC1 and
CUC2 genes in wild type. For any of these probes, we detected
aberrant expression patterns (expanded or reduced) in fewer than 5% of the
embryos (three out of 88 for FIL; four of 104 for PID; six
of 113 for CUC1; and six of 132 for CUC2).
ß-Glucuronidase (GUS) GUS staining
To detect GUS activity, embryos were stained with a solution described
previously (Takada et al.,
2001) at 37°C for 45 minutes. Stained embryos were dehydrated
in a graded ethanol series (30, 50, 70, 90 and 100%) for 15 minutes each.
Rehydration in a graded ethanol series (90, 70, 50, and 30%) was performed for
15 minutes each before observation.
Auxin treatment
Plants were grown under constant white light exposure until several
siliques started to develop (3 weeks). All developing siliques were cut
off before auxin treatment. The plants were subsequently sprayed with a heavy
mist of 10 µM 2,4-dichlorophenoxy-acetic acid with 0.01% Silwet L-77. Mock
treatments were performed with distilled water containing 0.01% Silwet L-77.
Auxin treatments were repeated once a week for 1 month. Seeds were collected
and germinated on plates for phenotypic analysis. For DR5::GUS
analysis, embryos were dissected from siliques 7 days after the treatment and
stained with GUS staining solution.
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Results |
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|
|
We analyzed the expression of these marker genes in pin1-3/+ or
pid-2/+ siliques because pin1-3 homozygous mutants were
completely sterile and the fertility of pid-2 homozygous mutants was
significantly low. ANT was expressed in a ring in all the embryos
examined in pin1-3/+ and pid-2/+ siliques (data not shown),
indicating that neither homozygous mutation affected the ANT
expression pattern. By contrast, FIL expression was disturbed in both
pin1-3 and pid-2 embryos
(Fig. 1I,J). In 26% of the
embryos in pin1-3/+ siliques (13 of 49), FIL was expressed
in a ring that surrounded the apex of the embryos
(Fig. 1I) or in an incomplete
ring with a breakage at one side. FIL expression in a ring was
detected also at the late heart stage (data not shown). In
21% (13 out of
62) of the embryos in pid-2/+ siliques, FIL expression was
asymmetric so that the strength and size of the signals differed between the
two domains of expression (Fig.
1J). Taken together, the radial expression pattern of ANT
is preserved, whereas the bilateral expression pattern of FIL is
disturbed in pin1 and pid embryos. In particular, the
pin1 mutation severely disrupts FIL expression, resulting in
a radially symmetric pattern.
Phenotype of pin1 pid double mutant
To examine the genetic interaction between PIN1 and PID
in cotyledon development, we constructed the pin1 pid double mutant.
Seedlings with the most severe phenotype completely lacked cotyledons,
displaying radial symmetry (Fig.
2A,D; Table 2).
Seedlings with the mild phenotype developed small bulges that were most likely
rudimentary cotyledons. The epidermal cells of these bulges were small and
round compared with those of wild-type cotyledons
(Fig. 2B,E;
Table 2). Seedlings that
displayed the weak phenotype produced small flat structures with a ridge along
the margin of the adaxial side (Fig.
2C; Table 2). All
pin1 pid seedlings developed a functional SAM that produced leaf
primordia, although these primordia displayed abnormal phyllotaxis and were
often fused with each other (Fig.
2D, arrowheads). Mutant SAMs continued to produce leaves and
eventually developed pin-like inflorescences similar to those of pin1
or pid single mutant. These phenotypes were observed in three
different combinations of pin1 and pid alleles, including
putative null mutants (Table 2;
see Materials and methods). Because the observed genetic interaction was
allele-nonspecific, we conclude that PIN1 and PID
redundantly promote cotyledon development, but are not essential for SAM
formation and maintenance.
|
|
Expression pattern of PID
We next examined the expression of pattern of PID in the embryo.
Although previous studies have reported that PID expression is
detected mainly in developing cotyledon primordia
(Christensen et al., 2000;
Benjamins et al., 2001
), a more
detailed expression study is required to assess PID function during
embryogenesis.
At the globular stage, PID mRNA expression was detectable in two
domains at opposite sides, each encompassing approximately three-quarters of
the embryo along the longitudinal axis
(Fig. 3A,H). In early
heart-stage embryos, PID mRNA expression was again detected in two
opposite domains that mainly included the boundary between cotyledon primordia
and the basal part of the primordia (Fig.
3B,C,H). No expression was detected in the presumptive SAM region
or at the top part of cotyledon primordia. In the late heart to torpedo
stages, PID mRNA expression was found in the adaxial side of
cotyledon primordia (Fig. 3D,H)
as well as in the boundary between cotyledon primordia
(Fig. 3E,H). Typically, the
signal in the cotyledon boundaries was stronger than those in the other
regions. Our results thus demonstrate PID mRNA expression at the
boundaries, which has not been described in previous studies
(Christensen et al., 2000;
Benjamins et al., 2001
).
|
CUC1 and CUC2 in pid single and pin1 pid double mutants
In the wild type, the CUC1 and CUC2 genes, which promote
cotyledon separation by preventing growth at the boundaries, were expressed in
a stripe between cotyledon primordia (Fig.
4A,B). We previously have shown that, in pin1 embryos,
CUC1 expression expands to almost the entire apex, whereas
CUC2 expression is restricted to a central spot at the early heart
stage (Aida et al., 2002). In
this study, we further analyzed the expression of these genes at the late
heart stage. At this stage, the expansion of CUC1 expression to the
periphery was partial, as revealed by the restricted spots of signals in
cotyledon primordia (Fig. 5A).
At the same stage, CUC2 expression was mainly detected at the center
of the apex and occasionally in part of the cotyledon primordia
(Fig. 5B).
|
|
To analyze CUC1 and CUC2 expression in the pin1
pid double mutant, we examined embryos developing in pin1-3/+
pid-2/pid-2 siliques. At the early heart stage, CUC1 expression
was expanded to include nearly the entire apex in 24% of the embryos (13
out of 55; Fig. 4E). This
pattern was essentially the same as that in the pin1 single mutant.
At the late heart stage, however, CUC1 expression remained in almost
the entire apex of the pin1 pid double mutant
(Fig. 5C), in contrast to that
of the pin1 single mutant. CUC2 expression was detected in
the central region and occasionally at part of the periphery in 19% of early
heart stage embryos (eight out of 42; Fig.
4F). This expression pattern was maintained at the late heart
stage (Fig. 5D).
Collectively, these results indicate that the pid mutation, when combined with pin1, causes a prolonged and complete expansion of CUC1 expression into the peripheral region of the embryonic apex. This is in contrast to the pin1 single mutant, in which the CUC1 expression is expanded only transiently and soon becomes restricted at the late heart stage because of the action of the PID gene. The pin1 pid double mutations also cause a slight expansion of CUC2 expression, in contrast to the pin1 single mutation in which CUC2 expression is reduced.
Phenotypes of pin1 pid cuc1, pin1 pid cuc2 and pin1 pid cuc1 cuc2 mutants
The prolonged expansion of CUC1 expression as well as the slight
expansion of CUC2 expression in cotyledon primordia may account for
the loss of cotyledon formation in the pin1 pid double mutant. To
test this possibility, we constructed pin1 pid cuc1 and pin1 pid
cuc2 triple mutants, as well as pin1 pid cuc1 cuc2 quadruple
mutant to genetically eliminate CUC1 and/or CUC2 function
from the pin1 pid background.
The pin1 pid cuc1 triple mutations markedly recovered cotyledon development, resulting in the formation of cup-shaped fused cotyledons (Fig. 5H). The extent of cotyledon fusion varied among seedlings, ranging from a partial fusion at the base to a nearly complete fusion. By contrast, most of the pin1 pid cuc2 triple mutants were indistinguishable from the pin1 pid double mutants, except for a few seedlings that produced partially fused cotyledons, the sizes of which were larger than those of the rudimentary cotyledons observed in the pin1 pid double mutants (Fig. 5I). These results indicate that ectopic CUC1 activity mainly prevents cotyledon formation in the pin1 pid double mutant and CUC2 partially contributes to this process. The fusion phenotype observed in each triple mutant combination may be due to the reduced activities of cotyledon separation caused by the cuc1 or cuc2 mutation at least in part.
In the pin1 pid cuc1 cuc2 quadruple mutant, all seedlings developed cotyledon with a fused cup shape (Fig. 5J). The extent of fusion was more pronounced and complete than that in the pin1 pid cuc1 triple mutant. Thus, the ectopic activities of CUC1 and CUC2 fully account for the repression of cotyledon growth in the pin1 pid double mutant.
To examine symmetry in the quadruple mutant, we observed the vascular
pattern of cotyledons, a suitable marker for seedling symmetry
(Aida et al., 1997). Both
wild-type (data not shown) and the cuc1 cuc2 double mutant
(Fig. 5G) displayed bilaterally
symmetrical arrangement of vascular strands
(Fig. 5K,M). By contrast, the
pin1 pid cuc1 cuc2 quadruple mutant displays a radially symmetrical
arrangement (Fig. 5L,N),
similar to the arrangement described for the pin1 cuc1 cuc2 triple
mutant (Aida et al., 2002
).
These results are consistent with the loss of bilateral symmetry in the
pin1 pid double mutant and indicate that the addition of
cuc1 and cuc2 mutations does not rescue the symmetry
defect.
Finally, we examined the effect of cuc1 or cuc2 mutation on pid mutation alone. In the pid cuc1 double mutant, the extent of cotyledon fusion was slightly enhanced compared with that in the pid single mutant, whereas the frequency of fusion was not changed (data not shown). By contrast, seedlings of the pid cuc2 double mutant were phenotypically indistinguishable from those of the pid single mutant (data not shown). These results show that neither cuc1 nor cuc2 markedly affects the phenotype of the pid single mutant.
STM expression and its activity in pin1 pid double mutant
We next examined the effect of the pin1 pid double mutations on
the expression of the STM gene, which is involved in SAM formation
and cotyledon separation downstream of the CUC1 and CUC2
genes (Aida et al., 1999;
Takada et al., 2001
).
STM was expressed in a stripe between cotyledon primordia at the
heart stage of the wild type (Fig.
5E), whereas it was expanded to include almost the entire apex in
the pin1 pid double mutant (Fig.
5F).
To eliminate STM activity from the pin1 pid background, we constructed the pin1 pid stm triple mutant. Addition of the strong stm-1 allele (Fig. 5O) to the pin1 pid double mutant partially recovered cotyledon development, as evidenced by the formation of large cotyledons compared with those of pin1 pid seedlings with mild phenotypes (Fig. 5P). The recovery of cotyledon growth by stm, however, was much less pronounced compared with that by the cuc1 mutation. These results indicate that ectopic STM expression in the pin1 pid double mutant is also responsible for the growth inhibition of cotyledon primordia, although its contribution is partial.
Effects of exogenous auxin treatment on wild-type and cuc mutant embryos
If the observed effects of the pin1 and pid mutations on
the CUC1 and CUC2 genes were caused by changes in auxin
distribution, the exogenous application of auxin could also affect these
genes, thereby perturbing normal cotyledon development. Consistently, previous
studies have shown that auxin treatment causes various cotyledon defects
including fusion, a phenocopy of the cuc1 cuc2 double mutant
(Liu et al., 1993;
Hadfi et al., 1998
;
Friml et al., 2003
). To
strengthen this hypothesis, we further tested the effect of auxin on
cuc1 and cuc2 single mutant embryos (Materials and
methods).
When wild-type embryos were treated with synthetic auxin,
2,4-dichlorophenoxyacetic acid (2,4-D), a fraction of the embryos developed
into seedlings with abnormal cotyledons: 3.0% of the seedlings displayed weak
fusion and 6.4% displayed complete fusion of cotyledons
(Fig. 6A;
Table 3). The vascular pattern
of the latter class was considerably irregular and not bilaterally symmetric
(Fig. 6B). In 2,4-D-treated
embryos, the activity of an auxin-responsive reporter gene, DR5::GUS
(Ulmasov et al., 1997b;
Sabatini et al., 1999
;
Friml et al., 2003
), was
detected in a broader region compared with that in mock-treated embryos
(Fig. 6C,D). These results
suggest that the application of auxin to the embryo changes the auxin
distribution in the apical region and causes cotyledon fusion, possibly by
reducing the activities of the CUC genes.
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Discussion |
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PIN1 and PID function in apical patterning of cotyledon primordia and their boundaries
The apical region of the embryo can be divided into three subregions, each
of which follows different developmental fates
(Fig. 7A)
(Aida et al., 1999). In the
wild type, CUC1 and CUC2 are expressed in both the
presumptive SAM (PS) and the boundary of cotyledon margins (BCM), whereas
FIL is expressed in the cotyledon primordia (CP). The expression
patterns of these three genes are bilaterally symmetric. By contrast,
ANT is expressed in both CP and BCM, reflecting radial symmetry. The
single and double mutants of pin1 and pid all develop a
functional SAM, suggesting that PIN1 and PID are not
essential for the establishment of PS. Consistent with this, none of the
expressions of the above markers reveal any abnormalities in PS of the
pin1 and pid mutants.
|
As embryogenesis proceeds, the area of BCM becomes partially excluded from CP in the pin1 single mutant (Fig. 7C), as indicated by the partial exclusion of the ectopic CUC1 expression at the late heart stage. This exclusion is dependent on PID, as the ectopic CUC1 expression remains in the entire peripheral region in the pin1 pid background at the same stage (Fig. 7D). This finding indicates that the late activity of PID can partially compensate for the failure caused by the loss of PIN1 activity.
Analysis of the pin1and pid mutants in the inflorescence
meristem has suggested a difference between the functions of these genes
(Reinhardt et al., 2003). When
a large amount of auxin is applied locally to the inflorescence meristem of
pin1, a fused, color-like flower primordium is induced at a site
close to the application. By contrast, the same amount of auxin applied to the
pid meristem induces multiple primordia having a normal size but no
fusion. The observed response of pin1 meristems is consistent with
the idea that PIN1 is involved in organ partitioning in both the
embryo and the inflorescence meristem. However, the response of pid
inflorescence meristems does not reveal any involvement of the PID
gene in flower primordium partitioning, in contrast to its proposed function
based on our analysis of embryogenesis. This difference may reflect different
functions of the PID gene between embryo and flower development.
Alternatively, pid inflorescence meristems may also display
partitioning defects that can be detected only by molecular markers during the
early stages of primordium formation.
PIN1 and PID promote cotyledon growth by repressing CUC1, CUC2 and STM activities
The prolonged expansion of CUC1 and CUC2 expression in
the apex of the pin1 pid embryos suggests that cotyledon growth is
suppressed by the ectopic activities of these genes. The elimination of both
CUC1 and CUC2 activities from pin1 pid (i.e.
quadruple mutant) results in the complete recovery of cotyledon growth, as
evidenced by a fused cup-shaped cotyledon that surrounds the seedling apex.
These results indicate that PIN1 and PID function to repress
CUC1 and CUC2 expression and/or activity in cotyledon
primordia, thereby allowing the primordia to develop. In contrast to the
pin1 pid double mutant, the pin1 single mutant does not
display severe reduction in cotyledon growth, despite the expansion of
CUC1 expression. This is probably because that the expansion of
CUC1 occurs only transiently. The later exclusion of CUC1
expression from the peripheral region may be sufficient for the primordia to
develop fully.
Although the pin1 pid embryos display severe reduction or complete
loss of cotyledon growth, they express both ANT and FIL,
each of which promotes different aspects of shoot organ development
(Long and Barton, 1998;
Sawa et al., 1999
;
Siegfried et al., 1999
). This
observation suggests that the double mutant initiates developmental programs
for cotyledon development at least in part. This notion is consistent with the
recovery of cotyledon growth in the pin1 pid cuc triple and quadruple
mutants, which indicates that the embryos are competent to form cotyledons
even when both PIN1 and PID activities are reduced.
The expression patterns of marker genes have also been examined in the
inflorescence meristem of the pin1 mutant
(Vernoux et al., 2000).
Similar to the embryo, the expression of primordium-specific genes such as
LFY and ANT is still present, and their expression domains
overlap with that of the boundary-specific CUC2 gene, again
suggesting that pin1 maintains competence for primordium growth
although the growth is suppressed by the ectopic expression of boundary
specific factors. Taken together, these results indicate that PIN1
and PID promote shoot organ growth by repressing negative factors for
organ formation such as the CUC1, CUC2 and STM genes, rather
than promoting positive factors, in both the embryo and the inflorescence
meristem.
Auxin and apical patterning of embryo
Recent studies have shown that an auxin gradient maximum is present in
initiating organ primordia (Benková
et al., 2003; Friml et al.,
2003
). In the embryo, the maxima of auxin gradients are present at
the tips of cotyledon and root primordia, and those at the cotyledon primordia
are likely to be dependent mainly on PIN1, although other members of the PIN
family are also suggested to have partially redundant functions
(Benková et al., 2003
)
(Fig. 7E). Complementary
distributions of the auxin gradient maxima and the domain of CUC1 and
CUC2 expression suggest that auxin negatively regulates the
expression of these genes (Fig.
7F). This idea is consistent with our auxin application
experiment, showing that an increased concentration of auxin in the apical
region induces the cotyledon fusion phenotype. The frequency of the phenotype
is higher in the cuc1 mutant than in the cuc2 mutant.
Because each mutation does not affect auxin response per se
(Daimon et al., 2003
), this
result may suggest that CUC2 is more effectively repressed by auxin
than is CUC1.
Our results demonstrate that PID has an overlapping function with
PIN1 in patterning the periphery of the embryonic apex. PID
transcripts accumulate mainly at the boundaries of cotyledon primordia and
slightly in regions that surround the base of cotyledon primordia
(Fig. 7E). We also found that
PID expression at the early heart stage is dependent on
PIN1. Considering that PID is an auxin-inducible gene
(Benjamin et al., 2001), we
speculate that the initial PID expression is induced in response to
the auxin distribution established by PIN1. Although the precise
cellular function of the PID protein is still unclear, previous studies have
suggested its role in promoting auxin transport
(Benjamin et al., 2001
). As
PID and CUC expression domains are overlapping in the boundary of
cotyledon primordia, we suggest that PID, by promoting auxin
transport, reduces the level of auxin at the boundary and increases it in the
primordia (Fig. 7E) to limit
CUC1 and CUC2 expression to the boundary
(Fig. 7F). Detailed analysis of
the effects of PID on auxin transport and distribution and
identification of the cellular process in which PID functions are
required for uncovering the mechanism by which PID regulates
patterning in the apical region of the embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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