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The Plant Cell, Vol. 19: 3578–3592, November 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
Cell-Fate Switch of Synergid to Egg Cell in Arabidopsis eostre
Mutant Embryo Sacs Arises from Misexpression of the
BEL1-Like Homeodomain Gene BLH1
W
Gabriela Carolina Pagnussat,a Hee-Ju Yu,a,1 and Venkatesan Sundaresana,b,2
a Section
of Plant Biology, University of California, Davis, California 95616
of Plant Sciences, University of California, Davis, California 95616
b Department
In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and
two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female
gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is
caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that
BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs).
We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that
KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of
KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELLKNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the
Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a
positional mechanism might be directing establishment of cell fates in early megagametophyte development.
INTRODUCTION
In Arabidopsis thaliana, the female gametophyte is a seven-cell
structure consisting of four cell types: one egg cell and two
synergids localized at the micropylar end of the mature embryo
sac, one central cell, and three antipodal cells of undetermined
function at the chalazal end (Figures 1A and 1B). All of the cells
within the embryo sac are highly polarized. While the egg cell’s
nucleus is located toward the chalazal end of the embryo sac, the
synergid and central cells have the opposite polarity (Willemse
and van Went, 1984; Huang and Russell, 1992). The formation of
the egg cell, synergids, and one of the polar nuclei can be traced
back to the four-nucleate stage of embryo sac development,
when the two pairs of nuclei migrate to opposite ends of the
coenocytic embryo sac, remaining separated by a large vacuole
(Pagnussat et al., 2005). At the chalazal pole, the nuclei are
positioned one above the other with respect to the micropylarchalazal axis. At the micropylar end, the nuclei generally locate
side by side (Christensen et al., 1997; Pagnussat et al., 2005). A
second round of mitotic nuclear division generates the eight
nuclei of the mature embryo sac (Pagnussat et al., 2005). No
1 Current
address: National Horticultural Research Institute, Rural Development Administration, I-Mok Dong 475, Jang-An Gu, Suwon, GyeonggiDo, 440-706 Republic of Korea.
2 Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Venkatesan
Sundaresan ([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.107.054890
genetic predisposition seems to guide the fate for any of the
embryo sac nuclei to form the egg cell. However, the precise
migration and positioning of the nuclei along the embryo sac at
the four to eight nucleate stages suggests an early distinction
between these nuclei as the third mitotic division progresses and
the embryo sac undergoes cellularization (Webb and Gunning,
1994). Although the mechanism of cellularization remains not
well understood (Russell, 1993; Brukhin et al., 2005), the microtubular cytoskeleton appears to establish and maintain organelle
polarity and to function in migration and arrangement of the
nuclei during embryo sac development (Webb and Gunning,
1990, 1994). In agreement with this model, radiating perinuclear
microtubules have been observed during megagametophyte
cellularization (Russell, 1993; Webb and Gunning, 1994).
Although in the last few years a large number of mutants
showing defects during megagametogenesis have been isolated
in Arabidopsis (Christensen et al., 1997, 1998, 2002; Moore et al.,
1997; Pagnussat et al., 2005), the cellular and molecular basis of
cell specification in the embryo sac remains unknown. Recently,
a mutant called lachesis (lis), which affects cell specification, has
been described (Gross-Hardt et al., 2007). In lis mutant embryo
sacs, accessory cells (synergids and antipodals) begin to display
characteristics of gametic cells (egg cell and central cell). It has
been proposed that LIS, which is homologous to the yeast
splicing factor PRP4, might be involved in a signaling pathway
operating in gametic cells that prevents accessory cells from
adopting a gametic cell fate. Although the lis study indicates that
accessory cells in the embryo sac might have the competence to
differentiate into gametic cells, the mechanisms underlying the
initial establishment of cell identity in the female gametophyte are
Embryo Sac Cell Specification
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Figure 1. eostre Embryo Sacs Show Two Egg Cells and Only One Synergid Cell.
(A) Differential interference contrast (DIC) image showing a wild-type embryo at stage FG7. Nuclei have been artificially colored in red.
(B) Scheme indicating the different cell types present in a wild-type embryo sac at stage FG7.
(C) DIC image showing eostre mutant embryo sac phenotype at FG7. Nuclei have been artificially colored in red.
(D) Scheme indicating the cells that morphologically resemble egg cells and synergid cells in eostre mutant embryo sacs.
(E) Expression of the specific egg cell marker ET1119 in a wild-type embryo sac.
(F) The egg cell marker ET1119 is expressed in two micropylar cells in eostre embryo sacs.
(G) Distribution of ovules showing different ET1119 expression patterns in eostre-1/EOSTRE pistils. Error bars indicate SE (n ¼ 168). The white bar
represents the percentage of ovules showing no GUS staining. The bar labeled ‘‘Gus (þ) 1 Ec’’ represents the percentage of ovules showing GUS
expression in only one cell of the embryo sac. The bar labeled ‘‘Gus (þ) 2 Ec’’ represents the percentage of ovules showing GUS expression in two cells
of the embryo sac. When ovules from wild-type pistils were analyzed, GUS staining was always detected in only one micropylar cell. Out of 264
observed, 47.8% of the ovules showed GUS staining.
(H) Expression of the specific synergid cell marker ET884 in a wild-type embryo sac.
(I) The synergid cell marker ET884 is expressed in only one micropylar cell in eostre embryo sacs.
(J) Distribution of ovules showing different ET884 expression patterns in eostre-1/EOSTRE pistils. Error bars indicate SE (n ¼ 127). The white bar
represents the percentage of ovules showing no GUS staining. The bar labeled ‘‘Gus (þ) 1 syn’’ represents the percentage of ovules showing GUS
expression in only one cell of the embryo sac. The bar labeled ‘‘Gus (þ) 2 syn’’ represents the percentage of ovules showing GUS expression in two cells
of the embryo sac. When ovules from wild-type pistils where analyzed, GUS staining was detected in two micropylar cells in 46.8% of the ovules
observed and in only one of the micropylar cells in 1.1% of the ovules (n ¼ 247).
Ccn, central cell nucleus; Ec, egg cell; EcL, egg cell–like cell; mi, micropylar pole; S, synergide; Slc, synergid-like cell.
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not known. In this report, we describe the characterization of a
novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac.
RESULTS
The Cell Identity of Micropylar Cells Is Altered in the
eostre Mutant
The eostre-1 mutant was identified from a large collection of
female gametophyte mutants generated by Ds insertions and
was originally called EDA12 (for Embryo Sac Development Arrest) (Pagnussat et al., 2005). As the Ds insertion carries an NPTII
gene, gametophytic defects can be identified by the decreased
transmission of resistance to kanamycin. The EDA12/eostre-1
mutant showed no sporophytic defects but exhibited an aberrant Kanr:Kans segregation ratio of 0.5513:1 in selfed progeny
(Pagnussat et al., 2005). As this low ratio (<1:1) suggested that
both female and male gametophytes might be affected by the
Ds insertion, we performed reciprocal crosses between eostre-1
and Landsberg erecta (Ler) wild-type plants. Our results show
that both the female and the male gametophytes were severely
affected by the mutation (Table 1). The defect in the male transmission was shown to arise from a defect in pollen development
(L. Boavida and S. McCormick, personal communication). To
analyze the basis of the female transmission deficiency, we
examined the terminal phenotype of the gametophytes by emasculating flowers from plants heterozygous for the Ds insertion,
when wild-type embryo sacs are at the final developmental stage
(stage FG7; Christensen et al., 1998; Figure 1) by emasculating
heterozygous flowers. We found that the mutant gametophytes
showed diverse defects ranging from embryo sacs completely
collapsed showing absolute degeneration (see Supplemental
Figure 1 online) or arrested very early (at FG1 to FG2), to embryo
sacs that progressed to stage FG7, but presented an abnormal
display of cells at the micropylar end. Specifically, we found that
29% of the embryo sacs in heterozygous pistils exhibited two
cells with a polarity that resembles wild-type egg cells, while only
one of the micropylar cells showed the morphology that corresponds to a synergid cell (Figures 1A to 1D, Table 2). Due to the
excess of presumptive egg cells, the EDA12 mutant was renamed eostre, after the Anglo-Saxon goddess of the spring.
Table 1. Transmission Efficiency of eostre Mutants
Parental Genotypes
(Female 3 Male)a
Kanr
Kans
Transmission
Efficiencyb
eostre-1/EOSTRE 3 EOSTRE/EOSTRE
EOSTRE/EOSTRE 3 eostre-1/EOSTRE
eostre-3/EOSTRE 3 EOSTRE/EOSTRE
EOSTRE/EOSTRE 3 eostre-3/EOSTRE
77
56
76
34
137
272
83
144
35.98%
17.07%
47.79%
19.10%
a Plants
were crossed manually, and seeds of the resulting cross were
collected and grown on selective Murashige and Skoog (MS) plates to
determine the efficiency in which the mutant allele was transmitted to
the next generation by the male or female gametes.
b Transmission efficiency ¼ Kanr/Kans 3 100%.
To further characterize the identities of the cells present in
eostre-1 embryo sacs, we tested the expression of different cellspecific molecular markers. The marker lines were crossed with
both wild-type plants and eostre-1 mutant plants, and the F1
progeny was analyzed for expression of the marker gene. The
expression of an egg cell–specific marker (ET119; Gross-Hardt
et al., 2007) was tested both in wild-type and in eostre-1 embryo
sacs. While wild-type embryo sacs express the marker only in the
egg cell, the marker was found to be strongly expressed in two of
the eostre-1 micropylar cells in ;15% of the embryo sacs
(Figures 1E to 1G). This result suggested that two of the eostre-1
embryo sac cells might have egg cell attributes. On the other
hand, while two micropylar cells were b-glucuronidase (GUS)
positive when the expression of a synergid-specific marker
(ET884; Gross-Hardt et al., 2007) was tested in wild-type embryo
sacs, its expression was downregulated in many eostre-1 embryo sacs and ;13% of the female gametophytes of eostre-1
pistils showed GUS expression in just one of the cells composing
the embryo sac (Figures 1H to 1J), indicating that only one of the
micropylar cells of the eostre-1 embryo sac might present
synergid features. Markers for the central cell and the antipodal
cells were also tested in eostre-1 embryo sacs. For both cases,
similar GUS staining patterns were observed in wild-type and
eostre-1 mutant embryo sacs (data not shown).
Fertilization of eostre Embryo Sacs Reveals Two Functional
Egg Cells
In view of the above results, we decided to test whether the
eostre-1 mutation was affecting embryo sac functions such as
pollen tube guidance or fertilization. Fertilization in Arabidopsis
requires controlled growth of the pollen tube until it enters the
micropyle to penetrate the female gametophyte. Since the
synergid cells are responsible for pollen tube attraction (Ray
et al., 1997; Shimizu and Okada, 2000) and eostre-1 mutants
appear to have only one synergid cell instead of the two present
in wild-type embryo sacs, we analyzed the pollen tube growth
patterns in eostre-1 by aniline blue staining. Although ;80% of
the mutant ovules were able to attract pollen tubes, abnormal
pollen tube growth patterns (i.e., spinning and curling pollen
tubes) were observed in ;22% of the ovules in eostre-1/
EOSTRE pistils (Figures 2A and 2B; see Supplemental Table
1 online). Also, ;11% of the ovules showed a pollen tube on the
funiculus but not at the micropyle. (The same situation could also
be observed when wild-type pistils were analyzed, but the
percentage of ovules without pollen tubes was only of 5.72;
see Supplemental Table 1 online). Furthermore, a significantly
higher fraction of mutant ovules was found to attract two pollen
tubes to the micropyle compared with wild-type ovules (4.26
versus 0.96%, respectively; see Supplemental Table 1 online;
P < 0.001, x2 test). Together, the results indicated a defect in
pollen tube attraction and guidance, and as a consequence, part
of the pollen tubes either cannot reach the micropyles of the
ovules or growth toward them following a wavering pattern.
These defects were also observed when wild-type plants were
used as pollen donors (see Supplemental Table 1 online; no
significant differences x2 test), indicating that the problem arises
due to a disruption of the female gametophytic functions.
Embryo Sac Cell Specification
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Table 2. Analysis of eostre/EOSTRE Pistils at Different Stages of Development
Stage and Phenotype of Ovules found in the Pistils Analyzed
Pistil Stagea
Collapsed Ovules
FM
FG2-3
FG4
FG5
FG6
FG7
Total Analyzed
Abnormal (%)
FG1
FG2-3
FG3-4
FG4
FG4-5
FG6
FG7
0
3
0
10
7
23
47
125
0
33
1
0
5
0
0
96 (0)
56 (0)
5 (3)
2 (1)
5 (5)
0
0
12 (0)
32 (6)
21 (7)
13 (5)
17 (14)
0
0
0
0
2 (1)
27 (6)
27 (14)
0
0
0
0
0
1 (0)
55 (6)
21 (21)
0
0
0
0
0
0
296 (107)
125
111
121
49
41
132
364
0.00
2.70
4.95
42.85
46.34
46.96
48.07
a Assignment
of pistil stage was done according to Christensen et al. (1998). An FG stage was assigned to pistils showing the majority (>75%) of the
wild-type female gametophytes at that particular stage. If a single stage does not predominate, a transition between two stages was assigned.
Values within parenthesis indicate number of abnormal embryo sacs observed.
To study if the two micropylar cells expressing the specific egg
cell marker in the eostre-1 embryo sac (Figure 1F) are able to
become fertilized, we studied the phenotype of eostre-1 embryo
sacs after pollinating eostre-1 pistils using wild-type plants as
pollen donors. When pistils were analyzed 24 h after pollination,
49.7% of the ovules showed an elongated zygote and endosperm development, 3.28% of the ovules were found unfertilized, 4.22% showed a zygote but have endosperm development
affected, and ;24% of the ovules looked fertilized, showing two
zygote-like cells, characterized by a relatively rounded shape
and a centrally located nucleus (Figure 2D). There were also
ovules showing only one zygote-like cell and an egg cell that also
exhibited some extent of endosperm development (four to six
endosperm nuclei, Figure 2C). For those ovules showing two
zygote-like cells, they did not show any endosperm development
(Figure 2D). Also, 18.3% of the ovules were found collapsed at
this stage. Although the two micropylar cells observed in the
mutant ovules after fertilization resemble the characteristics of
zygotic cells, we decided to investigate whether those cells were
actually fertilized by pollinating eostre-1 pistils using transgenic
plants carrying the PFAC1IE:GFP-GUS:TFAC1 construct as pollen donors. FAC1 (Embryonic Factor 1) encodes a putative AMP
Figure 2. Functional Study of Egg Cells and Synergids in eostre Embryo Sacs.
(A) Abnormal pollen tube growth patterns observed at the micropyle of an eostre mutant ovule.
(B) Pollen tube growing toward the micropyle of a wild-type embryo sac present in the pistil of eostre-1/EOSTRE plants.
(C) eostre embryo sac after fertilization showing one zygote-like cell, an egg cell–like cell, and endosperm development (endosperm nuclei are indicated
by arrowheads).
(D) eostre embryo sac after fertilization showing two zygote-like cells and no endosperm development.
(E) to (H) GUS expression in both wild-type ([E] and [G]) and eostre ([F] and [H]) ovules after an eostre-1/EOSTRE plant was pollinated with a transgenic
plant carrying the PFAC1IE:GFP-GUS:TFAC1 construct.
(E) GUS expression observed in the zygote of a wild-type ovule.
(F) GUS expression observed in both of the zygote-like cells present in this eostre embryo sac.
(G) GUS expression observed in the embryo and in the endosperm of a wild-type ovule.
(H) GUS expression observed in one zygote-like cell and in the endosperm of an eostre embryo sac. The arrows point at endosperm nuclei.
Ec, egg cell; EcL, egg cell–like cell; En, endosperm; EP, embryo proper; F, funiculus; mi, micropylar pole; PT, pollen tube; Z, zygote; ZL, zygote like.
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deaminase (EC:3.5.4.6) and is expressed in both the developing
embryo and endosperm from the paternal allele (Xu et al., 2005).
Two different patterns of GUS expression were observed in
eostre-1 mutants after pollination: (1) GUS staining was observed
either in both of the zygote- like cells or (2) GUS staining was
found in only one of the zygote-like cells and in the endosperm.
These results suggested that either fertilization of both of the egg
cells or fertilization of only one of the egg cells and of the central
cell can occur (Figures 2F and 2H; see Supplemental Table 2
online). GUS expression was not detected in both zygote-like
cells and endosperm at the same time, suggesting that even
when some eostre-1 mutant sacs are able to attract more than
one pollen tube (see Supplemental Table 1 online), only one of
them would deliver sperm cells into the embryo sac. On the other
hand, no autonomous endosperm development was observed
when the two egg cells are fertilized, suggesting that the
unfertilized central cell is not dividing after the fertilization event.
This result was intriguing since it was previously shown that a
positive signal from the fertilization of the egg cell initiates
proliferation of the unfertilized central cell in wild-type plants
(Nowack et al., 2006). We therefore considered whether the
fertilization of two egg cells rather than one in the embryo sac
was interfering with the signaling after fertilization, preventing
both embryo and endosperm development in the eostre-1 mutant. To test that possibility, we used the cdc-2 mutant, carrying a
mutation in the Arabidopsis Cdc2 homolog CDC2A, to pollinate
eostre-1/EOSTRE siliques. In cdc2a mutant pollen, only one
sperm cell, instead of two, is produced. Mutant pollen is viable
and the single sperm fertilizes only the egg cell, but this single
fertilization event appears to trigger endosperm development in
the central cell (Nowack et al., 2006). If the response of the
central cell of an eostre-1 embryo sac is similar to that of a wildtype embryo sac, fertilization with cdc2a pollen should result in
an increase in the fraction of ovules that show both embryo
development and some extent of endosperm development. However, we did not observe any difference in endosperm development in the siliques analyzed after pollinating eostre-1 with cdc2a
pollen (see Supplemental Table 3 online).
eostre-1 Shows Nuclei Migration and Cellularization
Abnormalities during Megagametogenesis
To analyze the developmental stage at which eostre-1 female
gametophytes start to show defects, we observed the embryo
sacs of heterozygous pistils at different phases of development.
Since female gametophyte development within a pistil is generally synchronous (Christensen et al., 1997), we used the wildtype embryo sacs siblings as a guide to ascribe delays to the
mutant embryo sacs present within the same pistil.
As shown in Table 2, eostre-1 female gametophytes begin to
develop normally, as no differences can be observed at FG1
stage among any of the embryo sacs present in the heterozygous
pistils analyzed. However, from stage FG3 on, the developmental
asynchrony was evident. While the majority of the embryo sacs
was already between FG3 and FG4 stages, ;27% of the embryo
sacs were still at FG1 stage. Moreover, some of the embryo sacs
already at FG4 stage showed abnormal nuclear position. While in
the wild-type embryo sacs at stage FG4 the two pairs of nuclei
are separated by a large central vacuole and showed a typical
arrangement (Figures 3A and 3C), in eostre-1 female gametophytes, the vacuole is positioned on a side of the embryo sac, the
two pairs of nuclei are not completely separated, and they look
aligned along the coenocytic embryo sac (Figure 3I). When pistils
were analyzed at stages FG4 to FG5, 10% of the embryo sacs
showed this defect (Table 2). Approximately 18% of the embryo
sacs also showed to be collapsed at this stage (Table 2) probably
arising from those that were previously observed to be severely
delayed. During the cellularization process that begins following
the third round of mitosis (FG5 and FG6), not only the developmental asynchrony was obvious but also ;30% of the embryo
sacs showed abnormal distribution of nuclei along the embryo
sac or cells with aberrant shapes and polarities at the micropylar
end (Table 2). At mid stage FG5, a wild-type embryo sac shows
four micropylar nuclei with a distinctive distribution, in which the
two nuclei that will further become synergid nuclei are located
side by side at the embryo sac mycropylar edge, while the future
egg cell nuclei is located toward the embryo sac micropylar half
(Figure 3D). However, an atypical arrangement of nuclei at the
micropylar pole was observed in eostre-1 embryo sacs at this
stage (Figure 3J). Only one nuclei instead of the two observed in
wild-type embryo sac is located at the micropylar embryo sac
edge, two nuclei are located side by side at the middle region of
the micropylar pole, and one nucleus is located closer to the
center of the embryo sac (Figures 3D and 3J). As cellularization
progresses in wild-type embryo sacs, cellular morphology in the
cells at the embryo sac’s micropylar pole become clearly identifiable. At FG6 stage, the egg cell shows a characteristic pear
shape and a highly polarized cytoplasm; its nucleus and most of
its cytoplasm are at its chalazal end and a large vacuole occupies
the remaining space of the cell (Figures 3A and 3E). Similarly,
wild-type synergid cells also have a distinguishing shape and
polarity, which is the opposite of the egg cell cytoplasm (Figures
3A and 3E). By contrast, eostre-1 female gametophytes showed
two cells in which the nuclei occupied a chalazal position and
only one cell in which the nucleus showed a micropylar orientation. Thus, at the end of FG7, eostre-1 embryo sacs appear to
contain two egg cells instead of one and only one synergid cell
(Figures 3K and 3L). This asynchrony in embryo sac development
was not observed when wild-type pistils were analyzed, and
abnormal or aborted embryo sacs were rarely observed (only 1 to
2.3%; see Supplemental Table 4 online).
A Ds Element Is Inserted Inside an Intergenic Region and
Promotes Misexpression of a BEL1-Like Gene in eostre
Embryo Sacs
Thermal asymmetric interlaced PCR (Liu et al., 1995) was used to
identify the flanking sequence surrounding the Ds insertion in
eostre-1 mutants using Ds-specific primers and arbitrary degenerated primers. The flanking sequence obtained revealed that
the single Ds insertion was inserted in the intergenic region
between the genes At2g35940 (BEL1-like homeodomain
1 [BLH1]) and At2g35950 (unknown protein) 1862 nt upstream
of the At2g35950 start codon (Figure 4A). This location was
further confirmed by PCR using sequence-specific primers for
the putative insertion site in combination with the corresponding
Embryo Sac Cell Specification
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Figure 3. Wild-Type and eostre Female Gametophyte Development in Arabidopsis.
(A) Scheme showing the developmental stages leading to the wild-type embryo sac formation.
(B) Wild-type embryo sac at FG3 stage, showing two nuclei separated by a vacuole.
(C) Wild-type embryo sac at FG4 stage, showing two pairs of nuclei separated by a vacuole.
(D) Eight-nucleated wild-type embryo sac. One of the chalazal end nuclei is migrating toward the micropylar end (indicated by arrows).
(E) Wild-type embryo sac containing seven cells and eight nuclei.
(F) Wild-type embryo sac containing seven cells and seven nuclei.
(G) Scheme showing the developmental stages leading to the eostre mutant female gametophyte formation.
(H) eostre embryo sac at FG3 stage, showing two nuclei separated by a small vacuole.
(I) eostre embryo sac at FG4 stage. The vacuole is positioned on a side of the embryo sac, the two pairs of nuclei are not completely separated, and they
look aligned along the embryo sac.
(J) eostre embryo sac at FG5 stage showing an atypical arrangement of nuclei at the micropylar end. One of the chalazal end nuclei is migrating toward
the micropylar end (indicated by arrows).
(K) eostre embryo sac containing seven cells and eight nuclei. eostre embryo sacs appear to contain two egg cells instead of one and only one synergid
cell.
(L) eostre embryo sac at FG7 stage. Nuclei have been artificially colored in red.
Cc, central cell; Ccn, central cell nucleus; Ch, chalazal pole; Ec, egg cell; EcL, egg cell–like cell; mi, micropylar pole; Pn, polar nuclei; S, synergide; SL,
synergid-like cell.
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Figure 4. The eostre Mutant Phenotype Is Caused by a Ds Insertion within the Intergenic Region between the Genes At2g35940 and At2g35950.
(A) Position of the Ds element in eostre-1 and of the T-DNA insertion in eostre-2 and eostre-3. The arrow indicates the GUS gene within the Ds insertion.
The Ds insertion is located 11,272 bp upstream of the At2g35940 (BLH1) start codon.
(B) DIC image showing eostre-2 mutant embryo sac phenotype at FG7.
(C) DIC image showing eostre-3 mutant embryo sac phenotype at FG7.
(D) Phenotype of an eostre-2/EOSTRE silique. The insert shows an enlarged section of the silique, and the arrow points to an aborted ovule.
(E) Phenotype of an eostre-3/EOSTRE silique. The insert shows an enlarged section of the silique, and the arrowhead points to an aborted ovule.
Ccn, central cell nucleus; EcL, egg cell–like cell; mi, micropylar pole of the embryo sac; SL, synergid-like cell.
Ds element primers. A DNA fragment of the predicted size
was amplified from the genomic DNA of eostre-1 but not from
wild-type Arabidopsis genomic DNA. The DNA sequence of the
PCR product verified the junction between the genomic DNA and
the Ds element and showed a direct repeat of 8 bp at the point of
insertion (Figure 4A). To verify if this insertion site was responsible for the phenotype observed in the eostre-1 mutant, we
obtained lines containing T-DNA insertions around this region
from the Salk Institute Genomic Analysis Laboratory collection
(Alonso et al., 2003). We analyzed two independent T-DNA alleles with insertions in the intergenic region between At2g35940
and At2g35950 that were named eostre-2 and eostre-3. In
eostre-2, the T-DNA is inserted 1161 nucleotides upstream of
the At2g35950 start codon, and in eostre-3, the T-DNA is inserted 3132 nucleotides upstream of the At2g35950 start codon.
No homozygous plants could be detected for either of these
alleles. However, the mutation seemed to be less penetrant in
these mutants. eostre-2 siliques contained ;27% of aborted/
desiccated ovules, while eostre-3 siliques contained ;32% of
aborted ovules (Figures 4B and 4C). To study whether the eostre
allele mutations affect the female gametophyte as in the original
eostre-1 mutation, we analyzed the phenotype of embryo sacs in
both eostre-2 and eostre-3 pistils. In both cases, ;15% of the
mutant ovules showed micropylar cells with abnormal polarities
as was observed for eostre-1 (Figures 4D and 4E). A fraction of
the ovules collapsed very early during development (;30% for
both alleles; see Supplemental Table 5 online). The remaining
fraction, even when they looked normal at stage FG7, seemed to
have problems attracting pollen tubes and remain unfertilized
(see Supplemental Table 5 online). We also noticed that the
number of unfertilized or aborted ovules in eostre-2 and eostre-3
varied widely from silique to silique even within the same plant, a
characteristic that was not observed in the eostre-1 mutant. Also,
eostre-3 exhibited reduced transmission through both the female and the male gametophyte (Table 1).
As At2g35950 was the nearest gene to these intergenic
insertions, we examined two lines obtained from the Salk
Institute Genomic Analysis Laboratory collection (Alonso et al.,
2003) carrying a T-DNA insertion within this gene to study if a
putative deficiency in At2g35950 expression was causing the
observed phenotype. For both lines, we were able to identify
homozygous null mutants plants for At2g35950, as no transcripts for this gene were detected by RT-PCR (data not shown),
indicating that disruption of At2g35950 does not lead to gametophytic defects.
In an attempt to identify genes of noncoding RNAs, the intergenic region was screened for the presence of microRNAs
using the microRNA candidates database (http://sundarlab.
ucdavis.edu/mirna/search_candidates.html/; Adai et al., 2005)
and for the presence of small RNAs using the small RNA
database (http://asrp.cgrb.oregonstate.edu/db), but no matches
were found around the insertion site. We also looked for transcription units across the intergenic region in an effort to detect
the expression of nonannotated genes using the Arabidopsis
Transcriptome Genomic Express Database (http://signal.salk.edu/
cgi-bin/atta; Yamada et al., 2003) and by checking the putative
transcripts by RT-PCR, without positive results. As no new genes
or noncoding RNAs across the region of the insertions studied
Embryo Sac Cell Specification
were detected, the expression of the upstream gene At2g35940
(BLH1) and the expression of two additional downstream genes
(At2g35960 and At2g35970, both encoding a harpin-induced
family protein) was studied in wild-type and eostre-1 ovules by
RT-PCR experiments using ovule total mRNA as a template. For
the downstream genes studied, we were not able to detect any
expression in wild-type or eostre-1 ovules (data not shown).
Although no expression of BLH1 was detected in wild-type
ovules either, its transcripts were consistently detected in
eostre-1 ovules (Figure 5A). This result suggested that BLH1
might be misexpressed in the mutant ovules. Accordingly, expression of BLH1 was also found in the eostre-1 alleles eostre-2
and eostre-3 (Figure 5B). To study if the overexpression detected
in eostre-1 was ovule specific, BLH1 expression was also analyzed in leaves. BLH1 expression was higher in eostre-1 leaves
compared with the wild-type ones (Figure 7D), although no
defects were observed in leaf development or morphology. This
result indicated that although the deregulation of BLH1 gene
might be general, it only causes a defect in reproductive tissues.
We used a gene trap line (GT9784) carrying an insertion in BLH1
gene from the Cold Spring Harbor collection (http://genetrap.
cshl.org/) to analyze the BLH1 expression pattern in Arabidopsis.
As observed in Figure 5G, when dissected pistils of heterozygous
plants were analyzed for GUS expression, GUS staining was only
detected in the transmitting tract and in the base of the funiculus
but not in the ovule or the embryo sac. Thus, the normal BLH1
expression pattern seems to exclude both the ovule and the
female gametophyte.
3585
The eostre Phenotype Is Caused by Misexpression of BLH1
in the Embryo Sac
As BLH1 was shown to be misexpressed in eostre ovules, we
further investigated if this might be the cause of the observed
phenotype by driving the expression of BLH1 in the embryo sacs
of wild-type plants using the pOp/LhG4 transactivation system
(Moore et al., 1998). We generated transgenic lines expressing
the chimeric transcription factor LhG4 under the control of two
different promoters: At5g40260 (pES1 [for Embryo Sac promoter
1]), which encodes a protein of the nodulin MtN3 family and is
expressed in the embryo sac from FG1 to FG7 (Yu et al., 2005),
and At1g26795 (pES2 [for Embryo Sac promoter 2]), which
encodes a self-incompatibility protein and it is strongly expressed from FG3 in the dividing nuclei of the embryo sac to
FG7 (Yu et al., 2005). A reporter construct carrying the BLH1
coding region under the control of the p10Op promoter, which is
activated by LhG4, was introduced in plants that were crossed to
pES:LhG4 plants. As the pES:LhG4 constructs were directly
introduced into transgenic plants carrying a pOp-GUS reporter
construct, we were able to confirm that the promoters used were
directing the expression of the reporter genes to the embryo sacs
by studying GUS staining patterns (Figure 5C). To verify if BLH1
was in fact expressed in these plants, RT-PCR experiments were
performed using total RNA from ovules of plants carrying the
Op:BLH1 reporter construct alone as well as plants carrying both
the driver and reporter constructs. BLH1 expression was only
detected in ovules from plants carrying both constructs (Figure
Figure 5. Misexpression of BLH1 in the Embryo Sac Recapitulates the eostre Phenotype.
(A) RT-PCR showing BLH1 overexpression and actin expression as a positive control (bottom) in eostre-1 ovules.
(B) Overexpression of BLH1 was also observed in eostre-2 and eostre-3 ovules in the Col background. Actin expression is shown as a positive control
(bottom).
(C) GUS staining pattern in a pES1BLH1 ovule showing that pES1 specifically directs expression of the reporter genes to the female gametophyte.
(D) BLH1 expression in pES1BLH1 ovules detected by RT-PCR. Actin expression is shown as a positive control (bottom).
(E) Phenotype of a pES1BLH1 embryo sac at FG4 stage. Arrowheads indicate the unusual position of nuclei.
(F) Phenotype of a pES1BLH1 embryo sac at FG7 stage.
(G) Expression pattern of BLH1 in the pistil of the BLH1 gene trap line GT9784.
Ccn, central cell nucleus; EcL, egg cell–like cell; Es, embryo sac; F, funiculus; mi, micropylar pole of the embryo sac; SL, synergid-like cell;
Tt, transmitting track.
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The Plant Cell
5D). These results indicated that BLH1 was successfully and
specifically expressed in the embryo sac of the plants under
study. When the pistils of F1 plants expressing BLH1 in the
embryo sacs were analyzed, approximately one-fourth of the
ovules showed to be defective, which was the proportion expected for a double heterozygote for the driver and reporter
construct. The defects observed range, as in eostre-1 mutants,
from early aborted embryo sacs to embryo sacs that progress to
the terminal stage showing two egg cells and a single synergid
(Figures 5E and5F). When the phenotype of these embryo sacs
was studied after fertilization, both embryo sacs showing only
one arrested zygote, one egg cell–like cell and four to six
endosperm nuclei or two zygotic-like structures without endosperm development were observed (see Supplemental Figure 2
online). Plants carrying the reporters or the driver constructs
alone did not show any phenotype, indicating no background
activity.
blh1 Loss-of-Function Mutants Have Normal and
Functional Gametophytes
To examine the effects of loss of function of BLH1, we analyzed
three independent T-DNA lines with insertions in At2g35940
(BLH1) that are predicted to be null alleles. For all three lines, we
were able to identify homozygous mutant plants with blh1 null
alleles (verified by RT-PCR; data not shown), indicating that
BLH1 alone is not essential for gametophyte development or
function. As BLH1 belongs to the BELL family of homeodomain
transcription factors (Chan et al., 1998; Roeder et al., 2003), a
large family that includes 13 proteins, we could not eliminate the
possibility that a functional redundancy might be obscuring
BLH1 function in the embryo sac. As BLH5 is the BEL1-like
protein that shows the closest homology to BLH1 (Roeder et al.,
2003), double gene knockout lines were generated and were
statistically analyzed in segregating F2 populations (blh1 3 blh5).
Homozygous plants for both insertions were recovered, and
plants were found to look like wild-type plants, showing no
obvious defects either in the sporophytic or the gametophytic
organs (data not shown). This result suggested either that BLH1
or BLH5 is not involved in gametophyte development or that a
broader functional redundancy among the members of the
BELL1 family exists, requiring additional mutations in other
BLH genes to reveal their functions in the embryo sac.
A Mutation in the Class II Knox Gene KNAT3 Reverts the
eostre Phenotype
Biochemical studies have shown that BELL proteins associate
with KNOX proteins to form heterodimers that are thought to
compose functional complexes regulating plant development
(Muller et al., 2001). Specifically, BLH1 has been reported to
interact with the KNOX proteins KNAT3, KNAT5, and KNAT6
(Hackbusch et al., 2005). If overexpression of BLH1 in the
embryo sac requires these KNOX proteins to cause the eostre
phenotype, we might expect that loss of function of the corresponding KNAT gene would suppress the eostre mutation. To
test this possibility, we generated double mutants by crossing
knox gene knockout lines to eostre-1. The segregating F1 population was analyzed by genotyping and analysis of the siliques.
No significant differences in the number of aborted seeds
per silique were observed between eostre-1 single mutant and
eostre-1 knat5 or eostre-1 knat6 double mutants (Figures 6A and
6B). On the other hand, heterozygous plants for both a T-DNA
insertion in knat3 gene and for the eostre-1 Ds insertion showed a
significant reduction in the number of aborted seeds per silique
when compared with the eostre-1 single mutant (Figure 6; t test,
P < 0.005). The suppression of eostre-1 cannot be attributed to
the different Arabidopsis ecotype of the knat3 mutant (Columbia
[Col]), as no suppression was detected when eostre-1 (Ler
background) was crossed to the knat5 or knat6 mutants (Col
Figure 6. A Mutation in the Class II Knox Gene KNAT3 Suppresses the eostre Phenotype.
(A) Mature siliques of eostre-1/EOSTRE plants and double mutants eostre1 knat6, eostre1 knat5, and eostre1 knat3. The bottom panel shows a mature
silique of the wild type.
(B) The aborted seeds per silique were quantified for each genetic background. Values represent average number of aborted seeds per silique (mean 6
SE, n ¼ 20). Light-gray bars correspond to ovules from plants heterozygous for the insertion in knox genes. The dark-gray bar shows data corresponding
to ovules from plants that were homozygous for the insertion in the knat-3 gene.
Embryo Sac Cell Specification
background). When the F2 population was analyzed, plants that
were homozygous for the T-DNA insertion in the knat3 gene and
heterozygous for the eostre-1 Ds insertion were recovered and
its siliques studied. Only ;13% of the ovules observed looked
aborted in these siliques, a number significantly lower than the
one observed for pistils from heterozygous plants for both the
knat3 gene and eostre-1 insertions (Figure 6; t test, P < 0.05).
Thus, the knat3 mutation is able to suppress the eostre phenotype, but the suppression is not complete, suggesting the
possibility that other KNATs might be interacting with BLH1 in
the embryo sac. Previous results have reported KNAT3 expression during early organ development in leaves, buds, and pedicels at the junction between two organs during development,
including the ovule-funiculus boundaries, and in maturing tissues, such as siliques, petioles of maturing leaves, and the root
(Serikawa et al., 1997; Truernit et al., 2006). Although no data
regarding KNAT3 expression in Arabidopsis embryo sac have
been reported so far, the closely related class II knox gene
KNOX6 was shown to be expressed in the maize (Zea mays)
embryo sac after cellularization (Evans, 2007). To examine
KNAT3 expression in Arabidopsis ovules, total RNA from wildtype ovules and from spl mutant ovules, which lack embryo sacs
(Yang et al., 1999), was used to study KNAT3 expression by RTPCR. KNAT3 expression was consistently detected in both wildtype and spl mutant ovules, indicating that KNAT3 is at least
expressed sporophytically in the ovule, although expression in
the embryo sac remains to be confirmed (Figure 7). Furthermore,
and to test the possibility of an upregulation of KNAT3 in eostre
ovules, the level of KNAT3 expression was assessed in eostre-1,
wild-type, and spl ovules by RT-PCR. No differences were found
among the samples, indicating that KNAT3 is not upregulated in
eostre-1 ovules (Figure 7B). As BLH1 overexpression was also
detected in somatic tissues (Figure 7D), we also studied KNAT3
expression in wild-type and eostre-1 leaves by RT-PCR. Again,
no differences were detected (Figure 7E).
At OFP5, a Putative Regulator of BLH1 Activity, Is Required
for Normal Embryo Sac Development
An investigation of Arabidopsis TALE protein interactions revealed a heavily connected network of interactions of TALE
proteins with each other and with members of the recently
discovered OVATE protein family (At OFP) (Hackbusch et al.,
2005). Particularly, it was shown that members of this family
interact both with KNAT3 and BLH1 in a yeast two-hybrid system
(Hackbusch et al., 2005). Furthermore, At OFP1 and At OFP5
regulate subcellular localization of BLH1, relocalizing it from the
nucleus, where it would be active as a transcription factor, to the
cytoplasmic space (Hackbusch et al., 2005). Although little
information is available about the specific expression patterns
of both TALE and At OFP genes in the female gametophyte, the
results with eostre and knat3 suggested that complexes involving both TALE and At OFP might be functional during embryo sac
development. To study this possibility, we analyzed Arabidopsis
lines with T-DNA insertions in four At OFP genes (At OFP1-1,
At OFP1-2, At OFP2-1, At OFP4-1, and At OFP5-1) obtained
from the Salk Institute Genomic Analysis Laboratory collection
3587
Figure 7. At OFP5, an Interactor of BLH1 and KNAT3, Is Required for
Normal Embryo Sac Development.
(A) At OFP5-1 mutant embryo sac at FG7 showing a phenotype similar to
the one observed in eostre-1 ovules.
(B) RT-PCR showing KNAT3 expression in wild-type, eostre-1, and spl
ovules. Actin expression is also shown as a positive control (bottom). The
picture corresponds to 20 PCR cycles.
(C) RT-PCR showing At OFP5 expression in wild-type and spl ovules.
Actin expression is shown as a positive control (bottom). The picture
corresponds to 20 PCR cycles. At OFP5 expression was detected in spl
ovules after 25 PCR cycles.
(D) RT-PCR showing BLH1 expression in wild-type and eostre-1 leaves.
The picture corresponds to 25 PCR cycles. BLH1 expression was
detected in wild-type leaves at 30 cycles.
(E) RT-PCR showing KNAT3 expression in wild-type and eostre-1 leaves.
The picture corresponds to 25 PCR cycles.
Ccn, central cell nucleus; EcL, egg cell–like cell; mi, micropylar pole of
the embryo sac; SL, synergid-like cell.
(Alonso et al., 2003). Although homozygous plants were recovered from selfing of heterozygous plants for At OFP1-1, At
OFP1-2, At OFP2-1, and At OFP4-1 mutants, no homozygous
plants were detected for At OFP5-1 mutants, suggesting that
At OFP5 might be required for essential processes in gametophyte or sporophyte development. When female gametophyte
development was investigated in At OFP5-1 pistils, out of 256
embryo sacs studied, 54% looked normal, while ;38% of the
ovules seemed to collapse very early during development
around stage FG2. The remaining ;8% of the embryo sacs
(20/256) showed abnormal micropylar cells, with embryo sacs
showing two egg cells as was observed in eostre-1 mutants
(Figure 7). These results strongly indicate a requirement for At
OFP5 during female gametophyte development and also that
TALE-OVATE protein complexes might be functional during
embryo sac early developmental stages. RT-PCR experiments
were performed to evaluate At OFP5 expression in the embryo
sac. At OFP5 expression levels were found to be higher in wildtype ovules compared with spl ovules, indicating that At OFP5 is
expressed both in the embryo sac and in the sporophytic tissues
of the ovule (Figure 7).
3588
The Plant Cell
DISCUSSION
Regulation of BELL-KNOX TALE Complexes Is Essential for
Normal Development of the Arabidopsis Embryo Sac
The eostre gametophytic mutant shows synergid to egg cell fate
switch in Arabidopsis embryo sacs, a phenotype that could be
recapitulated when BLH1 was misexpressed in the embryo sac.
Hence, misexpresion of BLH1 seems sufficient to generate the
eostre phenotype, probably by interfering with the normal patterning during female gametophyte development. Although no
loss-of-function mutants were found for BELL proteins that might
be involved in the normal embryo sac development pathway,
functional redundancy might be obscuring a substantial role for
these proteins during normal megagametogenesis. BELL and
KNOX proteins are members of the MEINOX-TALE superfamily
of eukaryotic transcriptional regulators that function as heterodimers and are known to regulate developmental processes both
in plants and animals (Knoepfler et al., 1997; Rieckhof et al.,
1997; Bellaoui et al., 2001; Muller et al., 2001; Smith et al., 2004).
Biochemical studies have shown that BELL proteins associate
with KNOX proteins to form functional complexes regulating
plant development (Muller et al., 2001). In particular, BLH1 has
been reported to interact with the KNOX proteins KNAT3,
KNAT5, and KNAT6 (Hackbusch et al., 2005). As a common
feature of KNOX-BELL interactions is that the KNOX protein
partner often interacts with a subset of BELL proteins (Cole et al.,
2006), BLH1 misexpression in the embryo sac might be preventing functional KNOX–BELL interactions to take place and, as a
result, interfering with normal patterning. Supporting this idea,
KNAT3, a gene belonging to the class II subfamily of knox genes
(Reiser et al., 2000) and an already described interactor of BELL
proteins (including BLH1; Hackbusch et al., 2005), appears to be
essential for the phenotypic effect of BLH1 overexpression in the
embryo sac (Figure 6). This result suggested that KNAT3 must be
expressed and functionally competent in the embryo sac. In
agreement, KNAT3 expression was detected in Arabidopsis
ovules (Figure 7B). In addition, the class II gene Knox6, which
is the nearest maize homolog of KNAT3, was the only Knox gene
found to be consistently expressed in isolated maize embryo
sacs (Evans, 2007). As no functions for class II Knox genes in
plant development have been reported in any plant species to
date (Reiser et al., 2000), knat3 suppression of the embryo sac
defects of the eostre mutant revealed a phenotype for a class II
Knox mutant. Emphasizing the importance of regulation of knox
genes during megagametophyte development, the maize gene
INDETERMINATE GAMETOPHYTE1 (IG1), which represses the
expression of meristem-specific knox genes in leaf primordia,
has been shown to be expressed during early embryo sac
development, and ig1 mutants exhibit embryo sac abnormalities
that include extra egg cells, extra polar nuclei, and extra synergids (Evans, 2007). In Arabidopsis, mutation of the putative Ig1
ortholog AS2 does not result in embryo sac defects (Semiarti
et al., 2001). However, At OFP5, a member of the Arabidopsis
ovate family, which was shown to interact with KNAT3 and to
regulate subcellular localization of BLH1 (Hackbusch et al.,
2005), appears to play an essential role during embryo sac
development (Figure 7). At ofp5 embryo sacs displayed pheno-
types that resemble those observed in eostre-1 mutants, consistent
with At OFP5 acting as a key negative regulator of BELL-KNOX
activity during early embryo sac development in Arabidopsis.
Factors Affecting Cell Fate Specification during Female
Gametophyte Development
The atypical migration and positioning of nuclei observed in
eostre mutant embryo sacs lead to a variety of morphological
and functional defects, including the production of an extra
functional egg cell and only one synergid cell. Our observations
suggest that the specification of cell fate, as egg cell or synergid,
appears to rely on a position-based mechanism. However, we
cannot rule out that the altered migration of nuclei could also be a
consequence of an altered fate. At FG5, only one nucleus is
located at the micropylar edge of eostre mutant embryo sacs, a
position at which the pair of nuclei that will further form part of the
synergid cells are normally positioned in wild-type embryo sacs.
Furthermore, two nuclei are located side by side at the middle
region of the micropylar pole, where typically the future egg cell
nucleus is placed in wild-type embryo sacs. As cellularization
progresses, eostre embryo sacs clearly showed an abnormal
pattern of cells at their micropylar edge, with only one cell
displaying synergid features and two cells showing egg cell
characteristics (Figure 1). These specific cells were also shown to
be functional, as the synergid cell was able to attract pollen tubes
and both egg cells in eostre mutant embryo sacs are able to
become fertilized (Figure 2). Recently, the description of the lis
mutant, which shows extra egg cells in detriment of both synergids and central cell identities, has led to a model that upon
differentiation, gametic cells would generate an inhibitory signal
that transmitted to the adjacent cells would prevent excess
gametic cell formation (Gross-Hardt et al., 2007). Although this
mechanism of lateral inhibition can explain the maintenance of
only one egg cell in the embryo sac after the initial specification
of cell fate, the mechanisms involved in the early establishment
of different cell fates in the embryo sac are not addressed by the
model. A positional mechanism would explain how cell fate is
specified in early megagametophyte development. Although
when all the nuclei present in the embryo sac might be competent to form gametic cells upon cellularization, those nuclei that
are at the micropylar or chalazal poles acquire the accessory cell
fates. However, once the egg cell is differentiated, a lateral inhibition mechanism seems to be necessary to maintain cell fates,
as accessory cells can yet differentiate later on into gametic cells
if this inhibitory mechanism is not present (Gross-Hardt et al.,
2007). The idea of a positional mechanism is also supported by
studies in maize, where ig1 embryo sacs undergo extra rounds of
free nuclear divisions, resulting in extra egg cells, extra central
cells, and extra polar nuclei (Guo et al., 2004; Evans, 2007).
The migration and position of nuclei during megagametogenesis in Arabidopsis has been shown to be highly regular
(Webb and Gunning, 1990, 1994). Previous studies by Brown and
Lemmon (1991, 1992) suggested that cytoplasmic domains may
determine the fate of cells during cell partitioning. This idea is
supported by our findings studying eostre mutants, as the
unusual position of the nuclei along the embryo sac might
expose them to different cytoplasmic domains compared with
Embryo Sac Cell Specification
the wild-type embryo sac nuclei. An abnormal microtubular
behavior (i.e., a change in microtubule dynamics) during and
following the second and third rounds of mitotic divisions (FG2 to
FG4) or a failure in the nuclei interactions with components of the
microtubular cytoskeleton might explain the unusual pattern of
nuclear migration and the resulting abnormal nuclear positioning
observed in eostre embryo sacs. The importance of specific
positions of nuclei along the embryo sac for cell fate determination might rely on the asymmetric distribution of still unknown
morphogenetic determinants, perhaps in the form of gradients,
that might be responsible for determining the fate of the different
cells composing the embryo sac as described for numerous
multicellular systems (Adler, 2000; Betschinger and Knoblich,
2004; Harris and Peifer, 2005).
METHODS
Plant Growth Conditions
The origin of the Ds insertion lines used in this study has been previously
described (Sundaresan et al., 1995). Typically 50 to 200 seeds were
sterilized in 20% (v/v) sodium hypochlorite, washed with sterile water, and
plated on MS medium with 50 mg L1 kanamycin in Percival growth
chambers (Percival Scientific), with a 16-h-light/8-h-dark cycle at 228C.
Resistant (green) seedlings were then transferred onto soil and grown
under the conditions described above with 60% relative humidity. For
crosses, flowers of the female parent were manually emasculated 2 d
before anthesis and cross-pollinated 2 d later.
Histology and Microscopy
To prepare cleared whole-mount preparations, pistils containing at least
20 ovules were dissected and cleared overnight in Hoyer’s solution (Liu
and Meinke, 1998). The dissected pistils were observed on a Zeiss
Axioplan imaging 2 microscope under DIC optics. Images were captured
on an Axiocam HRC CCD camera (Zeiss) using the Axiovision program
(version 3.1). For GUS staining, developing carpels and siliques were
dissected and incubated in GUS staining buffer [5 mM EDTA, 0.1% Triton
X-100, 5 mM K4Fe(CN)6, 0.5 mM K3Fe (CN)6, and 1 mg mL1 X-Gluc [Rose
Scientific] in 50 mM sodium phosphate buffer, pH 7.0) overnight at 378C.
Individual ovules were dissected from the pistils/siliques and cleared
overnight with Hoyer’s solution. The ovules were observed under DIC
optics. For pollen tube staining, 10 pistils containing at least 20 ovules
each were manually pollinated and opened longitudinally 24 h after
pollination for each mutant. The pistils were cleared in 10% chloral
hydrate at 658C for 5 min and washed with water, softened with 5 M NaOH
at 658C for 5 min, and washed again with water. The pistils were then
treated with 0.1% aniline blue in 0.1 M K3PO4 buffer, pH 8.3, for 3 h in
darkness and washed with 0.1 M K3PO4 buffer. The pistils were mounted
on a microscope slide using a drop of glycerol and carefully squashed
under a cover slip. The pistils were observed using a fluorescence
microscope.
Image Processing
All images were processed for publication using Adobe Photoshop CS
(Adobe Systems).
Segregation Analysis
For self-cross analysis, heterozygous plants were allowed to self-pollinate
and progeny seed was collected. The progeny seed was germinated on
3589
growth medium containing the proper markers (kanamycin, BASTA, or
sulfadiazine) as indicated in each case. The F1 seed was germinated on
growth medium containing 50 mg/mL kanamycin, and the number of
resistant and sensitive plants was scored. For analysis of reciprocal
crosses, we crossed the heterozygous mutant lines carrying a resistant
marker gene, with wild-type plants using the insertional lines as egg donor
and wild-type Ler as the sperm donor and vice versa. Quantification of
resistant plants in the progeny was performed by collecting the seeds
derived from these crosses and plating them in selective media according
to the selection marker carried by each of the insertional lines tested.
Molecular Analysis of the Sequences Flanking the Ds Insertion
The sequences flanking the Ds element were isolated with thermal
asymmetric interlaced PCR as described by Liu et al. (1995) and sequenced with an Applied Biosystems sequencer. The flanking sequences
obtained were run against BLASTN to identify the genomic location of the
Ds element. The junctions were then confirmed by PCR using sequencespecific primers for the putative insertion site in combination with the
corresponding Ds element primers. To verify the left border–genomic
sequence junction, we used the DS-specific primer Ds59-1A (59-ACGGTCGGGAAACTAGCTCTAC-39) combined with the genomic sequence
primer EDA12-5 (59-CCATCCTATGTATTTAGAGTTCCTGC-39). For the
right border–genomic sequence junction, we used the DS-specific primer
Ds39-2A (59-CGATTACCGTATTTATCCCGTTTC-39) combined with the
genomic sequence primer EDA12-3 (59-TGATACGATGGTTAGATCACG-39).
Both junctions were corroborated by sequencing.
Molecular Characterization of Insertional Lines around
the Ds Insertion
eostre-2 (SALK_033866) and eostre-3 (SAIL_582_F02) insertional lines
were obtained from the Salk Institute Genomic Analysis Laboratory
collection (Alonso et al., 2003). For eostre-2, the left border–genomic
sequence junction was determined by PCR using the T-DNA–specific
primer LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) combined with
the genomic sequence-specific primer eostre1RP (59-CAGTGGATATGGGAATGCAAC-39). For eostre-3, the left border–genomic sequence
junction was determined also by PCR in plants showing BASTA
resistance using the T-DNA–specific primer 59-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-39) combined with the genomic sequencespecific primer eostre2 RP (59-AATCCGATCGGTATTACGAGG-39). Lines
35950-1 (SALK_086362) and 35950-2 (SALK_086364) were obtained
from the Salk Institute Genomic Analysis Laboratory collection (Alonso
et al., 2003). For both lines, the left border–genomic sequence junction
was determined by PCR using the T-DNA–specific primer LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) combined with genomic sequencespecific primers. For both lines, the specific genomic primers were
35950-1/2 RP (59-TTGTACCGAGAAAGGCTCAAG-39) and 35950-/21 LP
(59-TGGGAATTTTGGATTTAGCCC-39). Line blh1-1 (SALK_089095) was
obtained from the Salk Institute Genomic Analysis Laboratory collection
(Alonso et al., 2003), while blh1-2 (GK-114D09) and blh1-3 (GK-475C05)
were obtained from the Nottingham Arabidopsis Stock Centre (Scholl
et al., 2000). For blh1-1, the left border–genomic sequence junction was
determined by PCR using the T-DNA–specific primer LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) combined with genomic sequencespecific primers blh1-1RP (59-TTCCAGCCGCTTAAGCATAC-39) and
blh1-1LP (59-TATGAATCCCAATCACAACGG -39). For blh1-2, the left
border–genomic sequence junction was determined by PCR using the
T-DNA–specific primer LBGK (59-TGGTTCACGTAGTGGGCCATCG-39)
combined with genomic sequence-specific primers blh1-2RP (59-CCGTCGGATCCGGCAGAGATCT-39) and blh1-2LP (59-GATCTTTGAGTCTGACACAGAGACC -39). For blh1-3, the left border–genomic sequence
junction was determined by PCR using the T-DNA–specific primer LBGK
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(59-TGGTTCACGTAGTGGGCCATCG-39) combined with genomic
sequence-specific primers blh1-3RP (59-CCAGCCGCTTAAGCATACATGT-39) and blh1-3LP (59-GGCGTCACTGGAATGCAAGGAA-39).
Molecular Characterization of Insertional Lines for KNOX and
OVATE Genes
Insertional lines for KNAT3 (SALK_136464), KNAT5 (SALK_088589), and
KNAT6 (SALK_140566) genes were obtained from the Salk Institute
Genomic Analysis Laboratory collection (Alonso et al., 2003). For the
three lines, the left border–genomic sequence junction was determined
by PCR using the T-DNA–specific primer LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) combined with genomic sequence-specific primers.
For knat3, the DNA-specific primers used were knat3LP (59-GTTAAACACAGCGCTTCTTCG-39) and knat3RP (59-TCACAGGATTCATTTTCTCACC-39). For knat5, the DNA-specific primers used were knat5LP
(59-ATTCGGGGTTTTGATTACCAG-39) and knat5RP (59-GAACAGCAACTCTTCCACGTC-39). For knat6, the DNA-specific primers used were
knat6LP (59-GTTTTAGCCATGGGATTAGGG-39) and knat6RP (59-TGATTTGTTATGGCCACAATG-39). T-DNA insertional lines for At OFP1
(SALK_111492 and SALK_127550), At OFP2 (SALK_122550), At OFP4
(SALK_014905), and At OFP5 (SALK_010386) were obtained from the
Salk Institute Genomic Analysis Laboratory collection (Alonso et al.,
2003). For all the lines, the left border–genomic sequence junction was
determined by PCR using the T-DNA–specific primer LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) combined with genomic sequencespecific primers. For At OFP1-1, the DNA-specific primers used were
At OFP1-1LP (59-AGAGATCCCAGATCTCGAAGC-39) and At OFP1-1RP
(59-AAGGTTGCGGTTTTGGATAAC-39). For At OFP1-2, the DNA-specific
primers used were At OFP1-2LP (59-ATTGGGCCGAAAACATATAGG-39)
and At OFP1-2RP (59-AAGGTTGCGGTTTTGGATAAC-39). For At OFP2-1,
the DNA-specific primers used were At OFP2-1LP (59-ACCAAATTCAAAGAAGCATCG-39) and At OFP2-1RP (59-TGGTGAGTTATGGTGAGGAGG-39). For At OFP4-1, the DNA-specific primers used were At
OFP4-1LP (59-TCGTTAGGGTTGTGACTGACC-39) and At OFP4-1RP
(59-CCTAGATTCAAAGGATGTGCG-39). For At OFP5-1, the DNA-specific
primers used were At OFP5-1LP (59-GCTTCTATATCCCCATTTTAATGTG-39) and At OFP4-1RP (59-CCTAGATTCAAAGGATGTGCG-39).
Molecular Characterization of a Gene Trap Line with an
Insertion in BLH1
The gene trap line GT9784, carrying an insertion in the BLH1 gene, was
obtained from the Cold Spring Harbor collection (http://genetrap.cshl.
org/). The junctions of the Ds element with the genomic DNA were
confirmed by PCR using sequence-specific primers for the insertion site
in combination with Ds element primers. The DS-specific primer Ds59-1A
(59-ACGGTCGGGAAACTAGCTCTAC-39) was used combined with the
genomic sequence primer GT9784RP (59-TCATCAATCTTGTAAGTTGTCA-39), and the DS-specific primer Ds39-2A (59-CGATTACCGTATTTATCCCGTTTC-39) was used combined with the genomic sequence
primer GT9784LP (59-GGTGGCGTACTAAGGACTGTGGGA-39).
Constructs and Plant Transformation
Total RNA was isolated from dissected Ler ovules using TRIzol (Invitrogen). cDNAs of BLH1 were generated by SuperScript II reverse
transcriptase (Invitrogen). After sequence verification, 10op::BLH1 was
constructed by inserting BLH1 cDNA behind an OP array (10OP-TATABJ36) and subsequently subcloned into the binary vector pCAMBIA 1300.
The plasmid was introduced into Agrobacterium tumefaciens strain
GV3101 by electroporation, and Ler wild-type plants were transformed
using the floral dip method (Clough and Bent, 1998). Transformants were
selected based on their ability to survive in MS medium with 20 mg L1
hygromicyn. Resistant (green seedlings with true leaves) were then transferred onto soil and grown under the conditions described above.
At5g40260 (pES1), At1g26795 (pES2), and promoter fragments were
previously cloned and its activity characterized as described (Yu et al.,
2005). pES:Lhg4 constructs were prepared by inserting the promoter
fragments upstream the coding sequence of the quimeric transcription
factor LhG4 and then subcloned into the binary vector pMLBART. The
plasmid was introduced into A. tumefaciens strain GV3101 by electroporation, and wild-type Ler plants containing the transgene 6OP:GUS were
transformed using the floral dip method (Clough and Bent, 1998). Transformants were selected based on their ability to survive in MS medium
containing 50 mg L1 kanamycin and 6 mg Ll of ammonium glufosinate.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative database under the following accession numbers: At2g35940
(BLH1), At5g40260 (pES1), At1g26795 (pES2), AT4G18830 (ATOFP5),
and AT5G25220 (KNAT3).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. DIC Image Showing a Collapsed Embryo Sac.
Supplemental Figure 2. Misexpression of BLH1 Expression in
pES2BLH1 Ovules Recapitulates the eostre Phenotype.
Supplemental Table 1. Pollen Tube Growth Patterns toward eostre
and Wild-Type Female Gametophytes.
Supplemental Table 2. Patterns of GUS Expression Observed when
Both Wild-Type Plants and eostre-1 Mutants Were Pollinated Using
Transgenic Plants Carrying the PFAC1IE:GFP-GUS:TFAC1 Construct
as Pollen Donors.
Supplemental Table 3. Embryo and Endosperm Development in
Plants Fertilized with cdc2a Pollen.
Supplemental Table 4. Analysis of Wild-Type Pistils at Different
Stages of Development.
Supplemental Table 5. Phenotype of Embryo Sacs in eostre-2 and
eostre-3 Pistils.
Expression Analysis
ACKNOWLEDGMENTS
Total RNA was isolated from dissected ovules using TRIzol (Invitrogen).
cDNA was generated by SuperScript II reverse transcriptase (Invitrogen).
This was used for RT-PCR analysis using 0.5 mL of cDNA template, and
intron-spanning primer pairs were used for 30 cycles at 948C for 30 s,
588C for 30 s, and 728C for 30 s. RT-PCR results were normalized using
Arabidopsis thaliana actin-2 controls for PCR. BLH1 cDNA was amplified
using the following primers: 59-CCGGGTCGGGTCTGGCTCTA-39 and
59-CGGCTCGTTCTGGGAGACCACG-39. ACTIN2 was amplified using
59-CCTGAAAGGAAGTACAGTG-39 and 59-CTGTGAACGATTCCTGGAC-39.
We thank John Bowman for providing 10OP-TATA-BJ36 and LhG4BJ36 vectors and seeds of 6OP-GUS transgenic plants, Joe Simorowski
for providing seeds of the BLH1 gene trap line, and members of the
Gasser and Sundaresan labs for helpful discussions. This work was
funded by National Science Foundation 2010 Grant 0313501.
Received August 8, 2007; revised October 29, 2007; accepted November
8, 2007; published November 30, 2007.
Embryo Sac Cell Specification
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Cell-Fate Switch of Synergid to Egg Cell in Arabidopsis eostre Mutant Embryo Sacs Arises from
Misexpression of the BEL1-Like Homeodomain Gene BLH1
Gabriela Carolina Pagnussat, Hee-Ju Yu and Venkatesan Sundaresan
Plant Cell 2007;19;3578-3592; originally published online November 30, 2007;
DOI 10.1105/tpc.107.054890
This information is current as of June 15, 2017
Supplemental Data
/content/suppl/2007/11/16/tpc.107.054890.DC1.html
References
This article cites 46 articles, 21 of which can be accessed free at:
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