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First published online 12 September 2007
doi: 10.1242/dev.009027
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1 Institute of Plant Sciences and Zürich-Base Plant Science Center, Swiss
Federal Institute of Technology, ETH Centre, CH-8092 Zürich,
Switzerland.
2 Institute of Biology II, University of Freiburg, Schänzlestr. 1, 79104
Freiburg, Germany.
* Author for correspondence (e-mail: koehlerc{at}ethz.ch)
Accepted 2 August 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Arabidopsis, Epigenetics, FERTILIZATION INDEPENDENT SEED genes, Imprinting, Polycomb group proteins
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
embryo passes through morphologically defined stages, characterized as
preglobular, globular, heart, torpedo, walking stick, early maturation and
maturation (Goldberg et al.,
1994; Laux and Jürgens,
1997).
Endosperm development differs dramatically from embryo development. In
Arabidopsis, the first divisions of the primary endosperm nucleus are
not followed by cytokinesis, giving rise to the formation of a syncytium.
Distinct nuclear-cytoplasmic domains form: the chalazal endosperm at the
posterior pole, the micropylar endosperm at the anterior pole and peripheral
endosperm domains (Brown et al.,
1999; Boisnard-Lorig et al.,
2001). Endosperm cellularization is initiated around the globular
to early heart stage of embryo development and starts in the micropylar
endosperm, which surrounds the embryo, to progress through the peripheral
endosperm to the chalazal region (Brown et
al., 1999; Boisnard-Lorig et
al., 2001). As the embryo matures, most of the endosperm is
degraded and absorbed by the embryo, and only a thin aleurone layer remains.
The endosperm is considered to support embryo growth and to regulate nutrient
transfer from the mother to the developing seeds
(Lopes and Larkins, 1993).
Mutations in genes of the FERTILIZATION INDEPENDENT SEED
(FIS) class can form diploid endosperm in the absence of
fertilization (Ohad et al.,
1996; Chaudhury et al.,
1997). Thus far, four FIS-class genes are known:
MEDEA (MEA), FERTILIZATION INDEPENDENT ENDOSPERM
(FIE), FERTILIZATION INDEPENDENT SEED2 (FIS2) and
MULTICOPY SUPPRESSOR OF IRA1 (MSI1)
(Grossniklaus et al., 1998;
Kiyosue et al., 1999;
Luo et al., 1999;
Ohad et al., 1999;
Köhler et al., 2003a;
Guitton et al., 2004). Among
fis mutants, the msi1 mutant has the strongest penetrance of
the autonomous endosperm development phenotype
(Köhler et al., 2003a;
Guitton et al., 2004) and also
forms parthenogenetic embryos (Guitton and
Berger, 2005). The FIS-class genes encode proteins with
homology to animal Polycomb group (PcG) proteins. Plant FIS proteins and
animal PcG proteins both form multisubunit complexes with a core size of 600
kDa called Polycomb repressive complex 2 (PRC2)
(Köhler et al., 2003a;
Chanvivattana et al., 2004;
Schwartz and Pirrotta, 2007).
Animal PRC2 complexes possess histone methyltransferase activity specific for
lysine 27 on histone H3 (H3K27) and possibly also H3K9
(Schwartz and Pirrotta, 2007).
Similarly, plant PRC2 complexes, such as the FIS complex and the EMF2 complex,
are required for H3K27 methylation and transcriptional repression of target
genes (Gehring et al., 2006;
Makarevich et al., 2006;
Schönrock et al., 2006;
Schubert et al., 2006).
Mutations in FIS genes cause parent-of-origin-dependent seed
abortion. All seeds that inherit a mutant fis allele from the mother
abort, regardless of the presence of a wild-type paternal allele. Development
of fis mutant seeds is delayed and seeds abort with embryos arrested
at late heart stage and displaying non-cellularized endosperm with strongly
overproliferated chalazal endosperm domains
(Grossniklaus et al., 1998;
Kiyosue et al., 1999;
Köhler et al., 2003a;
Guitton et al., 2004). The
maternal-effect parent-of-origin-dependent seed abortion in mea and
fis2 mutants can be explained by the findings that MEA and
FIS2 are imprinted genes, with the paternal allele of both genes
being specifically silenced in the endosperm
(Vielle-Calzada et al., 1999;
Kinoshita et al., 1999;
Luo et al., 2000;
Jullien et al., 2006a).
Similarly, the paternal FIE allele is not expressed during early
stages of seed development, providing an explanation for the maternal effect
of fie mutants (Yadegari et al.,
2000).
It is likely that MEA and FIS2 are subunits specific to the FIS complex,
whereas FIE and MSI1 are part of several distinct PRC2-like complexes
(Hennig et al., 2005;
Schubert et al., 2005).
Furthermore, MSI1 is potentially part of several different complexes, such as
chromatin assembly factor CAF-1, histone deacetylases and chromatin-remodeling
machines, which are likely to play a role during early embryogenesis
(Hennig et al., 2005). Similar
to mea, fis2 and fie mutants, lack of MSI1 function causes
parent-of-origin-dependent seed abortion. However, in addition to the
gametophytic effect, it has been proposed that lack of MSI1 function also
causes a sporophytic effect on seed development
(Guitton et al., 2004). Thus,
lack of both maternal and paternal MSI1 alleles causes a
significantly stronger defect than lack of the maternal MSI1 allele
alone. This implies that the paternal allele of MSI1 is active, but
fails to complement the maternal gametophytic msi1 defect.
To test this idea, we investigated the temporal requirements of MSI1 during seed development. We specifically addressed the question of whether early paternal expression of MSI1 is sufficient to rescue the maternal-effect msi1 seed abortion phenotype. Our data clearly show that MSI1 is not an imprinted gene and that early paternal MSI1 expression is not sufficient to rescue msi1 mutant seeds. By contrast, expression of MSI1 in msi1 female gametophytes is necessary to restore normal seed development, revealing that the female gametophytic effect of fis mutants is caused by a functional requirement for an intact FIS complex in the female gametophyte. Thus, FIS complex function in the female gametophyte before fertilization determines seed development after fertilization, establishing fis mutants as true epigenetic female gametophytic maternal-effect mutants.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The silent MSI1* allele, which encodes a wild-type MSI1
protein, is a TILLING (Induced Local Lesions IN Genomes) mutant obtained from
the Nottingham Arabidopsis Stock Centre, stock number CS92951. The
Q0990, M0221 and M0223 enhancer-trap lines expressing the GFP reporter protein
were generated in the laboratory of J. Haseloff
(http://www.plantsci.cam.ac.uk/Haseloff)
and obtained from the Nottingham Arabidopsis Stock Centre. The
SCR::YFP reporter line was kindly provided by Dr B. Scheres (Utrecht
University, The Netherlands). The DR5rev::GFP reporter line was
generously given by Dr J. Friml (University of Tübingen, Germany). The
WOX8::YFP reporter line contains 2511 bp of the WOX8
promoter and 1775 bp of WOX8 coding sequence fused to the
YFP reporter and recapitulates the WOX8 expression pattern
(Haecker et al., 2004) (H.B.
and T.L., unpublished). Marker lines were introduced into the msi1
background by crossing. Plants were grown in a greenhouse at 70% humidity and
daily cycles of 16 hours light at 21°C and 8 hours darkness at 18°C.
Developed gynoecia were emasculated and hand-pollinated 1 day after
emasculation. For RNA expression analysis, three gynoecia or siliques were
harvested at the indicated time points. Dissection of seeds into embryo and
endosperm plus seed coat fractions was performed under a dissection
microscope. Dissected material from 100 seeds was collected in RNAlater
(Ambion, Austin, USA) solution and processed as indicated below. For
transmission analysis of the msi1 mutant allele, which is tagged with
a phosphinothricin resistance marker, seeds derived from reciprocal crosses
were harvested 3 weeks after pollination. T1 seeds were plated on Murashige
and Skoog (MS) medium containing 30 mg/l phosphinothricin, and after
approximately 2 weeks the ratio of resistant to non-resistant seedlings was
scored.
Plasmid constructs and generation of transgenic plants
To generate the PHE1::MSI1 construct, the 3.0 kb of sequence
upstream of the PHE1 translational start was amplified by PCR
introducing EcoRI and NcoI restriction sites. The
MSI1 cDNA was amplified by PCR introducing NcoI and
BglII restriction sites. Both fragments were ligated into pCAMBIA
1380 using EcoRI and BglII restriction sites. To generate
the DD46::GUS construct, the region 900 bp upstream of the
DD46 translational start was amplified by PCR introducing
BamHI and NcoI restriction sites and the fragment introduced
into pCAMBIA 1381z using BamHI and NcoI. To generate the
DD46::MSI1 construct, pCAMBIA 1380 was opened with BamHI and
BglII and the DD46 promoter fragment flanked by
BamHI and NcoI restriction sites and the MSI1 cDNA
flanked by NcoI and BglII sites were introduced by ligation.
All primers are listed in Table
1. Heterozygous msi1 plants were transformed by floral
dip, and transgenic plants were selected on MS medium containing 30 mg/l
hygromycin. T1 plants were treated with BASTA to select for the msi1
mutation and resistant lines were assayed for complementation of seed
abortion.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Guitton et al., 2004).
Self-fertilized msi1 mutant plants form two classes of aborting
seeds: an early-aborting class, which contains grossly abnormal embryos, and a
late-aborting class, which contains embryos that phenotypically closely
resemble fis-class mutant embryos
(Köhler et al., 2003a;
Guitton et al., 2004). It has
been suggested that lack of MSI1 function has a gametophytic as well as a
sporophytic zygotic effect, causing the formation of early- and late-aborting
seeds, respectively (Guitton et al.,
2004). This model predicts that 50% of the seeds inherit a
maternal MSI1 allele and develop normally, whereas 50% of the seeds
inherit a maternal msi1-1 allele and abort early if also inheriting a
paternal msi1-1 allele or abort late if inheriting a paternal
MSI1 allele. We observed that the msi1-1 allele has 17%
early-aborting and 33% late-aborting seeds, which deviates from the ratio of
25% early- to 25% late-aborting seeds predicted by the model [n=583,
2=29.72> 20.05(2)=5.991;
Fig. 1A]. One reason for this
discrepancy could be a reduced transmission of the paternal msi1-1
allele. We tested this hypothesis by determining the transmission of the
msi1-1 allele through pollen. Indeed, we found that the transmission
of the paternal msi1-1 allele is reduced to 72% (n=500).
Taking the reduced transmission of the paternal msi1-1 allele into
account, only 18% homozygous msi1 mutant seeds can be expected. This
number closely matches the observed number of 17% early-aborting seeds [50%
wild type: 32% msi1/MSI1: 18% msi1/msi1;
n=583, 2=0.536<
20.05(2)=5.991]. To unequivocally test the
hypothesis that early-aborting seeds require a paternally inherited
msi1 allele, we pollinated heterozygous msi1 mutant plants
with wild-type pollen. In this experiment, 51% of the seeds were
phenotypically wild type and 49% of the seeds were late aborting with a
fis-like phenotype [50% wild type: 50% msi1/MSI;
n=487, 2=0.166<
20.05(1)=3.841;
Fig. 1B,C;
Table 2]. We did not observe
any early-aborting seeds, clearly proving that loss of both maternal and
paternal MSI1 alleles is the prerequisite for early seed abortion.
This result suggests that the paternal MSI1 allele is expressed and,
consequently, that MSI1 is not regulated by genomic imprinting, in
contrast to the fis-class mutants mea and fis2.
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).
As MSI1 is also expressed in sporophytic tissues
(Hennig et al., 2003;
Köhler et al., 2003a),
transcripts of maternal MSI1 or MSI1* alleles are
contributed by zygotic tissues as well as maternal sporophytic tissues.
Therefore, the maternal alleles always yielded signals of higher intensity
than the paternal alleles.
Imprinting of several genes has been shown to occur specifically in the
endosperm, whereas the same genes are biallelically expressed in the embryo
(Kinoshita et al., 1999;
Kinoshita et al., 2004;
Haun et al., 2007).
MSI1 is expressed in the embryo
(Köhler et al., 2003a);
therefore, we investigated whether expression of the paternal MSI1
allele is confined to the embryo and is silenced in the endosperm, or whether
the paternal MSI1 allele is also expressed in the endosperm. For this
purpose, we performed crosses of wild-type plants with
MSI1* plants and dissected F1 seeds at 6 days after
pollination (DAP) into embryo and endosperm plus seed coat fractions. As shown
in Fig. 2C, we could clearly
detect expression of the paternal MSI1* allele in the
embryo as well as in the endosperm. Thus, we conclude that MSI1 is
not imprinted, but is biallelically expressed in both embryo and
endosperm.
The female gametophytic defect of msi1 mutants does not affect embryo patterning
Heterozygous msi1 mutant seeds abort with embryos arrested at late
heart stage and displaying strongly overproliferated chalazal endosperm
domains (Köhler et al.,
2003a; Guitton et al.,
2004). However, it remains elusive why msi1 mutant
embryos arrest development and abort despite expression of the paternal
MSI1 allele. It is possible that developmental defects start to
accumulate early during embryogenesis when most of the paternal genome,
including MSI1, is still inactive and cause gross developmental
abnormalities later in embryogenesis, culminating in seed abortion. Therefore,
we tested whether marker genes that define major developmental steps during
early embryogenesis are correctly expressed in msi1 mutant as
compared with wild-type embryos. We tested markers for auxin distribution
[DR5 (Friml et al.,
2003)], the developing suspensor [WUSCHEL-RELATED HOMEOBOX
8 (WOX8) (Haecker et al.,
2004)], provascular tissue [enhancer-trap line Q0990
(Weijers et al., 2006)], the
quiescent center [SCARECROW
(Blilou et al., 2005)], and for
cells within the region to form the shoot apical meristem [enhancer-trap lines
M0221 and M0223 (Cary et al.,
2002)].
The auxin-reporting DR5::GFP marker was confined to the root pole,
cotyledon tips and provascular tissue of heart stage wild-type embryos
(Fig. 3). This pattern was
similar in msi1 embryos, suggesting that auxin distribution is mostly
normal in msi1. Expression of the WOX8 reporter was confined
to the suspensor in wild-type and msi1 mutant embryos, indicating
that the basal derivatives of the zygote forming the suspensor are correctly
established. The enhancer-trap line Q0990 from the publicly available Haseloff
collection
(http://www.plantsci.cam.ac.uk/Haseloff/construction/catalogFrame.html)
is expressed in pro - vascular cells of the central region immediately
adjacent to the hypophysis (Weijers et
al., 2006). Because this expression pattern remained in
msi1, specification of provascular cells seems to occur properly in
msi1 mutant embryos.
Establishment of root apical meristems was monitored using
SCR::YFP, which is expressed only in the quiescent center and
derivatives of the ground meristem
(Wysocka-Diller et al., 2000).
Expression of SCR::YFP in msi1 closely resembled expression
in wild type, suggesting that initiation of the root apical meristem is
largely normal in msi1. To monitor formation of shoot apical
meristems, enhancer-trap lines M0221 and M0223 were used. Both lines show GFP
reporter activity in cells within the region forming the shoot apical
meristem, and M0223 reflects expression of CUP-SHAPED COTYLEDON1
[CUC1 (Cary et al.,
2002)]. As with the other markers used, activity of M0221 and
M0223 was similar in wild type and msi1, indicating that progenitor
cells for the shoot apical meristems are properly specified.
Based on these findings, we conclude that defects established in the msi1 female gametophyte do not affect basic embryo pattern formation, and embryo arrest at late heart stage is caused by mechanisms that remain to be identified.
Paternal expression of MSI1 immediately after fertilization cannot rescue the msi1 female gametophytic defect
We considered two possible explanations for the female gametophytic defect
of msi1 mutants: (1) delayed expression of the paternal MSI1
allele at only 3 DAP (Fig. 2B)
is responsible for the female gametophytic defect; or (2) lack of functional
MSI1 causes a defect in the female gametophyte and the consequences of this
defect become obvious during later stages of seed development. We tested the
first possibility by expressing paternal MSI1 immediately after
fertilization. We made use of the PHERES1 (PHE1) promoter,
which is one of the few promoters escaping early paternal silencing and is
expressed immediately after fertilization
(Köhler et al., 2005). We
tested whether expression of MSI1 under control of the PHE1
promoter (referred to as PHE1::MSI1) could be detected immediately
after fertilization, by crossing wild-type plants with pollen derived from
PHE1::MSI1 transgenic plants. Indeed, expression of the paternal
MSI1 allele was detected at 1 DAP
(Fig. 4A). We compared the
expression level of the PHE1::MSI1 transgene with the endogenous
MSI1 allele starting at 3 DAP by semi-quantitative RT-PCR. Taking
into account the different amplification efficiencies of the different primer
pairs, expression of the PHE1::MSI1 transgene was about 3.5-fold
higher than that of the endogenous paternal MSI1 allele. In
heterozygous msi1 plants, 50% of the seeds carry a maternal
msi1 allele and thus suffer from the female gametophytic defect. If
early paternal expression of MSI1 could rescue this female
gametophytic msi1 phenotype, then we would expect that a hemizygous
PHE1::MSI1 construct would rescue 25% of the seeds and lead to 75%
normal seeds. However, among 11 independent transgenic PHE1::MSI1
lines in an msi1 mutant background, we did not identify any plant
with more than 50% normal seeds, indicating that early paternal expression is
not sufficient to rescue the gametophytic msi1 mutant defect.
Instead, we observed a reduction in the number of early-aborting seeds by
about half, suggesting that paternally expressed PHE1::MSI1 is
sufficient to promote development of early-aborting homozygous msi1
mutant seeds up to the stage of late-aborting heterozygous msi1 seeds
(data not shown). This hypothesis was tested by pollinating msi1
mutant plants with pollen of three independent homozygous PHE1::MSI1
transgenic lines in an msi1 mutant background and scoring subsequent
seed development. In contrast to pollination with pollen from heterozygous
msi1 plants, which led to 17% early-aborting seeds, after pollination
with pollen from PHE1::MSI1; msi1 transgenic lines no early-aborting
seeds were observed (Fig. 4B,D;
Table 2). Thus, early paternal
MSI1 expression is sufficient to establish prolonged development of
homozygous msi1 mutant seeds. We analyzed seeds of this cross by
clearing and found no significant change of seed development as compared with
seeds developing on msi1 plants pollinated with wild-type pollen
(Fig. 4C). To unequivocally
test whether early paternal MSI1 expression can rescue the
msi1 mutant phenotype, we tested transmission of the maternal
msi1 allele after pollination of msi1 plants with pollen of
PHE1::MSI1 plants. We crossed msi1 plants with pollen of
three independent PHE1::MSI transgenic lines in a wild-type
background. Using more than 100 seedlings for each line, we found no
significant maternal transmission of msi1
(Table 3). Thus, we conclude
that early paternal MSI1 expression is not sufficient to rescue the
female gametophytic defect of msi1 mutant seeds.
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). We
established transgenic plants containing the DD46 promoter fused to
the ß-GLUCURONIDASE (GUS) reporter gene (referred to as
DD46::GUS) and investigated the temporal and spatial expression
pattern of this reporter construct. Before fertilization, we detected GUS
activity in the central cell, in the synergids and in the egg cell. After
fertilization, GUS expression ceased and was almost undetectable within the
seed when the embryo had reached the globular stage, at about 2 DAP
(Fig. 5A). We confirmed this
expression pattern using microarray data obtained from different reproductive
stages of Arabidopsis development
(Hennig et al., 2004). Whereas
DD46 was highly expressed before fertilization, reduced transcript
levels were detectable after pollination
(Fig. 5B). Thus, the
DD46 promoter is specifically active in the female gametophyte and
expression ceases after fertilization.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). A
central role of MSI1 in plant development is supported by the observation that
reduced MSI1 levels in MSI1 cosuppression plants affect many aspects
of sporophytic plant development (Hennig
et al., 2003). Consistent with this idea is the observation that
transmission of the msi1 mutant allele through the male gametophyte
is significantly reduced, suggesting that lack of MSI1 function also impairs
male gametophyte development. We failed to observe a transmission defect of
the msi1 mutant allele through pollen in previous investigations
(Köhler et al., 2003a).
However, we noticed that transmission of the msi1 mutant allele
differs between self-pollinated and manually pollinated plants. One possible
explanation for this finding is that pollen used for manual pollination is
more mature than pollen of self-pollinated plants, suggesting that
msi1 mutant pollen development is delayed. Therefore, transmission
analysis in this study was performed with freshly shed pollen.
In pollen of FIE cosuppression plants, the paternally silenced
MEA allele becomes reactivated
(Jullien et al., 2006b),
suggesting that FIE is necessary for repression of MEA and other
paternally silenced genes in pollen. It is conceivable that this repression
requires a functional PRC2-like complex and that MSI1 is part of this complex.
Therefore, one possible function of MSI1 during pollen development could be
the repression of paternally imprinted genes like MEA and
FIS2. Alternatively, MSI1 could be needed for activity of CAF-1
during pollen development. However, when testing fas1-4 and
fas2-4 mutants (Exner et al.,
2006), which lack one or other of the two CAF-1 subunits, we did
not observe any transmission defect (data not shown). Future studies are
needed to clarify which molecular function of MSI1 is required during pollen
development. Such functions could include participation in PRC2-like
complexes, in CAF-1 or in other, uncharacterized complexes.
MSI1 is biallelically expressed in the embryo and the endosperm
It has been hypothesized that the maternal effect of fis mutants
is caused by lack of expression of paternal FIS alleles and, indeed,
the paternal alleles of MEA and FIS2 are imprinted
(Vielle-Calzada et al., 1999;
Kinoshita et al., 1999;
Luo et al., 2000;
Jullien et al., 2006a).
However, our results demonstrate that this does not apply to all fis
mutants. We show that MSI1 is not paternally imprinted, but is
clearly biallelically expressed in embryo and endosperm. The accumulation of
transcripts of the paternal MSI1 allele is delayed relative to that
of the maternal MSI1 allele. However, the timing of paternal
MSI1 expression is comparable to that of a large number of genes
investigated thus far (Vielle-Calzada et
al., 2000). Thus, MSI1 is not paternally imprinted.
Expression of the paternal allele of the FIS-class gene FIE also
occurs around 2-3 DAP, and it has been discussed that the parent-of-origin
effect on seed development in fie and mea mutants is caused
by different mechanisms (Yadegari et al.,
2000). However, it has not been investigated whether delayed
expression of the paternal FIE allele is responsible for the
parent-of-origin effect of fie mutants. We tested whether delayed
expression of the paternal MSI1 allele is responsible for the
msi1 mutant phenotype by expressing MSI1 under the control
of a promoter that is paternally active immediately after fertilization. As
early paternal MSI1 expression did not rescue seed development, we
conclude that MSI1 functions in the female gametophyte and establishes gene
expression patterns that are required for development of the seed after
fertilization. Interestingly, we also did not observe rescue of seed
development when expressing the FIS2 gene under control of the
PHE1 promoter (data not shown). In contrast to the biallelically
expressed MSI1 gene, the paternal allele of FIS2 is not
active in the endosperm; thus, FIS2 is paternally imprinted
(Luo et al., 2000;
Jullien et al., 2006a).
Nonetheless, early paternal expression is not sufficient to rescue the
fis2 mutant phenotype. Therefore, we conclude that paternal
imprinting of FIS genes does not cause the parent-of-origin effect on
seed development. Instead, the parent-of-origin effect of fis mutants
is caused by lack of expression of FIS genes in the female
gametophyte.
MSI1 activity in the female gametophyte affects seed development
PRC2-like complexes have histone methyltransferase activity, and this
activity of the FIS complex appears necessary for normal seed development
(Gehring et al., 2006;
Makarevich et al., 2006). It
is likely that genes marked by histone methylation in the female gametophyte
need to be kept silent after fertilization. Indeed, the FIS target gene
PHE1 is methylated in the female gametophyte before fertilization and
lack of FIS function causes strong overexpression of PHE1 after
fertilization (Köhler et al.,
2003b; Makarevich et al.,
2006). Thus, the FIS complex establishes epigenetic modifications
on its target genes that cause stable silencing during subsequent cell
divisions. This function is consistent with the proposed role of PRC2
complexes in animals to stably maintain established repressive transcriptional
states (Bantignies and Cavalli,
2006). A similar function has been assigned to the PRC2-like
complex containing the FIS2 homolog VERNALIZATION2 (VRN2)
(Gendall et al., 2001). VRN2
is required for the vernalization-dependent stable repression of the
FLOWERING LOCUS C (FLC) gene. In vrn2 mutants, the
initial repression of FLC after vernalization is not impaired;
however, FLC repression is not stably maintained during subsequent
periods of warm conditions (Gendall et
al., 2001).
Function of the FIS complex after fertilization
All FIS genes are also expressed after fertilization in the
endosperm (Kinoshita et al.,
1999; Vielle-Calzada et al.,
1999; Luo et al.,
2000; Köhler et al.,
2003a), suggesting that the FIS complex has additional functions
after fertilization, and it has been demonstrated that the FIS complex is
necessary for suppression of the paternal MEA allele in the endosperm
(Gehring et al., 2006;
Jullien et al., 2006b).
Comparing the PHE1::MSI1 and the DD46::MSI1 constructs, we
found that expression of MSI1 before fertilization in msi1
mutant gametophytes is necessary to restore wild-type seed development. As
DD46 is also active after fertilization, we could not address the
question of whether expression of MSI1 in the female gametophyte is
also sufficient to rescue the msi1 maternal gametophytic defect.
However, given that FIS genes are expressed after fertilization and
that the FIS complex is functionally active, we consider this possibility as
rather unlikely.
Seeds lacking a functional FIS complex have strongly overproliferated
chalazal endosperm domains similar to those of seeds resulting from
interploidy crosses of diploid maternal plants pollinated with pollen from
tetraploid plants (Scott et al.,
1998). Therefore, it has been hypothesized that the FIS complex
regulates genomic imprinting and represses transcription of loci in the
maternally derived genome that are normally expressed only when paternally
contributed (Spielman et al.,
2001). Consistent with this prediction is the expression of the
FIS target gene PHE1, which is maternally repressed and paternally
active (Köhler et al.,
2005). Furthermore, pollination of fis mutants mea,
fie and fis2 with pollen of the cdka;1 mutant that only
forms one generative cell causes the formation of viable seeds containing a
normal zygotic embryo and homodiploid endosperm. Thus, bypassing the paternal
contribution can rescue fis mutant seeds, providing strong support
for this hypothesis (Nowack et al.,
2007). Therefore, it is likely that FIS complex-mediated genomic
imprinting of PHE1 and other, as yet unidentified, genes is
established in the female gametophyte and is maintained by the FIS complex
after fertilization.
Embryo patterning is not affected in msi1 mutant embryos
After fertilization, the FIS complex mainly acts in the endosperm and
fis mutants, including msi1, have defects in endosperm
development (Grossniklaus et al.,
1998; Kiyosue et al.,
1999; Köhler et al.,
2003a; Guitton et al.,
2004). Abortion of fis seeds is preceded by an arrest in
embryo development, and we hypothesized that defects established in
msi1 mutant gametophytes affect embryo pattern formation and cause
developmental arrest of heterozygous msi1 mutant embryos. However,
all markers of early embryo development and cellular differentiation tested in
this study were expressed with similar patterns in wild-type and msi1
mutant embryos. We did not observe changes in expression of marker genes for
auxin distribution, shoot and root apical meristem regions, provascular
tissues and suspensor identity, indicating that there are no major defects in
the establishment of the apical-basal axis or radial pattern formation.
Therefore, we hypothesize that the female gametophytic defect caused by the
msi1 mutation does not directly impact on embryo pattern formation
and that embryo arrest occurs by as yet undefined mechanisms. By contrast,
homozygous msi1 mutant embryos arrest development after only a few
cell divisions and with severe developmental aberrations
(Köhler et al., 2003a),
consistent with the role of MSI1 in FIS-independent complexes.
There are two possible explanations for the developmental arrest of
heterozygous msi1 mutant embryos: (1) the arrest occurs after pattern
formation by an inherent defect of the embryo; or (2) embryo arrest is caused
by an external defect, i.e. in the endosperm. Several observations favor the
second hypothesis. Embryo arrest of fis mutant seeds occurs at late
heart stage. Whereas the endosperm of wild-type seeds starts to cellularize at
this stage and nuclei proliferation ceases
(Brown et al., 1999;
Boisnard-Lorig et al., 2001),
endosperm of fis mutants does not undergo cellularization and instead
continues to divide (Kiyosue et al.,
1999). The endosperm of many dicotyledonous species such as
Arabidopsis is non-persistent and is considered as a transient medium
supporting embryonic morphogenesis and early maturation by controlling the
flux of nutrients delivered from the maternal plant to the developing embryo
(Lopes and Larkins, 1993). The
embryo is surrounded by the endosperm and both embryo and endosperm need to
coordinate their development in order to produce viable seeds.
Hyperproliferation of the endosperm caused by an increased paternal dosage
also inhibits embryo growth, suggesting that increased proliferation of the
endosperm is detrimental for embryo development
(Scott et al., 1998).
Conversely, bypassing the paternal contribution in fis; cdka;1
double-mutant seeds restores almost wild-type-like embryo development
(Nowack et al., 2007). It is
conceivable that prolonged proliferation of the endosperm deprives the embryo
of nutrients or, alternatively, that the endosperm does not reach the
appropriate developmental stage to deliver nutrients to the developing embryo.
Therefore, we suggest that lack of the FIS complex in the female gametophyte
causes abnormal gene expression patterns in the central cell that persist
after fertilization and produce defects in the endosperm that ultimately
trigger arrest of embryo development and seed abortion.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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