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First published online October 26, 2007
doi: 10.1242/10.1242/dev.007310
1 Section of Plant Biology, University of California, One Shields Avenue, Davis,
CA 95616, USA.
2 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724,
USA.
3 Institute of Plant Biology and Zürich-Basel Plant Science Center,
University of Zürich, Zollikerstrasse 107, CH-8008 Zürich,
Switzerland.
4 Department of Plant Sciences, University of California, One Shields Avenue,
Davis, CA 95616, USA.
* Author for correspondence (e-mail: sundar{at}ucdavis.edu)
Accepted 27 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Embryogenesis, Autonomous endosperm, Maternal effect, Paternal allele activation, Fertilization, Plant reproduction, Seed development
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The developmental programs of embryo and endosperm require both parental
genomes. The extent to which each genome contributes to these two major seed
components might not be equivalent, especially during early seed development
before the embryo heart stage. In Arabidopsis thaliana, dominant
maternal control of endosperm and embryo development has been demonstrated by
the FERTILIZATION-INDEPENDENT SEED (FIS) class of genes,
which includes the homologs of the genes encoding the Drosophila and
mammalian Polycomb repressive complex 2 (PRC2) components MEA,
FIS2, FIE and MSI1 (reviewed by
Pien and Grossniklaus, 2007).
Mutations in any of these genes cause common mutant phenotypes of seeds with
autonomous endosperm, abnormal cellular proliferation of fertilized embryos
and endosperm, arrested heart-staged embryos, and ultimate seed abortion.
Mutant phenotypes result only when the genetic lesions are present in the
maternal allele inherited from the female gametophyte. In the case of
MEA and FIS2, the gene products are supplied during early
seed development only by the maternal alleles. Both genes are already
expressed before fertilization in the embryo sac but MEA, and
probably FIS2, also show imprinted maternal expression after
fertilization in the developing seeds
(Jullien et al., 2006b;
Kinoshita et al., 1999;
Luo et al., 2000;
Vielle-Calzada et al., 1999).
Gametophytic paternal effects on seed development have not been reported;
however, preferential paternal expression in early seeds has been documented
for PHERES1 (PHE1), a MADS box gene
(Köhler et al., 2003b).
Even in this case, only the expression of the maternal allele has been
demonstrated to affect embryo and endosperm development. Maternal
PHE1, a direct downstream target of maternal MEA repression,
is strongly upregulated in mea seeds and partially rescues
mea seed abortion when this deregulated expression is suppressed
(Köhler et al., 2003b;
Köhler et al., 2005).
In this study, we describe a gametophytic maternal effect mutant, glauce (glc), where the embryo develops in the absence of endosperm. In addition to affecting the fertilization of the central cell, glc genetically counteracts the mutants of the FIS class that control fertilization-independent endosperm formation and postfertilization embryo development. The maternally inherited glc mutant is defective in the embryonic paternal expression of PHE1 and the bi-parentally expressed genes RPS5a and FAC1, which are important for early embryo and endosperm development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). mea-8 was supplied by the ABRC stock center as
line SAIL_55_B04. Transgenic FIS2::GUS, FAC1::GFP-GUS, CYCB1;1::GUS,
PHE1::GUS and PIN7::PIN7-GUS seeds were gifts from Abed
Chaudhury (CSIRO, Canberra, Australia), Chun-Ming Liu (Plant Research
International, Wageningen, The Netherlands), Celia Baroux (Institute of Planty
Biology, Zürich, Switzerland), Claudia Köhler (Institute of Plant
Science, ETH Zürich, Switzerland) and Jirí Friml (University of
Tübingen, Germany), respectively. Except for
CYCB1;1::GUS and mea-8 lines, which were both in
Columbia (Col) background and FIS2::GUS line in C24 background, all
other lines in this study were in Landsberg erecta (Ler)
background. Plants with the glc, msi1-3
(Pagnussat et al., 2005),
mea-1 (Grossniklaus et al.,
1998) alleles were selected on 50 mg/l kanamycin, mea-8
on 10 mg/l glufosinate and PHE1::GUS
(Köhler et al., 2003b) on
20 mg/l hygromycin.
Whole-mount ovule clearing and GUS assays
These procedures were carried out as described
(Pagnussat et al., 2005;
Yu et al., 2005).
Confocal laser-scanning microscopy and DNA quantification of nuclei in seeds
Propidium iodide staining and confocal laser-scanning microscopy procedures
were performed as described (Baroux et al.,
2007) without the enzymatic treatment step, for
glc/GLC seeds 1-2 days after pollination (DAP).
Pollen staining
Pollen grains from anthers of glc/GLC late-13-staged
flowers (Bowman, 1994) were
stained with 1 µg/ml DAPI (4',6-diamino-2-phenylindole) in the dark
for 1 hour, washed briefly in distilled water, and observed under a Zeiss
Axioskop 2 microscope with both fluorescence and DIC optics.
DNA extraction, PCR and Southern blot hybridization
Genomic DNA was extracted from fresh leaves and flowers with the GenElute
Plant Genomic DNA kit (Sigma, USA) or the Phytopure kit (Amersham,
Switzerland) following the manufacturer's protocol. PCRs were performed with
0.5 units of Taq polymerase in 1xPCR buffer containing 1.5 mM
MgCl2, 200 µM each dNTP and 10 pmol each primer. PCR parameters
were as follows: 94°C for 2 minutes, 30 cycles of 94°C/30 seconds,
52°C/30 seconds, and 72°C/1 minute, with a final extension of 72°C
for 3 minutes. Thermal asymmetric interlaced (TAIL)-PCR procedures have been
described previously (Parinov et al.,
1999). Primers for Ds insertion site verification,
cleaved amplified polymorphisms (CAPS) markers for the deletion at the
Ds locus of SET2030, and for amplifying Southern probes will be
provided upon request. Southern blot analysis was performed with the DIG-Easy
Hyb Kit (Roche, Switzerland) following the manufacturer's protocol or as
described (Sambrook and Russell,
2001) with the DECAprime II labeling kit (Ambion, USA) and
[32P]dCTP (Perkin-Elmer, USA).
Tetraploid seed generation
Meristems of glc/GLC plants undergoing the transition from
vegetative to reproductive phase were treated with 0.25% colchicine. Seeds
from these treated plants were pooled and germinated on MS plates containing
50 mg/l kanamycin. Seedlings were scored for resistance or sensitivity at 12
days after plating.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
line displayed an aberrant kanamycin resistance:kanamycin sensitivity
(kanR:kanS) ratio of 0.64:1 instead of 3:1
(Table 1). Reciprocal crosses
of SET2030 heterozygous plants and wild-type plants showed a severe reduction
in female transmission (15%) and moderate reduction in male transmission (49%)
(Table 1). To examine the cause
of the female sterility, we cleared SET2030/+ siliques with Hoyer's solution
(Liu and Meinke, 1998) and
studied the ovule phenotype by light microscopy under Nomarski optics. Two
days after early-12-staged flowers (Bowman,
1994) of SET2030/+ plants were emasculated, the embryo sacs in all
ovules of the same siliques displayed wild-type morphology with four typical
cells of a wild-type mature embryo sac: a central cell, an egg cell and two
synergid cells (Fig. 1A). The
correct cell identities of the SET2030 embryo sac were confirmed with five
marker lines expressing GUS specifically in the central cell
[FIS2::GUS (Luo et al.,
2000) and MEA::GUS
(Spillane et al., 2004)], the
egg cell (ET1119 and ET1086), and the synergids (ET2634) in approximately 50%
of ovules from SET2030/+ plants hemizygous for these marker lines
(Fig. 1B-F).
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62% (n=158) of the seeds from the same silique appeared
wild-type, the remaining seeds were smaller, with embryos arrested at various
stages up to the globular stage with defective endosperm (see below) and
therefore were considered as mutant seeds
(Fig. 2). In general, embryos
of mutant seeds lagged one stage behind embryos of wild-type seeds in the same
silique. At 2.5 DAP, when most wild-type seeds of the heterozygous siliques
had reached the 8- and 16-cell embryo stages, mutant seeds mainly were at the
quadrant and octant stage (Table
2). Later, when wild-type seeds had reached the globular stage,
most mutant seeds had collapsed, but in about 8% of mutant seeds
(n=76), embryo development could proceed to the pre-globular or
globular stage. Embryos of mutant seeds up to the pre-globular stage looked
morphologically similar to wild-type pre-globular embryos
(Fig. 2B-F), but by the late
globular stage displayed some disorganization
(Fig. 2G,
Fig. 2I compared with
Fig. 2J). Notably, in most
mutant seeds, the central cell was totally devoid of endosperm development;
instead, there was a single large nucleus, which could be either the
unfertilized central cell nucleus or the fertilized, non-dividing primary
endosperm nucleus (Fig. 2B-G,
Table 2). A small proportion of
mutant seeds had 2-8 endosperm nuclei of equal or unequal size
(Fig. 2H,
Table 2). These embryo and
endosperm defects remained unchanged regardless of whether SET2030/+ flowers
were pollinated with self pollen or wild-type pollen.
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). We found
that the 3' end of the Ds element inserted into the first exon
of At1g65200, and the Ds 5' end at 800 bp
upstream of At1g66030, suggesting that 350 kb of DNA between
these two genes might have been deleted at this Ds locus
(Fig. 3A). To confirm this
deletion, we pollinated Col wild-type flowers with glc/GLC
pollen in a Ler background and tested for the presence or absence of
the Ler alleles in the F1 hybrid
glc(Ler)/GLC(Col) of nine genes distributed
throughout this putative deletion (see
Page et al., 2004). We used
the BlastDigester software (Ilic et al.,
2004) to design the cleaved amplified polymorphisms (CAPS) markers
for the single nucleotide polymorphisms (SNPs) in these genes between
Ler and Col ecotypes that can be distinguished by restriction
digestions of the PCR products. We found that for the five genes distributed
in the 215 kb of genomic DNA towards the 3' Ds, only the
Col alleles were present in the F1 hybrid
(Fig. 3A), indicating that this
DNA is deleted in glc. The other four genes in the 135 kb of
genomic DNA towards the 5' Ds showed both Ler and Col
alleles in the F1 hybrid (Fig.
3A), suggesting that this segment of DNA is either duplicated
elsewhere in the Ler genome or was transposed by the Ds
insertion to a new location. Further characterization was performed by
Southern blot analysis of glc/GLC and wild-type genomic DNA digested
with several restriction enzymes within the Ds element and the
regions flanking both sides of the Ds, using DNA probes that
hybridized to the Ds element or the flanking regions. The
Ds-specific probe indicated only one Ds copy in glc
plants (Fig. 3B). The
flanking-region probes revealed the restriction patterns consistent with the
predicted genomic sequences in the immediate vicinity (3-4 kb) on both sides
of the Ds (see Fig. S2 in the supplementary material). These results
suggest that the Ds insertion did not generate further rearrangements
flanking the site of insertion.
We then investigated the possibility that the glc phenotype does
not arise from the deletion but from a second site mutation linked to the
identified Ds locus, possibly arising from a Ds footprint.
We looked for recombinants between glc and the Ds insertion
in the F1 progeny of crosses between glc/GLC male and Ler
wild-type female plants. Several independent lines of two recombinant types
were recovered at rates of 1%. Type I recombinant plants were kanR
indicating the presence of the Ds element (see Fig. S3A in the
supplementary material), but were phenotypically wild type. Type II
recombinant plants were kanS indicating the absence of the Ds element
(see Fig. S3A in the supplementary material), but the mutant phenotype was
maintained. Next, we inspected whether the deletion at the Ds locus
was still present in these recombinants by examining the Ler CAPS
markers spanning the deletion in F1 hybrids of the recombinants with wild-type
Col, as for the original glc mutant line. We found that all
Ler CAPS markers in the deletion were now present in type I
recombinants but still absent in type II recombinants (see Fig. S3B in the
supplementary material). These data indicated that the glc mutant
phenotype is correlated with the absence of a DNA segment rather than the
presence of the Ds element. We further confirmed the recovery of the
deleted DNA in type I recombinant plants by Southern blot analysis with probes
hybridizing to the DNA corresponding to the deletion: the band signal
intensity for type I recombinant plants was in the intensity range of
wild-type Ler plant and approximately twice as strong as that of the
original glc mutant line and of the type II recombinant plants (see
Fig. S3C in the supplementary material). Taken together, these results
indicate that the glc phenotype in the original glc mutant
line and in the type II recombinant lines was caused by the deletion
associated with the Ds insertion, and the phenotypic rescue occurring
in type I recombinants resulted from the recovery of the DNA in this deletion,
possibly arising through unequal crossover (see Discussion). We also assessed
the glc mutation for recessivity/dominance in diploid
glc/GLC gametophytes from tetraploid flowers created by colchicine
treatment of the meristem of diploid glc/GLC plants. The kanR:kanS
ratios of plants grown from the mixed diploid and tetraploid seeds of three
independently treated plants were 2:1 (n=293), 1.5:1 (n=350)
and 3.6:1 (n=327) - much higher than, and significantly different
from, the 0.64:1 ratio of seeds of the original diploid glc/GLC
plants (P<0.0001 in all cases), suggesting a significantly
increased transmission of the glc mutant allele. This result
indicates that the presence of a wild-type GLC allele in heterozygous
glc/GLC gametophytes rescues the mutant effect of the glc
allele. Therefore, we conclude that glc is a recessive
loss-of-function mutation caused by the deletion of this DNA fragment.
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;
Baroux et al., 2001) pollen
expressed GUS from the paternal CYCB1:1 promoter
(Fig. 4A,B). This finding rules
out the possibility that glc embryos develop parthenogenically
because of the fertilization signals from the arriving sperm cells without
actually being fertilized. We did not observe GUS expression in endosperm of
wild-type seeds nor in the single nucleus of glc seeds. As in
wild-type embryos, the suspensor domain of glc embryos also properly
expressed the early embryo polarity marker gene PIN7
(Friml et al., 2003)
(Fig. 4C,D), indicating that
glc embryos initiate normal development.
Fertilization of the glc central cell is impaired
The central cells of glc embryo sacs appeared to differentiate
normally with correct cell identity, as evidenced by the GUS expression of the
two central cell-specific marker lines tested, FIS2::GUS and
MEA::GUS (Fig. 1B,C),
in about half of the ovules from glc/GLC siliques that were
hemizygous for either FIS2::GUS or MEA::GUS (119/240 and
117/250, respectively). However, glc central cells did not develop
into endosperm, raising the question of whether they were fertilized. The
single nucleus in the post-pollination glc central cell could be
either the unfertilized homo-diploid (2n) central cell nucleus or the
fertilized triploid (3n) primary endosperm nucleus. To distinguish
between these two possibilities, we examined the DNA content of this nucleus
in glc seeds stained with propidium iodide and optically sectioned by
confocal laser scanning microscopy (Barrell
and Grossniklaus, 2005). We used the DNA contents of 15 diploid
sporophytic nuclei in the integument cells of the same glc seed as
the reference for the diploid DNA content, for which the 2n values
range from 2C to 4C (Fig. 5,
and see Table S1 in the supplementary material). We then compared the DNA
content of the single nucleus in glc central cell of each seed with
this diploid standard reference of the same seed.
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To validate the accuracy of our DNA quantification method for nuclei, we also examined ten triploid endosperm nuclei from a fertilized wild-type seed at the one-cell embryo stage and compared their DNA contents with those of ten diploid integument nuclei of the same seed. We found that 9 out of 10 wild-type endosperm nuclei had DNA content higher than the 4C level of the integument nuclei and only one had this 4C level (Fig. 5G, and see Table S1 in the supplementary material). Of these nine nuclei, three were in the range of the 6C level (3n). None had higher than the 6C level or below 4C level, validating that our method authentically reflects the dynamic replication typical of wild-type syncytial endosperm. This result, therefore, supported the quantification of glc central cell nucleus and confirmed the non-fertilization status of the glc central cell in general, although fertilized glc central cells do occur infrequently.
glc suppresses autonomous endosperm development of mea and msi1 in the pre-fertilization central cell
The absence of endosperm development in glc seeds is in contrast
to the autonomous and over-proliferated endosperm phenotypes of the
gametophytic maternal effect fis class mutants mea, fis2,
fie and msi1 (reviewed by
Grossniklaus, 2005). This
prompted us to investigate the genetic interactions between glc and
these FIS class genes in the pre-fertilization central cell.
Specifically, we asked whether glc could suppress the autonomous
endosperm development of fis mutants. We generated doubly
heterozygous mutants of glc with mea-1
(Grossniklaus et al., 1998),
mea-8 (SAIL_55_B04, ABRC stock center) or msi1-3 [previously
reported as the mee70 mutant
(Pagnussat et al., 2005)] and
counted the number of whole-mount ovules 6-7 days after emasculation that
displayed more than one nucleus in the central cell. The proportions of
autonomous endosperm ovules in the single mutants of mea-1, mea-8,
and msi1-3 were 12.8%, 28.7% and 40.9%, respectively
(Table 3). If GLC
functions were not required in the endosperm repression pathway in the absence
of fertilization, these proportions should remain similar in the double
mutants. Conversely, if glc suppressed autonomous endosperm of
fis class mutants, the presence of glc in the double mutants
should reduce the fraction of ovules with autonomous endosperm because a
quarter of ovules in any silique is expected to carry both glc and
mea or glc and msi1, respectively. Consistent with
the second possibility, the proportions of ovules with autonomous endosperm
were reduced by nearly half in the double mutants of glc with
mea-1 (6.8%), mea-8 (16.8%), and msi1-3 (25.5%)
(Table 3). Therefore, we
conclude that GLC functions are required either downstream of the
FIS class genes, or independently in addition to the FIS
class genes for autonomous endosperm development in the central cell before
fertilization.
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). By contrast, in fertilized glc seeds, no endosperm
develops and glc embryo development progresses more slowly than in
the wild-type embryo, resulting eventually in embryo arrest
(Table 2). Therefore, we
investigated the epistasis between glc and mea in
whole-mount double mutant glc mea-1 seeds fertilized by wild-type
pollen at 2.5 DAP. At this time point, most seeds of mea-1 single
mutant and wild-type siliques (95% and 92%, respectively) were morphologically
normal, ranging from one-cell to pre-globular stages with normally
proliferating endosperm (Table
2). On the other hand, the glc single mutant had 62%
normal-looking seeds and 25% `no-endosperm' seeds
(Table 2). In the double mutant
glc mea-1, if glc is epistatic to mea at and after
fertilization, we expected the glc seed proportion to remain
unchanged. Conversely, if mea is epistatic to glc, we
predicted a reduction in the glc seed proportion and an increase in
normal-looking seed frequency. Compared with single mutants, we found no
evidence for significant changes in the double mutants, which had 65%
normal-looking seeds and 23% `no-endosperm' seeds
(Table 2), indicating that
glc is also epistatic to mea with respect to
post-fertilization seed development. Thus, we suggest that GLC
post-fertilization functions are also required either downstream of
MEA or, alternatively, in an independent antagonistic pathway.
Embryonic expression of the PHE1 gene is abolished in glc seeds
PHE1, a direct downstream repression target of MEA and
FIE, is expressed in both embryo and endosperm soon after
fertilization, peaks at the early globular stage and declines from the late
globular stage onwards (Köhler et
al., 2003b). Paternal PHE1 is unaffected by MEA,
but maternal PHE1 is partially repressed by maternal MEA
(Köhler et al., 2003b;
Köhler et al., 2005;
Makarevich et al., 2006). As
glc displays antagonistic effects to mea in both
unfertilized ovules (Table 3)
and fertilized seeds (Table 2),
we explored the effect of the glc mutation on embryonic PHE1
expression in fertilized seeds by monitoring PHE1 promoter activity
of either parental allele in glc seeds at the preglobular or early
globular stage, when PHE1 is most strongly expressed
(Köhler et al., 2003b;
Köhler et al., 2005). To
examine paternal PHE1, we pollinated glc/GLC
flowers with PHE1::GUS homozygous pollen. To observe
maternal PHE1, we crossed female glc/GLC
PHE1::GUS/- flowers with wild-type pollen. In siliques
from these crosses, wild-type seeds were easily distinguished from
glc seeds based on their size and the presence/absence of endosperm.
In both types of cross, we found GUS expression only in wild-type but not in
mutant seeds (Fig. 6),
indicating that maternal GLC functions are required for the
activation of both parental PHE1::GUS alleles in fertilized
seeds.
Paternal embryonic expression of the RPS5a and FAC1 genes is affected in glc seeds
Although GLC is required for embryonic PHE1 expression
(Fig. 6), the glc
mutation does not affect embryonic CYCB1;1 and PIN7
expression (Fig. 4). As
CYCB1;1 and PIN7 are expressed only in the embryo whereas
PHE1 is expressed in both embryo and endosperm, we assessed the
extent of the impact of GLC on two other genes, FAC1
(Xu et al., 2005) and
RPS5a (Weijers et al.,
2001), known to be expressed bi-parentally in both embryo and
endosperm from the zygotic and two-cell embryo stage onwards, respectively.
Embryos are arrested at the zygotic stage in fac1 mutant seeds when
both parental alleles are disrupted (Xu et
al., 2005), whereas embryo development proceeds as far as the
globular and walking stick stages in rps5a homozygous and
heterozygous mutants, respectively, the latter because of haploinsufficiency
(Weijers et al., 2001). We
examined the promoter activity of the FAC1 and RPS5a genes
in glc/GLC embryos, using plants carrying FAC1::GUS
or RPS5a::GUS fusions. We found that in glc/GLC embryos,
paternal expression of both FAC1 and RPS5a was severely
affected, whereas wild-type embryos at comparable stages showed strong
expression (Fig. 7). Paternal
FAC1::GUS was abolished (Fig.
7A,B), as was paternal RPS5a::GUS
(Fig. 7C,D), though the latter
showed sporadic expression in some glc embryos
(Fig. 7E). On the other hand,
maternal RPS5a promoter activity was not affected by glc
(Fig. 7F). Expression of
maternal FAC1::GUS could not be examined in embryos due to
interference by intensive FAC1::GUS expression in the
sporophytic integument tissue. These results suggest that maternal
GLC functions are required for the expression of the paternal alleles
of FAC1 and RPS5a.
We also assessed the requirement of maternal GLC functions for paternal PHE1, RPS5a, and FAC1 allele expression in the two types of recombinants described above (see Fig. S3 in the supplementary material). Similarly to the original glc mutant line, in mutant embryos of type II recombinant lines, where glc phenotype and the deletion at the Ds locus were still maintained (see Fig. S3 in the supplementary material), no paternal promoter activities of these three genes were detected (data not shown). Conversely, in embryos and endosperm of type I recombinant lines, where the phenotype was reverted to wild type and the DNA at the deletion was recovered (see Fig. S3 in the supplementary material), GUS expression reported by paternal promoters of these genes was detected in the majority of seeds as observed in seeds of wild-type plants (data not shown). Therefore, the recovery of the deleted DNA rescued not only the mutant phenotype but also the maternal activation of the paternal alleles of the examined genes, confirming that GLC functions reside within this deletion.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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215 kb at the Ds insertion site on chromosome 1. Phenotypic
rescue exhibited in the recombinants where the DNA in this deletion has been
recovered implies that glc is a recessive loss-of-function mutation.
Our recombinant data (see Fig. S3 in the supplementary material) are
consistent with an event in which the Ds insertion that generated the
deletion also transposed part of the DNA (135 kb) adjacent to the
telomeric side of the deletion to a centromeric site separated by 1 cM
(see Fig. S4 in the supplementary material). This model predicts that the two
types of recombinants observed will be generated at frequencies of 1%
through unequal crossover between the mutant chromosome and the wild-type
chromosome (see Fig. S4 in the supplementary material).
A deletion in another mutant, tons missing (tms), which
partially overlaps the centromeric side of the deletion in glc up to
At1g65330 (PHE1), results in embryo lethality
(Page et al., 2004). As
glc is a gametophytic mutant and primarily affects the endosperm,
this overlap region containing PHE1 cannot be the cause of
glc phenotype. In addition, we were unable to complement the
glc mutation using PHE1 alone (Q.A.N. and V.S.,
unpublished). However, we cannot rule out the fact that the glc
phenotype requires the function of more than one gene, for example, a gene
such as PHE1 that lies within the overlap with the tms
deletion and another gene that lies outside the region of overlap.
Communication flow between embryo and endosperm in glc early seed development
Successful fertilized seed development requires efficient coordination and
effective communication between the embryo, endosperm and seed coat. In
wild-type Arabidopsis, the seed initiation program commences with at
least three rounds of endosperm nuclear division before the zygote starts its
first division (Faure et al.,
2002; Mansfield and Briarty,
1990). This developmental progression could indicate that
endosperm formation is a requirement for the onset of embryogenesis. As was
proposed for agl80 mutant embryo sacs, a lack of endosperm might
result in an arrested zygote (Portereiko
et al., 2006b). However, other lines of evidence suggest that this
might not be the general rule, as the embryo can develop up to the globular or
heart stage in capulet2 (cap2) mutant seeds despite severely
retarded and abnormal endosperm (Grini et
al., 2002), or in wild-type seeds where four-nucleate endosperm is
ablated by the expression of diphtheria toxin
(Weijers et al., 2003).
glc embryo development up to the globular stage in the complete
absence of endosperm additionally provides an unequivocal argument for the
independence of early embryogenesis from the endosperm. Therefore, at the
earliest stage of seed development, communication from the early endosperm to
the zygote appears not essential for the coordinated development of the
embryo.
Evidence for the reverse communication - embryo-to-endosperm - has recently
been reported (Nowack et al.,
2006; Nowack et al.,
2007). Although the zygote/embryo is not required for autonomous
endosperm development as known from the fis class mutants (reviewed
by Chaudhury and Berger, 2001)
(Gehring et al., 2004;
Grossniklaus, 2005),
unfertilized endosperm proliferation in seeds having fertilized embryos
suggests that, when present, the zygote/early embryo triggers a signal to the
unfertilized central cell resulting in partial endosperm development
(Nowack et al., 2006).
Moreover, when the egg cell alone is fertilized, endosperm development of the
unfertilized central cell is further promoted by the mea mutation
(Nowack et al., 2006;
Nowack et al., 2007). However,
the apparently normally developing glc embryos fertilized by
wild-type pollen (Figs 2,
4) were not accompanied by
development of endosperm. The glc mutation prevents autonomous
endosperm development in mea and msi1 embryo sacs, where
fertilization is not a consideration (Table
3), and post-fertilization endosperm development was not
significantly different in mea glc double mutant versus glc
single mutant embryo sacs (Table
2). Thus, we conclude that the glc phenotype is probably
due to the inability of the central cell to undergo endosperm development in
glc mutant embryo sacs, rather than the failure of signaling
following egg cell fertilization.
Fertilization of the central cell is specifically impaired in the glc mutant
Double fertilization is ubiquitous in angiosperms (reviewed by
Friedman and Williams, 2004).
The second fusion event between a sperm cell nucleus and a ventral canal
nucleus has also been observed in the non-flowering seed plant genera
Ephedra (Friedman,
1990; Friedman,
1992) and Gnetum
(Carmichael and Friedman,
1995), which, together with Welwitchia, are collectively
referred to as the Gnetales (Bowe et al.,
2000; Chaw et al.,
2000). To date, three angiosperm mutants of known molecular
identity, DUO1 (Rotman et al.,
2005), GCS1 (Mori et
al., 2006; von Besser et al.,
2006) and NFD1
(Portereiko et al., 2006a),
have been reported to disturb both fertilization events, where neither gamete
fusion nor embryo and endosperm development are observed. Thus, common genetic
programs for the fertilization process shared by both the egg cell and the
central cell must have been compromised in these mutants.
How the fertilization event between the central cell and a sperm cell has
evolved during angiosperm evolution remains unknown. It could have resulted
from the co-option of existing genetic programs of the egg-sperm fertilization
event or as a genetic novelty. Until now, only the cdc2a mutant has
been reported to have preferential fertilization of the wild-type egg cell by
the single sperm present in cdc2a mutant pollen
(Nowack et al., 2006). In the
glc mutant, impaired fertilization is also specific to the central
cell, but here the defect arises from the female gamete. Although both
wild-type sperm cells released from wild-type pollen are present in
glc embryo sac and there is no evidence for sperm dimorphism in
Arabidopsis, the egg cell is the preferred choice of fertilization.
This observation suggests that there is specificity in the control of the
fertilization event by each female gamete, whether it lies in the instructive
signals for gamete recognition, the cell structures that facilitate
plasmogamy, or the nuclear structures that enable karyogamy. Furthermore,
although the glc central cell at the time the embryo sac matures
appears morphologically normal with correct cell identity, its disability in
accomplishing fertilization evokes the question: when is its fertilization
competency established? We are currently carrying out further studies on the
glc mutant to answer these questions.
Maternal antagonism between FIS and GLC functions in endosperm and embryo development
Sexually reproducing angiosperms repress seed development in the absence of
fertilization. In Arabidopsis, autonomous endosperm repression is
achieved by the FIS-PRC2 complex (Chaudhury
et al., 1997; Grossniklaus and
Vielle-Calzada, 1998; Guitton
et al., 2004; Köhler et
al., 2003a; Kiyosue et al.,
1999; Luo et al.,
1999; Ohad et al.,
1996; Ohad et al.,
1999; Wang et al.,
2006). Maternal MEA of this complex establishes repressive histone
methylation marks on the maternal allele of the target gene PHE1,
thus repressing maternal PHE1 expression in the central cell before
fertilization (Köhler et al.,
2005; Köhler and
Makarevich, 2006). The suppressive effect of glc on the
autonomous endosperm phenotype of fis mutants
(Table 3) qualifies maternal
GLC functions as a component of the positive regulation of the
fertilization-independent endosperm development pathway
(Fig. 8A).
The FIS genes also negatively regulate endosperm and embryo
proliferation after fertilization. This is evidenced in fertilized
fis seeds where the endosperm overproliferates
(Luo et al., 2000;
Vielle-Calzada et al., 1999;
Yadegari et al., 2000;
Guitton et al., 2004;
Köhler et al., 2003a),
mea-1 embryos over-grow aberrantly
(Grossniklaus et al., 1998)
and msi1 embryos develop abnormally
(Köhler et al., 2003a;
Guitton and Berger, 2005;
Pagnussat et al., 2005). This
post-fertilization FIS function is presumably accomplished by
repressing target genes that might positively regulate embryo and endosperm
development, such as PHE1 and MEIDOS
(Köhler et al., 2003b).
In wild-type fertilized seeds, maternal MEA activity partially
represses the maternal PHE1 allele, and the low level of maternal
PHE1 expression is hypothesized to result from activators not
identified so far (Köhler et al.,
2005; Makarevich et al.,
2006). In glc seeds, this residual promoter activity of
maternal PHE1 was totally abolished
(Fig. 6C,D), suggesting that
GLC functions could fulfil the role of the proposed additional
regulator. This requirement of maternal GLC for maternal
PHE1 activation, together with the pre- and post-fertilization
epistasis between glc and mea (Tables
2,
3), designates maternal
antagonism between GLC and FIS functions in seed
development.
Another mutant reported to have a similar epistatic relationship with
mea is cap2, which is mapped 5 Mb telomeric of
glc (Grini et al.,
2002). However, the post-fertilization interactions of
PHE1 and CAP2 are not known. The nature of the cap2
pre-fertilization interaction with mea appears to be different from
that of glc with mea. This dissimilarity is reflected in
double mutant mea cap2 seeds having the same proportion of
`autonomous seeds' as does mea, and the much more retarded
cap2 embryo development compared with glc embryos despite
partial endosperm development in cap2 seeds
(Grini et al., 2002).
Therefore, the different outcomes from the double mutant analysis with
mea might arise from the different functions of GLC and
CAP2.
GLC functions in the model of FIS-regulating seed development
Although maternal PHE1 is negatively regulated by maternal
MEA, paternal PHE1 expression is not affected by
MEA (Köhler et al.,
2005). How the paternal PHE1 allele is activated in
fertilized seeds has been a missing link in the downstream genetic regulatory
network of the FIS genes. GLC provides a plausible candidate
for this missing link because paternal PHE1 promoter activity was
also eliminated in glc embryos that maternally inherit the mutant
glc allele (Fig.
6A,B). The dependence of paternal RPS5a and FAC1
expression on the presence of the wild-type maternal GLC allele
(Fig. 7) suggests that maternal
GLC functions play a role in the activation of paternal
RPS5a and FAC1 in addition to paternal PHE1. In the
case of PHE1 and RPS5a
(Köhler et al., 2005;
Weijers et al., 2001), the
observed effect of maternal glc on these paternal alleles is unlikely
to be due to the developmental delay of glc embryos, since even the
few persistent globular glc embryos at 4 DAP still did not show
regular GUS expression from PHE1 and RPS5a paternal
promoters (Fig. 6A,B,
Fig. 7C-E). Nevertheless, we do
not rule out the idea that the activation effect of maternal GLC
functions could be an indirect influence of maternal GLC on other
maternal factors that operate on the paternal alleles of these genes.
|
).
PHE1 per se is not essential for seed development because homozygous
phe1/phe1 plants have no developmental defects
(Köhler et al., 2005). It
cannot be concluded that lethality in tms and glc is
attributable to the absence of only these two genes, because many other genes
are also deleted in both mutants. Although the deletion on the maternal
glc allele abolishes bi-parental PHE1 expression altogether
(Fig. 6), it is unable to
rescue the mea mutation because the double mutant glc mea
behaves like the single mutant glc
(Table 2). Yet, mea
seeds where PHE1 expression level is restored to the wild-type level
via antisense PHE1 under MEA promoter control or via a
ddm1 demethylation background, can be partially rescued
(Köhler et al., 2003b;
Köhler et al., 2005).
Therefore, although PHE1 alone is not necessary for seed development,
it appears that in the absence of MEA activity, a certain threshold of
PHE1 transcripts must not be exceeded to avoid seed abortion. We propose that GLC functions can be integrated into the model of seed development regulated by the FIS genes as shown in Fig. 8B. Maternal GLC could operate in a pathway to promote endosperm growth independently of the repressive activity of maternal FIS genes. It is also possible that maternal GLC functions downstream of MEA in the FIS-regulated pathway for embryo development and might be partially repressed by maternal MEA, either directly or indirectly. After fertilization, embryo and endosperm development are promoted by PHE1 and other seed-growth-promoting genes, such as MEIDOS, RPS5a and FAC1. Bi-parental PHE1 and paternal RPS5a and FAC1 are activated by maternal GLC, either directly or indirectly, at least in the embryo and possibly also in the endosperm. FIS genes counteract GLC action by negatively regulating the seed growth promoting genes PHE1 and MEIDOS and possibly GLC. This counteraction prevents the unchecked and imbalanced stimulation that leads to aberrant embryo and endosperm proliferation, which ultimately results in seed abortion.
A new perspective of maternal control in early seed development: paternal allele activation by a maternal factor
Although gene expression mechanisms of paternal alleles in plant early
embryogenesis have not been surveyed and documented, several mechanistic
scenarios can be postulated. Paternal expression could be a carry-over from
the pre-expressed state in the male gametophyte genome before fertilization.
It could also be induced by the pre-programmed self-activation of the paternal
alleles after fertilization. Another possibility is that paternal allele
activation requires embryonic regulators derived from both parental genomes.
The dependence of paternal PHE1, RPS5a and FAC1 expression
on maternal GLC demonstrates that paternal allele expression of
certain genes, at least in the embryo, is induced by some element(s) of the
maternal genome, which is derived from the female gametophyte. Of the five
genes with diverse functions that we examined for expression from the paternal
allele, two exhibit detectable expression only in the embryo and not in the
endosperm: CYC B1;1, which is one of the Arabidopsis mitotic
cyclins (Colon-Carmona et al.,
1999) and PIN7, which establishes early embryonic
polarity and patterning via effecting an auxin activity gradient
(Friml et al., 2003). The
other three genes, PHE1
(Köhler et al., 2003b),
RPS5a (Weijers et al.,
2001) and FAC1 (Xu et
al., 2005), also belong to different functional categories:
PHE1 is a transcription factor; the remaining two are considered
housekeeping genes that encode a ribosomal protein subunit (RPS5a) and an AMP
deaminase (FAC1). The paternal alleles of all these three genes are expressed
in both the embryo and endosperm from very early stages of seed development.
Therefore, the subset of paternally expressed genes that is positively
regulated by maternal factors might consist of those genes that have functions
in both embryo and endosperm, and not genes that function in the embryo alone.
This hypothesis is consistent with the observations that GLC
functions are required maternally for endosperm but not embryo development.
Further investigations of glc effects on other paternally expressed
genes will be needed to define the set of genes of which paternal allele
activation requires maternal GLC.
The concept of prevalent maternal control of early seed development in
plants has emerged during the past years, first evidenced by the
preferentially maternal expression of many genes in seeds during the first few
days after fertilization in both Arabidopsis
(Vielle-Calzada et al., 2000)
and maize (Grimanelli et al.,
2005). The gametophytic maternal effect of the FIS genes
(reviewed by Grossniklaus,
2005) and of the large class of MATERNAL EFFECT EMBRYO
ARREST (MEE) genes
(Pagnussat et al., 2005), of
which some display this early preferentially maternal expression
(Jullien et al., 2006b;
Kinoshita et al., 1999;
Luo et al., 2000;
Vielle-Calzada et al., 1999)
(Q. A. Ngo, PhD thesis, University of California, 2006), provides further
support for the concept. Factors of the maternal genome can regulate other
maternal factors negatively, as exemplified by the repression of maternal
PHE1 by maternal MEA and FIE
(Köhler et al., 2003a;
Köhler et al., 2003b;
Makarevich et al., 2006), or
positively, as demonstrated by the activation of maternal MEA by
maternal DEMETER (Choi et al.,
2002; Choi et al.,
2004). Moreover, the regulatory mechanism of the maternal genome
crosses its own genome boundary to affect the paternal genome. Recently, an
intriguing mechanism by which the maternal genome contributes to early seed
development has been revealed by the negative crossregulation executed by the
MEA protein produced from a maternal allele, which represses its own paternal
allele via histone methylation (Baroux et
al., 2006; Gehring et al.,
2006; Jullien et al.,
2006a). GLC has now added positive crossregulation to the
diverse repertoire of maternal control: the product from its maternal allele
activates the paternal alleles of certain other genes. In light of this
positive crossregulation, the nature of zygotic and early embryonic
bi-parentally expressed genes merits revisiting. The origin of paternal
expression of such genes might, if examined more carefully, depend upon the
maternal expression of other genes that are upstream regulatory factors. Thus,
in early seed development, a maternal contribution, which originates in the
female gametophyte, could ultimately control the paternal contribution.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/22/4107/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Baroux, C., Blanvillain, R. and Grossniklaus, U. (2001). Paternally inherited genes are down-regulated but retain low activity during early embryogenesis in Arabidopsis. FEBS Lett. 509,11 -16.[CrossRef][Medline]
Baroux, C., Gagliardini, V., Page, D. R. and Grossniklaus,
U. (2006). Dynamic regulatory interactions of Polycomb group
genes: MEDEA autoregulation is required for imprinted gene expression in
Arabidopsis. Genes Dev.
20,1081
-1086.
Baroux, C., Pecinka, A., Fuchs, J., Schubert, I. and
Grossniklaus, U. (2007). The triploid endosperm genome of
Arabidopsis adopts a peculiar, parental dosage-dependent chromatin
organization. Plant Cell
19,1782
-1794.
Barrell, P. J. and Grossniklaus, U. (2005). Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II. Plant J. 43,309 -320.[CrossRef][Medline]
Bowe, S. M., Coat, G. and dePamphilis, C. W.
(2000). Phylogeny of seed plants based on all three genomic
compartments: extant gymnosperms are monophyletic and Gnetales' closest
relatives are conifers. Proc. Natl. Acad. Sci. USA
97,4092
-4097.
Bowman, J. L. (1994). Arabidopsis: An Atlas of Morphology and Development. New York: Springer-Verlag.
Carmichael, J. S. and Friedman, W. E. (1995). Double fertilization in Gnetum gnemon: the relationship between the cell cycle and sexual reproduction. Plant Cell 7,1975 -1988.[Abstract]
Chaudhury, A. M. and Berger, F. (2001). Maternal control of seed development. Semin. Cell Dev. Biol. 12,381 -386.[CrossRef][Medline]
Chaudhury, A. M., Ming, L., Miller, C., Craig, S., Dennis, E. S. and Peacock, W. J. (1997). Fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 94,4223 -4228.[CrossRef]
Chaw, T. M., Parkinson, C. L., Cheng, Y., Vincent, T. M. and
Palmer, J. D. (2000). Seed plant phylogeny inferred from all
three plant genomes: monophyly of extant gymnosperms and origin of Gnetales
from conifers. Proc. Natl. Acad. Sci. USA
97,4086
-4091.
Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J. J., Goldberg, R. B., Jacobsen, S. E. and Fischer, R. L. (2002). DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110,33 -42.[CrossRef][Medline]
Choi, Y., Harada, J. J., Goldberg, R. B. and Fischer, R. L.
(2004). An invariant aspartic acid in the DNA glycosylase domain
of DEMETER is necessary for transcriptional activation of the imprinted MEDEA
gene. Proc. Natl. Acad. Sci. USA
101,7481
-7486.
Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999). Spatio-temporal analysis of mitotic ativity with a labile cyclin-GUS fusion protein. Plant J. 20,503 -508.[CrossRef][Medline]
Faure, J. E., Rotman, N., Fortune, P. and Dumas, C. (2002). Fertilization in Arabidopsis thaliana wild type: developmental stages and time course. Plant J. 30,481 -488.[CrossRef][Medline]
Friedman, W. E. (1990). Double fertilization in
Ephedra, a non-flowering seed plant: its bearing on the origin of endosperm.
Science 247,951
-954.
Friedman, W. E. (1992). Evidence of a pre-angiosperm origin of endosperm. Science 225,336 -339.
Friedman, W. E. and Williams, J. H. (2004).
Developmental evolution process of the sexual process in ancient flowering
plant lineages. Plant Cell
16,S119
-S132.
Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H., Hamann, T., Offringa, R. and Jürgens, G. (2003). Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426,147 -153.[CrossRef][Medline]
Gehring, M., Choi, Y. and Fischer, R. L.
(2004). Imprinting and seed development. Plant
Cell 16,S203
-S213.
Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B. and Fischer, R. L. (2006). DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell 124,495 -506.[CrossRef][Medline]
Grimanelli, D., Perotti, E., Ramirez, J. and Leblanc, O. (2005). Timing of the maternal-to-zygotic transition in early seed development in maize. Plant Cell 17,1061 -1072.
