RESEARCH ARTICLE 2031
Development 139, 2031-2039 (2012) doi:10.1242/dev.077057
© 2012. Published by The Company of Biologists Ltd
Endosperm cellularization defines an important
developmental transition for embryo development
Elisabeth Hehenberger1, David Kradolfer1,2 and Claudia Köhler1,2,*
SUMMARY
The endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endosperm
develops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occurs
at a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidy
crosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seed
development. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) and
endosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endosperm
cellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryo
development, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endosperm
cellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is a
consequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct target
gene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endosperm
cellularization.
INTRODUCTION
Seed development in flowering plants is initiated by double
fertilization of the female gametophyte. The female gametophyte
harbors two distinct gametic cells that will have distinct fates after
fertilization: the haploid egg cell will give rise to the diploid
embryo and the homodiploid central cell will form the triploid
endosperm (Drews and Yadegari, 2002). The endosperm is a
terminal tissue that supports embryo growth by delivering nutrients
acquired from the mother plant (Ingram, 2010). Like most
angiosperms, the endosperm of Arabidopsis thaliana follows the
nuclear type of development, in which an initial phase of free
nuclear divisions without cytokinesis (syncytial phase) is followed
by cellularization (Costa et al., 2004). In Arabidopsis, at the 16nuclei stage the syncytium begins to differentiate into three distinct
developmental domains: the micropylar, central and chalazal
domains. The presence of a large central vacuole forces the
cytoplasm in the central domain into a thin peripheral layer, while
syncytial cytoplasm surrounds the embryo in the micropylar
domain and the chalazal endosperm develops adjacent to the
vascular connection with the seed parent (Berger, 2003; Brown et
al., 1999; Brown et al., 2003). At the eighth mitotic cycle,
cellularization of the syncytial endosperm is initiated in the
micropylar domain around the embryo, coinciding with the early
heart stage of embryo development (Boisnard-Lorig et al., 2001).
Cellularization proceeds in a wave-like manner from the
micropylar to the chalazal domain, filling the central cell, except
1
Department of Biology and Zurich-Basel Plant Science Center, Swiss Federal
Institute of Technology, ETH Centre, CH-8092 Zurich, Switzerland. 2Department of
Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of
Agricultural Sciences and Linnean Center for Plant Biology, SE-75007 Uppsala,
Sweden.
*Author for correspondence ([email protected])
Accepted 21 March 2012
for near the cyst in the chalazal chamber, with successive layers of
endosperm cells (Brown et al., 1999; Berger, 2003; Brown et al.,
2003).
Compartmentalization of the developing seed into three distinct
endosperm domains is likely to regulate the uptake of nutrients into
the developing seed. Experiments with labeled sucrose in oilseed
rape seeds suggest that the transport of sucrose from the
integuments to the embryo occurs via the micropylar endosperm,
while sucrose uptake via the chalazal endosperm is thought to be
involved in filling the central endosperm vacuole, the main storage
pool for hexoses during seed development (Morley-Smith et al.,
2008). The large central vacuole determines sink strength during
early development by rapidly converting imported sucrose to
hexoses in the young seed. With progressing endosperm
cellularization, the size of the central vacuole decreases, correlating
with a decrease in the ratio of hexoses to sucrose (Ohto et al., 2005;
Morley-Smith et al., 2008; Ohto et al., 2009).
Endosperm cellularization is impaired in several mutants
affecting cytokinesis in the embryo, including knolle, hinkel, open
house, runkel and pleiade (Sorensen et al., 2002), implying that
endosperm cellularization and somatic cytokinesis share multiple
components of the same basic machinery. The spätzle mutant is
characterized by the absence of cellularization in the endosperm
but does not show cytokinesis defects in the embryo, indicating a
role for SPÄTZLE in a process specific to endosperm
cellularization (Sorensen et al., 2002). Similarly, in the endosperm
defective 1 (ede1) mutant, the endosperm fails to cellularize but the
effects on embryo patterning are less severe, implicating a main
function for EDE1 in endosperm cellularization (Pignocchi et al.,
2009). However, in both mutants, embryo development is delayed
after the heart stage and seed shrinkage or collapse occurs,
suggesting that endosperm cellularization is crucial for normal
embryo and seed development (Sorensen et al., 2002; Pignocchi et
al., 2009). Interploidy crosses furthermore support an important
role for endosperm cellularization in embryo development.
DEVELOPMENT
KEY WORDS: Arabidopsis, Endosperm, Polycomb group proteins
2032 RESEARCH ARTICLE
Development 139 (11)
Increased paternal genome dosage results in the delay or complete
failure of endosperm cellularization, correlating with impaired
embryo development (Scott et al., 1998; Dilkes et al., 2008; Erilova
et al., 2009). Developmental defects caused by interploidy crosses
are associated with a dysregulation of genes that are directly or
indirectly controlled by the FERTILIZATION INDEPENDENT
SEED (FIS) Polycomb group (PcG) complex, implicating that
developmental aberrations in response to interploidy crosses are
largely caused by dysregulated FIS target genes (Erilova et al.,
2009).
PcG proteins form chromatin-modifying complexes that ensure
the long-term controlled repression of specific target genes. PcG
proteins form several families of multiprotein complexes, including
the two main complexes Polycomb repressive complex 1 (PRC1)
and PRC2. PRC2-mediated gene repression is characterized by
trimethylation of lysine 27 of histone H3 (H3K27me3) and seems
to primarily control genes involved in developmental decisions
(Hennig and Derkacheva, 2009; Beisel and Paro, 2011). The
subunits of PRC2 complexes are evolutionary well conserved. In
plants, several PRC2 subunits are encoded by small gene families
that form specific complexes with distinct functions during plant
development (Hennig and Derkacheva, 2009). The FIS PRC2
complex plays a pivotal role in suppressing the initiation of
endosperm and seed development in the absence of fertilization
(Ohad et al., 1996; Chaudhury et al., 1997; Köhler et al., 2003a;
Guitton et al., 2004). After fertilization, loss of FIS function causes
endosperm overproliferation and cellularization failure, ultimately
leading to seed abortion (Chaudhury et al., 1997; Kiyosue et al.,
1999; Sorensen et al., 2001). A major regulator of endosperm
cellularization is the type I MADS-box transcription factor
AGAMOUS-LIKE 62 (AGL62) (Kang et al., 2008). Loss of
AGL62 causes precocious endosperm cellularization, indicating a
role for AGL62 as a suppressor of endosperm cellularization.
Whereas in wild-type seeds AGL62 expression declines abruptly
prior to cellularization, in fis mutants AGL62 expression does not
decline (Kang et al., 2008), suggesting that FIS PRC2 might
regulate endosperm cellularization by controlling the expression of
AGL62.
In this study we investigated the role of FIS PRC2 in endosperm
cellularization and the consequences of endosperm cellularization
failure. We show that failure of endosperm cellularization in
fertilization independent seed 2 (fis2) and ede1 mutants correlates
with impaired embryo development. Restoration of endosperm
cellularization in fis2 seeds by reducing AGL62 expression
promotes embryo development, strongly supporting an essential
role of endosperm cellularization in viable seed formation. We
furthermore reveal that endosperm cellularization failure in fis2
seeds correlates with increased hexose levels, suggesting that arrest
of embryo development is a consequence of failed nutrient
translocation to the developing embryo. Finally, we establish a
molecular link for FIS PRC2-mediated endosperm cellularization
by showing that AGL62 is a direct target gene of the FIS PRC2
complex.
For each genotype and time point, seeds of three siliques were collected,
corresponding to three biological replicates. Only undamaged seeds were
harvested on ice and counted during harvesting, allowing relation of the
measured amounts of carbohydrates to the number of seeds. The seeds
were ground frozen in a Silamat S5 mixer (Ivoclar Vivadent, Amherst,
USA) twice for 10 seconds and extracted with 200 l prewarmed 80%
ethanol for 4 minutes at 80°C under constant rotation. After centrifugation
for 5 minutes at 16,000 g, the supernatant was transferred to a new tube
and the extraction repeated. The combined supernatants were dried under
vacuum at 30°C.
The dried samples were resuspended in 50 l water and soluble sugars
were measured in a MWGt Discovery XS-R microplate reader (BioTek
Instruments, Winooski, USA) after adding 50 l of cocktail [25 mM
HEPES pH 7.5, 1 mM MgCl2, 1 mM ATP, 1 mM NAD, 9 U/ml hexokinase
(Roche Applied Science, Indianapolis, USA)] and 99 l of water. After
establishing a blind value, glucose, fructose and sucrose were measured by
consecutively adding 1 U glucose-6-phosphate dehydrogenase (Roche
Applied Science), 0.7 U phosphoglucose isomerase (Roche Applied
Science) and 5 U invertase (Fluka, St Louis, USA). Soluble carbohydrate
contents were calculated as described (Smith and Zeeman, 2006).
MATERIALS AND METHODS
RNA extraction and qPCR analysis
The fis2 mutant alleles used in this study are fis2-1 (Ler accession)
(Chaudhury et al., 1997) and fis2-5 (Col-0 accession) (Weinhofer et al.,
2010). The ede1-1, agl62-2 and osd1-1 mutant alleles are in the Col-0
accession background and have been described previously (Kang et al.,
2008; Pignocchi et al., 2009; d’Erfurth et al., 2009). For crosses, designated
female partners were emasculated, and the pistils hand-pollinated 2 days
after emasculation. Single- and double-mutant plants were characterized
Transmission analysis
Seeds were surface sterilized as above and plated on MS media
supplemented with 100 M gibberellic acid to facilitate germination. After
stratification at 4°C in the dark for 3 days, seedlings were grown in a
growth room under a long-day photoperiod (16 hours light and 8 hours
darkness) at 23°C. Seedlings were harvested for genotyping after 12-16
days using the primers listed in supplementary material Table S1.
Embryo rescue and in vitro culturing
Siliques of fis2 mutant plants were collected at ~8-10 DAP and surface
sterilized for 10 minutes in 1.5% sodium hypochlorite and 0.1% Tween 20
and then washed three times with sterile water. Embryos of fis2 seeds were
dissected under sterile conditions using insulin needles. The embryos were
plated on Murashige and Skoog medium (MS salts, pH 5.8, 0.8%
Bactoagar, 2.7 mM glutamine) containing either 30 mM or 340 mM
sucrose and cultivated under long-day conditions at 21°C.
Genomic DNA extraction from embryos was performed using the
REDExtract-N-Amp Plant PCR Kit (Sigma, Poole, UK) with the primers
listed in supplementary material Table S1.
Generation of plasmids
The 1.8 kb sequence upstream of the APL3 (AT4G39210) transcriptional
start was amplified by PCR using the primers listed in supplementary
material Table S1 and cloned into pCAMBIA1381Z (Cambia).
Analysis of carbohydrates
Seeds at indicated stages were harvested into RNAlater (Sigma-Aldrich,
St Louis, USA) and total RNA was extracted using the RNeasy Plant Mini
Kit (Qiagen, Valencia, USA). For quantitative RT-PCR, RNA was treated
on-column with DNase I and reverse transcribed using the First Strand
cDNA Synthesis Kit (Fermentas, Glen Burnie, USA). Gene-specific
primers and Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad,
USA) were used on a 7500 Fast Real-Time PCR System (Applied
Biosystems). Quantitative RT-PCR was performed using three replicates
DEVELOPMENT
Plant material and growth conditions
by PCR using the primers listed in supplementary material Table S1. Seeds
were surface sterilized (5% sodium hypochlorite, 0.01% Triton X-100) and
plated on MS medium (MS salts, 1% sucrose, pH 5.6, 0.8% Bactoagar).
After stratification for 1 day at 4°C, plants were grown in a growth room
under a long-day photoperiod (16 hours light and 8 hours darkness) at
23°C. Ten-day-old seedlings were transferred to soil and plants were grown
in a growth room at 60% humidity and daily cycles of 16 hours light at
23°C and 8 hours darkness at 18°C. The APL3::GUS line was generated
by Agrobacterium tumefaciens-mediated transformation (Clough and Bent,
1998) into heterozygous fis2-1 plants and six independent lines
homozygous for the transgene locus in the fis2-1 background were
analyzed.
and ACTIN11 as a reference gene. Results were analyzed as previously
described (Simon, 2003). Primers are listed in supplementary material
Table S1.
Chromatin immunoprecipitation (ChIP)
ChIP from endosperm nuclei and seedling tissue was performed as
described (Weinhofer et al., 2010). Gene-specific primers and the Fast
SYBR Green Master Mix were used on the 7500 Fast Real-Time PCR
System to test enrichment of H3K27me3-marked fragments. Quantitative
ChIP PCR was performed using three replicates and results were analyzed
as described (Simon, 2003) and are presented as percentage of input.
Microscopy
Seeds were harvested for periodic acid-Schiff staining at the indicated
time points and incubated in 50% ethanol, 5% acetic acid and 4%
formaldehyde at 4°C for 12 hours. The seeds were dehydrated in four 1hour steps, followed by an overnight incubation in 100% ethanol. After
two wash steps with 100% ethanol, the seeds were stepwise infiltrated
with Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) preparation
solution and incubated for 16 hours in 100% preparation solution. For
embedding, seeds were mixed with polymerization solution and allowed
to polymerize for at least 2 hours. The embedded samples were sectioned
into 4 m sections using an RM2255 microtome (Leica, Wetzlar,
Germany). The sections were collected on water-covered SuperFrost Plus
microscope slides (Menzel, Braunschweig, Germany) and dried on a
heating plate at 72°C. The sections were oxidized for 30 minutes with
1% periodic acid, washed and stained for another 30 minutes with
Schiff’s reagent (Sigma-Aldrich). The sections were embedded in
Entellan New Mounting Media (Electron Microscopy Sciences, Hatfield,
USA) for microscopy.
Seeds were harvested at the indicated time points and stained to detect
GUS activity as described (Köhler et al., 2003b). The seeds were
dehydrated and embedded as described above. Clearing analysis of seeds
was performed as described (Köhler et al., 2003b).
Microscopy imaging was performed using a Leica DM 2500 microscope
with DIC optics. Images were captured using a Leica DFC300 FX digital
camera, exported using Leica Application Suite version 2.4.0.R1 and
processed using Photoshop CS5 (Adobe Systems, San Jose, USA).
RESEARCH ARTICLE 2033
RESULTS
Endosperm cellularization failure correlates with
embryo arrest in fis2 and ede1 mutants
Loss of FIS function causes an endosperm cellularization failure
and arrest of embryo development, leading to seed abortion ~8
days after pollination (DAP) (Ohad et al., 1996; Chaudhury et al.,
1997; Grossniklaus et al., 1998; Kiyosue et al., 1999; Köhler et al.,
2003a; Guitton et al., 2004). We addressed the question of whether
arrest of embryo development upon loss of FIS function is a direct
consequence of endosperm cellularization failure or rather an
indirect consequence of dysregulated gene expression impacting
on embryo development. If embryo arrest is a direct consequence
of endosperm cellularization failure, mutants defective in
endosperm cellularization are expected to have a fis-like embryo
arrest phenotype. We tested this hypothesis by analyzing the ede1
mutant, which has a mutation in a plant-specific microtubuleassociated protein (Pignocchi et al., 2009). Loss of EDE1 function
causes endosperm cellularization failure, resulting in seeds that
contain uncellularized endosperm and in embryo arrest at late
heart stage (Pignocchi et al., 2009). Under our conditions, at 8
DAP 11.4% of the seeds derived from homozygous ede1-1
mutants had a fis-like phenotype, with uncellularized endosperm
and embryos arrested at heart stage (n340; Fig. 1A-C), whereas
88.6% of the seeds had cellularized endosperm and embryos
progressed in their development beyond heart stage (Fig. 1E).
Development of those ede1 embryos that progressed beyond heart
stage was still delayed compared with wild-type seeds; at 8 DAP,
ede1 mutant embryos had only reached the early torpedo stage of
development, whereas wild-type embryos had already entered the
bent cotyledon stage (Fig. 1E,D). Delayed embryo development
was accompanied by a delay in endosperm cellularization;
whereas cellularization of the peripheral endosperm was clearly
visible in wild-type endosperm at 6 DAP, ede1-1 seeds reached a
similar cellularization state only at ~8 DAP (Fig. 1A,E). Embryo
Fig. 1. Endosperm cellularization failure
correlates with embryo arrest in fis2 and
ede1 mutants. Schiff-stained Technovit sections
of wild-type, fis2-5 and ede1-1 Arabidopsis
seeds. (A)Wild-type seed with embryo at
torpedo stage and cellularizing endosperm at 6
DAP. (B)fis2-5 seed with embryo arresting at
heart stage and uncellularized endosperm at 8
DAP. (C)ede1-1 seed with embryo arresting at
heart stage and uncellularized endosperm at 8
DAP. (D)Wild-type seed with embryo at bent
cotyledon stage and fully cellularized endosperm
at 8 DAP. (E)ede1-1 seed with embryo at
torpedo stage and cellularizing endosperm at 8
DAP. (F)ede1-1 seed with embryo at bent
cotyledon stage and fully cellularized endosperm
at 10 DAP. CV, central vacuole. Scale bar:
100m.
DEVELOPMENT
Endosperm cellularization
development remained delayed and ede1 embryos reached the
bent cotyledon stage 2 days later than wild-type embryos, at 10
DAP (Fig. 1F).
Together, their close correlation in ede1 mutant seeds supports
the idea that embryo arrest is a consequence of endosperm
cellularization failure. It is however possible that a fraction of ede1
mutant embryos arrest development due to a direct requirement of
EDE1 function in the embryo.
Cellularization failure is connected with increased
hexose content in the endosperm
We considered the possible consequences of cellularization failure
impacting on embryo development. Endosperm cellularization
causes the central vacuole to become smaller, until it becomes
completely invaded by endosperm cells (Olsen, 2001). The central
vacuole of the endosperm is the main storage pool for hexoses in
the seed; therefore, a decrease in vacuole size in response to
cellularization is likely to cause a decrease in hexose content
(Morley-Smith et al., 2008). Indeed, the time of endosperm
cellularization is connected with a decrease in the ratio of hexoses
(glucose and fructose) to sucrose (Baud et al., 2002). Therefore,
hexose levels are expected to remain high in mutants that fail to
undergo endosperm cellularization.
We tested this hypothesis by measuring glucose, fructose
(referred to as hexoses) and sucrose levels in fis2 mutant seeds at
6, 7 and 8 DAP, whereby the fis2 phenotype allowed discrimination
and separate measurement of developing and arresting seeds in
fis2/+ siliques from 7 DAP onwards. Before 7 DAP, discrimination
between developing and arresting fis2 seeds was not possible,
Development 139 (11)
therefore we measured a mixture of 50% wild-type and 50% fis2
seeds. At 6 DAP, hexose levels in wild type and fis2 were
comparable (Fig. 2A), correlating with the presence of a large
central vacuole in wild-type seeds (Fig. 1A). At 7 DAP, the central
vacuole in wild-type seeds had strongly decreased in size
(supplementary material Fig. S1), correlating with decreased
hexose levels (Fig. 2A). Consistent with the predictions, hexose
levels in fis2 mutant seeds remained high at 7 and 8 DAP, whereas
wild-type hexose levels strongly decreased to ~25% of those
present at 6 DAP. At 8 DAP, arresting fis2 seeds contained hexose
levels that were more than five times higher than those in wild-type
seeds at the same time point (Fig. 2A). At 8 DAP, sucrose levels in
wild-type seeds decreased to about half the amount at 6 DAP,
whereas sucrose levels in fis2 remained unchanged over the
investigated time period (Fig. 2A).
The increased levels of hexoses and sucrose in fis2 seeds at 8
DAP correlated with increased expression of the APL3 gene, which
encodes one of the large subunits of the ADP-glucose
pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis
(Zeeman et al., 2010) (Fig. 2B-E). Expression of APL3 is highly
responsive to sucrose and glucose (Crevillen et al., 2005), making
the APL3 promoter fused to the -GLUCURONIDASE (GUS) a
suitable reporter to monitor the tissue-specific localization of
carbohydrates. Whereas in fis2 seeds at 8 DAP a strong GUS signal
could be observed in the seed coat, wild-type seeds from the same
silique had only very weak GUS staining in the seed coat (Fig. 2BE). By contrast, wild-type seeds at 5 DAP had similarly strong
GUS staining in the seed coat as fis2 seeds at 8 DAP (Fig. 2F),
correlating with a similar developmental stage of the embryo.
Fig. 2. Hexose content is increased in endosperm
cellularization mutants. (A)Enzymatic measurements
of soluble carbohydrates (glucose, fructose and sucrose)
in wild-type and isolated fis2-1/+ Arabidopsis seeds at
6, 7 and 8 DAP. Error bars indicate s.e.m. (n3
biological replicates) (B-H) Analysis of APL3::GUS
expression in fis2 seeds. Clearings (B,C) and Technovit
sections (D-H) of GUS-stained seeds expressing
APL3::GUS. (B,D)Wild-type seed at 7 DAP; (C,E) fis2-1
seed at 8 DAP; (F) wild-type seed at 5 DAP.
(G,H)Enlargements of the embryos shown in E,F,
respectively. (I)Enzymatic measurements of soluble
carbohydrates in isolated ede1-1/– seeds at 8 DAP. Error
bars indicate s.e.m. (n3 biological replicates). Gluc,
glucose; Fruc, fructose; Suc, sucrose; wt, wild type.
Scale bar: 100m.
DEVELOPMENT
2034 RESEARCH ARTICLE
Endosperm cellularization
RESEARCH ARTICLE 2035
Fig. 3. Reduced maternal genome dosage in the fis2-5/+ agl622/+ double mutant suppresses the fis2 phenotype. (A)Quantitative
RT-PCR analysis of AGL62 expression in wild-type and fis2-5
Arabidopsis seeds from 2-5 DAP. Error bars indicate s.e.m. (B)Seeds
from wild type (left), from the cross fis2-5 ⫻ wild type (center) and
from the cross fis2-5/+ agl62-2/+ ⫻ wild type (right) at 14 DAP. Insets
show isolated embryos from seeds of the same developmental stage
and genotype. (C)Distribution of seed phenotypes from the cross fis2-5
⫻ wild type and fis2-5/+ agl62-2/+ ⫻ wild type at 14 DAP, including
maternal transmission of the fis2-5 mutation in the respective crosses.
Numbers above bars indicate seeds analyzed per genotype. Numbers
for the transmission refer to the number of seedlings investigated.
(D)Schiff-stained Technovit sections at 9 DAP (top row) and 14 DAP
(bottom row). Wild-type seeds contain bent cotyledon stage (9 DAP)
and fully developed (14 DAP) embryos. fis2-5 seeds contain heart stage
embryos (9 and 14 DAP). fis2-5 agl62-2 seeds contain early torpedo (9
DAP) and bent cotyledon (14 DAP) stage embryos. Scale bars: 100m.
However, whereas wild-type embryos had a pronounced GUS
staining zone surrounding the embryo (Fig. 2H), this zone was
absent in fis2 embryos (Fig. 2G). This staining is likely to originate
from the endosperm surrounding the embryo, suggesting that the
embryo receives carbohydrates from the neighboring endosperm in
wild-type seeds, whereas the embryo-surrounding endosperm in
fis2 fails to perform this function.
If increased hexose levels are a consequence of cellularization
failure, uncellularized ede1 seeds should have similarly increased
hexose levels as fis2 seeds. To test this hypothesis, we isolated ede1
seeds that failed to undergo endosperm cellularization at 8 DAP.
Restoration of cellularization by maternal loss of
AGL62 normalizes fis2 seed development
If endosperm cellularization failure is the cause of embryo
developmental arrest, restoration of endosperm cellularization in
fis2 mutants should promote embryo development. Loss of the
MADS-box transcription factor AGL62 causes precocious
endosperm cellularization, implicating AGL62 as a major regulator
of endosperm cellularization in Arabidopsis (Kang et al., 2008). In
agreement with previous studies revealing prolonged expression of
an AGL62 reporter construct in fis mutant endosperm (Kang et al.,
2008), AGL62 transcript levels were strongly increased and
prolonged in fis2 seeds compared with wild-type transcript levels,
which were undetectable at 5 DAP (Fig. 3A). Reduced expression
of AGL62 correlates with the onset of endosperm cellularization in
Arabidopsis seeds (Kang et al., 2008), suggesting repression of
endosperm cellularization in fis2 seeds by extended and increased
AGL62 expression.
To test whether reduced dosage of AGL62 can suppress the fis2
mutant phenotype we generated fis2/+; agl62/+ double mutants.
When double-heterozygous mutants were pollinated with wild-type
pollen a new seed class was observed that was clearly
distinguishable from arresting fis2 seeds (Fig. 3B). At 14 DAP,
~23% of the seeds were not collapsed but instead phenotypically
distinguishable from wild-type seeds by their enlarged size (Fig.
3C; 25.8% aborted seeds, 23.2% non-aborted enlarged green seeds,
51% normal seeds; n635), suggesting that agl62 could partially
suppress the fis2 mutation. Although we also observed a fraction
of non-collapsed seeds in fis2/+ single mutants at 14 DAP (12.2%,
n433 total seeds), none of these seeds progressed beyond the heart
stage of development. By contrast, ~20% of fis2 agl62 embryos
developed up to the torpedo stage (n147 total fis2 agl62 seeds).
Sections of the enlarged green fis2 agl62 seeds at 14 DAP revealed
either progressing or full cellularization of the endosperm (Fig.
3D), whereby advanced levels of cellularization correlated with
advanced embryo development. Development of fis2 agl62
embryos was delayed compared with wild-type embryos: whereas
most wild-type embryos were in the bent cotyledon stage at 9 DAP,
fis2 agl62 embryos still had a torpedo-like shape (Fig. 3D). The
delayed embryo development in fis2 agl62 seeds correlated with a
delay of endosperm cellularization, which occurred 4-5 days later
than in wild-type seeds (Fig. 3D), suggesting that AGL62 is
important but might not be the only dysregulated FIS target
impacting endosperm development. Alternatively, it is possible that
the paternally contributed wild-type AGL62 allele remained
expressed at higher levels in fis2 seeds.
To further test the hypothesis that the agl62 mutation can
suppress the fis2 mutant phenotype, we examined whether the
concomitant presence of the agl62 and fis2 mutations would allow
transmission of fis2 through the maternal gametophyte. fis2/+
agl62/+ mutants were pollinated with wild-type pollen and F1
seedlings derived from this cross were tested for the presence of
DEVELOPMENT
These seeds were phenotypically clearly distinguishable from
cellularized wild-type seeds by their white and glossy appearance.
Uncellularized ede1 seeds had similarly increased hexose levels as
uncellularized fis2 seeds (Fig. 2I), supporting the hypothesis that
increased hexose levels are a consequence of cellularization failure
in the endosperm.
To conclude, endosperm cellularization failure correlates with
elevated hexose levels in the developing seeds, suggesting that
incoming sucrose is not properly delivered to the developing
embryo, causing embryo arrest.
the fis2 mutation. Out of 34 seedlings carrying the agl62 mutation,
nine were positively genotyped for the fis2 mutation, revealing
~50% transmission of the fis2 allele in the presence of the agl62
mutation. By contrast, out of 48 seedlings derived from the control
cross of fis2/+ pollinated with wild-type pollen, only one seedling
contained the fis2 mutation (Fig. 3C). However, this seedling was
not viable and arrested development immediately after germination.
We conclude that agl62 allows transmission of the fis2 allele
through the female gametophyte, albeit at reduced frequency.
Loss of maternal AGL62 normalizes triploid seed
development
Interploidy crosses of diploid maternal and tetraploid paternal
plants cause increased expression of AGL62, which correlates
with endosperm cellularization failure and seed abortion (Erilova
et al., 2009). We tested whether maternal loss of AGL62 could
suppress triploid seed failure by pollinating agl62/+ plants with
diploid pollen from the omission in second division 1 (osd1)
mutant. Pollination of wild-type plants with osd1 pollen leads to
the formation of 100% triploid progeny (d’Erfurth et al., 2009),
similar to interploidy crosses of diploid maternal and tetraploid
paternal plants. Triploid seeds in the Columbia (Col) accession
have similar phenotypic abnormalities as fis mutant seeds and
abort containing embryos arrested at heart stage surrounded by
largely uncellularized endosperm (Dilkes et al., 2008). Upon
pollination of wild-type plants with diploid osd1 pollen, 88% of
the seeds were dark brownish and collapsed, whereas 12% were
a mixture of phenotypically normal seeds (3%) and enlarged
abnormally shaped seeds that nonetheless contained developed
embryos (9%) (n461 total seeds; Fig. 4A). Pollination of
agl62/+ with osd1 pollen more than doubled the number of
Fig. 4. Triploid seed failure is suppressed by agl62. (A)Distribution
of mature seed phenotypes from the cross wild type ⫻ osd1-1 and
agl62-2/+ ⫻ osd1-1 including the germination rate of the respective
crosses. Numbers above bars indicate seeds analyzed per genotype.
Numbers for the germination refer to the number of seeds plated.
(B)Seed phenotypes corresponding to categories shown in A as normal
(top), non-aborted (middle) and aborted (bottom). (C)Cleared images
of seeds from cross agl62-2/+ ⫻ osd1-1 at 12 DAP corresponding to
normal (left), aborted (middle) and non-aborted (right) phenotypes.
Development 139 (11)
phenotypically normal seeds and enlarged seeds (7% and 20%,
respectively; n522 total seeds; Fig. 4A-C), supporting the view
that reduced levels of AGL62 can restore viable triploid seed
formation. We tested the germination capacity of triploid seeds
and, consistent with increased numbers of phenotypically normal
and enlarged triploid seeds derived from pollinated agl62 mutant
plants, the germination rate of those seeds was almost twice as
high as that of wild-type triploid seeds (19% versus 36%, n155
and 85 total seeds, respectively; Fig. 4A). Together, these
findings support the hypothesis that maternal loss of AGL62
partially restores triploid seed viability.
Restoration of endosperm cellularization is
connected with decreased hexose levels
If cellularization failure is causally connected with increased
hexose levels, restoration of endosperm cellularization should be
connected with decreased hexose levels. To test this hypothesis, we
performed carbohydrate measurements in fis2 agl62 and fis2 seeds
at 8 DAP. At this time point, fis2/+; agl62/+ double-mutant seeds
cannot be distinguished from the fis2/+ seeds by eye, resulting in
a mixture of 50% double-mutant and 50% fis2 seeds in the doublemutant sample. Nonetheless, in the double-mutant sample a
significant reduction in soluble carbohydrate levels was observed,
supporting the idea that endosperm cellularization is connected
with a decrease in hexose levels (Fig. 5A). Decreased hexose levels
were connected with decreased sucrose levels (Fig. 5A), suggesting
that endosperm cellularization caused decreasing sink strength, as
reflected by reduced sucrose levels.
Together, our data reveal that restoration of endosperm
cellularization and the resulting decrease in hexose levels allow
progression of embryo development, suggesting that, as a
consequence of endosperm cellularization, incoming sucrose can
be channeled to the embryo to support its growth. If so, we
expected that fis2 embryos should survive when cultured in vitro
on optimal sucrose concentrations (340 mM) (Eastmond et al.,
2002). Indeed, isolated fis2 embryos had high survival rates when
cultured in vitro on plates containing 340 mM sucrose (16
developing fis2 embryos out of 40 isolated embryos; Fig. 5B).
Genotyping of ten surviving embryos revealed that five of them
were homozygous for the fis2 mutation, four were heterozygous
and one embryo was wild type, confirming that fis2 mutant
embryos can indeed be rescued when cultured under appropriate
conditions. By contrast, fis2 embryos did not progress in their
development when cultured on low-sucrose medium (30 mM; no
developing fis2 embryos out of 12 isolated embryos), similar to a
failure of wild-type embryos to progress in their development when
cultured on low-sucrose medium (Eastmond et al., 2002).
AGL62 is a direct target of the FIS PRC2 complex
Increased and prolonged expression of AGL62 in fis2 seeds
suggests that AGL62 could be directly regulated by the FIS PRC2
complex. Although AGL62 is not a target of PRC2 complexes
during vegetative development (Zhang et al., 2007), recent data
from our laboratory revealed that the FIS PRC2 complex targets a
different set of genes to PRC2 complexes during vegetative
development (Weinhofer et al., 2010). Microarray profiling data of
H3K27me3 localization in the endosperm revealed substantial
levels of H3K27me3 at the AGL62 locus, suggesting that AGL62
is a specific FIS PRC2 target in the endosperm (Fig. 6A). We
experimentally tested the presence of H3K27me3 at the AGL62
locus by ChIP experiments using purified endosperm chromatin.
These experiments confirmed that AGL62 is substantially marked
DEVELOPMENT
2036 RESEARCH ARTICLE
Fig. 5. Restoration of endosperm cellularization is connected
with decreased hexose levels, and fis2 embryos can be rescued
on high-sucrose medium. (A)Enzymatic measurements of soluble
carbohydrates (glucose, fructose and sucrose) in wild-type seeds,
isolated fis2-5 mutant seeds, isolated wild-type sibling seeds in a fis2-5
silique, and isolated non-aborting fis2-5/+ agl62-2/+ seeds derived from
the cross fis2-5/+ agl62-2/+ ⫻ wild type at 8 DAP. *P<0.05 for glucose
and fructose, *P0.05 for sucrose, versus arresting fis2-5 seeds
(unpaired t-test). Error bars indicate s.e.m. (n3 biological replicates).
(B)fis2-5 mutant embryos immediately after isolation (top) and after 14
(middle) or 28 (bottom) days on high-sucrose medium. Scale bars:
200m.
by H3K27me3 in the endosperm, whereas AGL62 does not contain
H3K27me3 marks in seedlings, thus rendering AGL62 an
endosperm-specific PcG target gene (Fig. 6B).
If AGL62 acts directly downstream of the FIS complex, agl62 is
expected to be epistatic over fis2. To test this hypothesis, we
analyzed seed development in self-fertilized fis2/+; agl62/+ mutants.
As in the agl62/+ single mutant, ~25% of the seeds in self-fertilized
fis2/+; agl62/+ siliques were phenotypically indistinguishable from
agl62/– seeds (23.7% early arresting seeds; n654;
c20.59<c0.05[1]23.84; Fig. 6C), revealing that agl62 is epistatic over
fis2. Together, these data strongly support the hypothesis that AGL62
is a downstream target gene of the FIS PRC2 complex.
DISCUSSION
Our study has revealed that endosperm cellularization failure in fis2
as well as ede1 mutants is closely correlated with an arrest of
embryo development at the heart stage. Conversely, endosperm
RESEARCH ARTICLE 2037
Fig. 6. AGL62 is a specific target of the FIS PRC2 complex in the
endosperm. (A)H3K27me3 profiles of AGL62 (At5g60440) in
vegetative tissues or endosperm based on published data (Weinhofer et
al., 2010; Zhang et al., 2007). The gray bar represents the annotated
gene body from transcription start (left) to transcription end (right).
(B)ChIP analysis of H3K27me3 levels in isolated endosperm nuclei and
seedlings. Non-specific IgG antibodies served as a negative control.
Recovery of chromatin was determined by quantitative real-time PCR
and is shown as percentage of input. Error bars indicate s.e.m.
(C)Clearings of progeny of self-fertilized fis2-5/+ agl62-2/+. fis2 seeds
were clearly phenotypically distinguishable from fis2 agl62/+ seeds at
14 DAP (left and right panels). fis2 agl62/– seeds were phenotypically
indistinguishable from agl62/– seeds and arrested development at 3
DAP (center panel). Scale bars: 100m for left and right; 50m for
central.
cellularization in a fraction of ede1 seeds correlated with generally
normal, but slightly delayed, embryo development, leading to
viable seeds. Endosperm cellularization failure in response to
interploidy crosses also causes an arrest of embryo development at
the heart stage (Scott et al., 1998), supporting the idea that
cellularization of the endosperm is crucial for the embryo to
proceed beyond heart stage. Endosperm cellularization in wild-type
seeds was associated with reduced hexose content, which is likely
to be caused by a decrease in the size of the central vacuole.
Vacuolar hexoses are likely to serve as substrates for cell wall
biosynthesis or, alternatively, to be channeled to the growing
embryo. Embryo arrest in fis2 and ede1 mutants was accompanied
by strongly increased hexose levels, correlating with a failure of
the endosperm to cellularize.
The mechanism of endosperm cellularization failure is likely to
differ in fis2 and ede1. Loss of FIS functions causes strong
overexpression of glycosyl hydrolase-encoding genes (Weinhofer
et al., 2010). Glycosyl hydrolases preferentially hydrolyse the
major component of endosperm cell walls, callose (Minic and
Jouanin, 2006), suggesting that repression of cell wall-degrading
enzymes is a requirement for successful endosperm cellularization.
This furthermore implies that endosperm cellularization failure in
fis2 is caused by a failure to repress genes that encode cell walldegrading enzymes. By contrast, EDE1 is required for microtubule
organization (Pignocchi et al., 2009). Microtubules are part of the
DEVELOPMENT
Endosperm cellularization
cell wall-forming phragmoplast, suggesting that disturbed
phragmoplast formation is the cause for endosperm cellularization
failure in ede1.
Despite the distinct causes of endosperm cellularization failure
in each mutant, the effect on embryo development is similar,
strengthening the idea that endosperm cellularization is essential
for embryo development. Endosperm cellularization causes the
central vacuole to decrease in size. As a corollary, incoming
sucrose will no longer be predominantly channeled to the central
vacuole and can instead be partitioned to the embryo, which will
form the major sink in the developing seed (Morley-Smith et al.,
2008). Failure of endosperm cellularization will cause the central
vacuole to remain the major sink in the seed, consistent with
high hexose-to-sucrose ratios in fis2 and ede1 mutant seeds. As
a consequence, it is possible that sucrose is not sufficiently
channeled to the embryo, but remains to be transported to the
vacuole and is cleaved into hexoses. In agreement with this view,
delayed endosperm cellularization in the apetala 2 mutant is
similarly connected with increased hexose-to-sucrose ratios and
to a delay in embryo development (Ohto et al., 2005; Ohto et al.,
2009).
The tissue-specific localization of carbohydrates using the APL3
promoter supports the idea that, as a consequence of endosperm
cellularization, sucrose is channeled to the embryo through the
surrounding endosperm in wild-type seeds, in agreement with
sucrose tracer experiments in oilseed rape (Morley-Smith et al.,
2008). Consistently, APL3 expression could not be detected in the
region surrounding the fis2 embryo, suggesting that owing to the
lack of endosperm cellularization in fis2 the incoming sucrose
remains to be channeled to the central vacuole and does not reach
the embryo. Alternatively, it is possible that the embryosurrounding endosperm in fis2 and ede1 mutants fails to
appropriately differentiate to deliver incoming sucrose to the
embryo. The sucrose transporter AtSUC5 is expressed in the
micropylar region of the endosperm (Baud et al., 2005), indicating
that this region has to achieve a specific differentiation status to
deliver sucrose to the embryo. Together, we hypothesize that
reduced provision of the embryo with sucrose is the likely cause
for embryo arrest in mutants that fail to undergo endosperm
cellularization. In line with this view is the fact that the patterning
of heart stage embryos lacking FIS activity is undistinguishable
from that of wild-type embryos (Leroy et al., 2007), making a
defect in establishing organ identity rather unlikely as the cause for
embryo arrest.
Our data further reveal that the type I MADS-box transcription
factor AGL62 is a direct target gene of the FIS PRC2 complex,
establishing AGL62 as an endosperm-specific PcG target gene.
AGL62 is a negative regulator of endosperm cellularization (Kang
et al., 2008) and our data suggest that the endosperm cellularization
failure in fis2 is largely a consequence of increased AGL62
expression. The agl62 mutant is epistatic over fis2, in agreement
with the view that AGL62 is acting downstream of FIS2. The fis2
phenotype could partially be reversed by maternal loss of AGL62
function. Restoration of endosperm cellularization in fis2/+
agl62/+ double-mutant seeds occurred concomitantly with embryo
development, leading to the formation of viable fis2 embryos.
Delayed endosperm cellularization in fis2/+ agl62/+ mutant seeds
was accompanied by delayed embryo development, implying that
progression of embryo development is strictly coupled to
endosperm cellularization. As AGL62 is exclusively expressed in
the endosperm (Kang et al., 2008), it is most likely that the
normalization of fis2 embryo development in fis2/+ agl62/+
Development 139 (11)
mutant seeds is a consequence of restored endosperm
cellularization. Similarly, triploid seed failure could be suppressed
by maternal loss of AGL62 function, in agreement with the view
that endosperm cellularization failure is largely responsible for
triploid seed failure (Scott et al., 1998). Loss of maternal AGL62
function has previously been shown to cause partial rescue of
hybrid seeds derived from crosses of Arabidopsis thaliana and
Arabidopsis arenosa (Walia et al., 2009), suggesting that increased
AGL62 expression and consequently endosperm cellularization
failure are common mechanisms underlying interploidy and
interspecies seed failure.
Acknowledgements
We thank Sabrina Huber for excellent technical support; Wilhelm Gruissem for
sharing laboratory facilities; Samuel Zeeman and Sebastian Streb for sharing
materials and intellectual support; Abed Chaudhury and Max Bush for
providing fis2-1 and ede1-1 seeds, respectively; and Lars Hennig and members
of the C.K. laboratory for critical comments on this manuscript.
Funding
This research was supported by Swiss National Science Foundation grants
[PP00P3_123362 and CRSI33_127506/1 to C.K.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077057/-/DC1
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