endosperm cellularization defines an important ... · endosperm (drews and yadegari, 2002). the...

RESEARCH ARTICLE 2031

Development 139, 2031-2039 (2012) doi:10.1242/dev.077057© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONSeed development in flowering plants is initiated by doublefertilization of the female gametophyte. The female gametophyteharbors two distinct gametic cells that will have distinct fates afterfertilization: the haploid egg cell will give rise to the diploidembryo and the homodiploid central cell will form the triploidendosperm (Drews and Yadegari, 2002). The endosperm is aterminal tissue that supports embryo growth by delivering nutrientsacquired from the mother plant (Ingram, 2010). Like mostangiosperms, the endosperm of Arabidopsis thaliana follows thenuclear type of development, in which an initial phase of freenuclear divisions without cytokinesis (syncytial phase) is followedby cellularization (Costa et al., 2004). In Arabidopsis, at the 16-nuclei stage the syncytium begins to differentiate into three distinctdevelopmental domains: the micropylar, central and chalazaldomains. The presence of a large central vacuole forces thecytoplasm in the central domain into a thin peripheral layer, whilesyncytial cytoplasm surrounds the embryo in the micropylardomain and the chalazal endosperm develops adjacent to thevascular connection with the seed parent (Berger, 2003; Brown etal., 1999; Brown et al., 2003). At the eighth mitotic cycle,cellularization of the syncytial endosperm is initiated in themicropylar domain around the embryo, coinciding with the earlyheart stage of embryo development (Boisnard-Lorig et al., 2001).Cellularization proceeds in a wave-like manner from themicropylar to the chalazal domain, filling the central cell, except

for near the cyst in the chalazal chamber, with successive layers ofendosperm cells (Brown et al., 1999; Berger, 2003; Brown et al.,2003).

Compartmentalization of the developing seed into three distinctendosperm domains is likely to regulate the uptake of nutrients intothe developing seed. Experiments with labeled sucrose in oilseedrape seeds suggest that the transport of sucrose from theinteguments to the embryo occurs via the micropylar endosperm,while sucrose uptake via the chalazal endosperm is thought to beinvolved in filling the central endosperm vacuole, the main storagepool for hexoses during seed development (Morley-Smith et al.,2008). The large central vacuole determines sink strength duringearly development by rapidly converting imported sucrose tohexoses in the young seed. With progressing endospermcellularization, the size of the central vacuole decreases, correlatingwith 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 mutantsaffecting cytokinesis in the embryo, including knolle, hinkel, openhouse, runkel and pleiade (Sorensen et al., 2002), implying thatendosperm cellularization and somatic cytokinesis share multiplecomponents of the same basic machinery. The spätzle mutant ischaracterized by the absence of cellularization in the endospermbut does not show cytokinesis defects in the embryo, indicating arole for SPÄTZLE in a process specific to endospermcellularization (Sorensen et al., 2002). Similarly, in the endospermdefective 1 (ede1) mutant, the endosperm fails to cellularize but theeffects on embryo patterning are less severe, implicating a mainfunction for EDE1 in endosperm cellularization (Pignocchi et al.,2009). However, in both mutants, embryo development is delayedafter the heart stage and seed shrinkage or collapse occurs,suggesting that endosperm cellularization is crucial for normalembryo and seed development (Sorensen et al., 2002; Pignocchi etal., 2009). Interploidy crosses furthermore support an importantrole for endosperm cellularization in embryo development.

1Department of Biology and Zurich-Basel Plant Science Center, Swiss FederalInstitute of Technology, ETH Centre, CH-8092 Zurich, Switzerland. 2Department ofPlant Biology and Forest Genetics, Uppsala BioCenter, Swedish University ofAgricultural Sciences and Linnean Center for Plant Biology, SE-75007 Uppsala,Sweden.

*Author for correspondence ([email protected])

Accepted 21 March 2012

SUMMARYThe endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endospermdevelops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occursat a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidycrosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seeddevelopment. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) andendosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endospermcellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryodevelopment, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endospermcellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is aconsequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct targetgene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endospermcellularization.

KEY WORDS: Arabidopsis, Endosperm, Polycomb group proteins

Endosperm cellularization defines an importantdevelopmental transition for embryo developmentElisabeth Hehenberger1, David Kradolfer1,2 and Claudia Köhler1,2,*

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Increased paternal genome dosage results in the delay or completefailure of endosperm cellularization, correlating with impairedembryo development (Scott et al., 1998; Dilkes et al., 2008; Erilovaet al., 2009). Developmental defects caused by interploidy crossesare associated with a dysregulation of genes that are directly orindirectly controlled by the FERTILIZATION INDEPENDENTSEED (FIS) Polycomb group (PcG) complex, implicating thatdevelopmental aberrations in response to interploidy crosses arelargely caused by dysregulated FIS target genes (Erilova et al.,2009).

PcG proteins form chromatin-modifying complexes that ensurethe long-term controlled repression of specific target genes. PcGproteins form several families of multiprotein complexes, includingthe two main complexes Polycomb repressive complex 1 (PRC1)and PRC2. PRC2-mediated gene repression is characterized bytrimethylation of lysine 27 of histone H3 (H3K27me3) and seemsto primarily control genes involved in developmental decisions(Hennig and Derkacheva, 2009; Beisel and Paro, 2011). Thesubunits of PRC2 complexes are evolutionary well conserved. Inplants, several PRC2 subunits are encoded by small gene familiesthat form specific complexes with distinct functions during plantdevelopment (Hennig and Derkacheva, 2009). The FIS PRC2complex plays a pivotal role in suppressing the initiation ofendosperm 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 causesendosperm overproliferation and cellularization failure, ultimatelyleading to seed abortion (Chaudhury et al., 1997; Kiyosue et al.,1999; Sorensen et al., 2001). A major regulator of endospermcellularization is the type I MADS-box transcription factorAGAMOUS-LIKE 62 (AGL62) (Kang et al., 2008). Loss ofAGL62 causes precocious endosperm cellularization, indicating arole for AGL62 as a suppressor of endosperm cellularization.Whereas in wild-type seeds AGL62 expression declines abruptlyprior to cellularization, in fis mutants AGL62 expression does notdecline (Kang et al., 2008), suggesting that FIS PRC2 mightregulate endosperm cellularization by controlling the expression ofAGL62.

In this study we investigated the role of FIS PRC2 in endospermcellularization and the consequences of endosperm cellularizationfailure. We show that failure of endosperm cellularization infertilization independent seed 2 (fis2) and ede1 mutants correlateswith impaired embryo development. Restoration of endospermcellularization in fis2 seeds by reducing AGL62 expressionpromotes embryo development, strongly supporting an essentialrole of endosperm cellularization in viable seed formation. Wefurthermore reveal that endosperm cellularization failure in fis2seeds correlates with increased hexose levels, suggesting that arrestof embryo development is a consequence of failed nutrienttranslocation to the developing embryo. Finally, we establish amolecular link for FIS PRC2-mediated endosperm cellularizationby showing that AGL62 is a direct target gene of the FIS PRC2complex.

MATERIALS AND METHODSPlant material and growth conditionsThe 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-0accession background and have been described previously (Kang et al.,2008; Pignocchi et al., 2009; d’Erfurth et al., 2009). For crosses, designatedfemale partners were emasculated, and the pistils hand-pollinated 2 daysafter emasculation. Single- and double-mutant plants were characterized

by PCR using the primers listed in supplementary material Table S1. Seedswere surface sterilized (5% sodium hypochlorite, 0.01% Triton X-100) andplated 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 roomunder a long-day photoperiod (16 hours light and 8 hours darkness) at23°C. Ten-day-old seedlings were transferred to soil and plants were grownin a growth room at 60% humidity and daily cycles of 16 hours light at23°C and 8 hours darkness at 18°C. The APL3::GUS line was generatedby Agrobacterium tumefaciens-mediated transformation (Clough and Bent,1998) into heterozygous fis2-1 plants and six independent lineshomozygous for the transgene locus in the fis2-1 background wereanalyzed.

Transmission analysisSeeds were surface sterilized as above and plated on MS mediasupplemented with 100 M gibberellic acid to facilitate germination. Afterstratification at 4°C in the dark for 3 days, seedlings were grown in agrowth room under a long-day photoperiod (16 hours light and 8 hoursdarkness) at 23°C. Seedlings were harvested for genotyping after 12-16days using the primers listed in supplementary material Table S1.

Embryo rescue and in vitro culturingSiliques of fis2 mutant plants were collected at ~8-10 DAP and surfacesterilized for 10 minutes in 1.5% sodium hypochlorite and 0.1% Tween 20and then washed three times with sterile water. Embryos of fis2 seeds weredissected under sterile conditions using insulin needles. The embryos wereplated on Murashige and Skoog medium (MS salts, pH 5.8, 0.8%Bactoagar, 2.7 mM glutamine) containing either 30 mM or 340 mMsucrose and cultivated under long-day conditions at 21°C.

Genomic DNA extraction from embryos was performed using theREDExtract-N-Amp Plant PCR Kit (Sigma, Poole, UK) with the primerslisted in supplementary material Table S1.

Generation of plasmidsThe 1.8 kb sequence upstream of the APL3 (AT4G39210) transcriptionalstart was amplified by PCR using the primers listed in supplementarymaterial Table S1 and cloned into pCAMBIA1381Z (Cambia).

Analysis of carbohydratesFor each genotype and time point, seeds of three siliques were collected,corresponding to three biological replicates. Only undamaged seeds wereharvested on ice and counted during harvesting, allowing relation of themeasured amounts of carbohydrates to the number of seeds. The seedswere 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 centrifugationfor 5 minutes at 16,000 g, the supernatant was transferred to a new tubeand the extraction repeated. The combined supernatants were dried undervacuum at 30°C.

The dried samples were resuspended in 50 l water and soluble sugarswere measured in a MWGt Discovery XS-R microplate reader (BioTekInstruments, Winooski, USA) after adding 50 l of cocktail [25 mMHEPES 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. Afterestablishing a blind value, glucose, fructose and sucrose were measured byconsecutively adding 1 U glucose-6-phosphate dehydrogenase (RocheApplied Science), 0.7 U phosphoglucose isomerase (Roche AppliedScience) and 5 U invertase (Fluka, St Louis, USA). Soluble carbohydratecontents were calculated as described (Smith and Zeeman, 2006).

RNA extraction and qPCR analysisSeeds at indicated stages were harvested into RNAlater (Sigma-Aldrich,St Louis, USA) and total RNA was extracted using the RNeasy Plant MiniKit (Qiagen, Valencia, USA). For quantitative RT-PCR, RNA was treatedon-column with DNase I and reverse transcribed using the First StrandcDNA Synthesis Kit (Fermentas, Glen Burnie, USA). Gene-specificprimers and Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad,USA) were used on a 7500 Fast Real-Time PCR System (AppliedBiosystems). Quantitative RT-PCR was performed using three replicates

RESEARCH ARTICLE Development 139 (11)

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and ACTIN11 as a reference gene. Results were analyzed as previouslydescribed (Simon, 2003). Primers are listed in supplementary materialTable S1.

Chromatin immunoprecipitation (ChIP)ChIP from endosperm nuclei and seedling tissue was performed asdescribed (Weinhofer et al., 2010). Gene-specific primers and the FastSYBR Green Master Mix were used on the 7500 Fast Real-Time PCRSystem to test enrichment of H3K27me3-marked fragments. QuantitativeChIP PCR was performed using three replicates and results were analyzedas described (Simon, 2003) and are presented as percentage of input.

MicroscopySeeds were harvested for periodic acid-Schiff staining at the indicatedtime points and incubated in 50% ethanol, 5% acetic acid and 4%formaldehyde at 4°C for 12 hours. The seeds were dehydrated in four 1-hour steps, followed by an overnight incubation in 100% ethanol. Aftertwo wash steps with 100% ethanol, the seeds were stepwise infiltratedwith Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) preparationsolution and incubated for 16 hours in 100% preparation solution. Forembedding, seeds were mixed with polymerization solution and allowedto polymerize for at least 2 hours. The embedded samples were sectionedinto 4 m sections using an RM2255 microtome (Leica, Wetzlar,Germany). The sections were collected on water-covered SuperFrost Plusmicroscope slides (Menzel, Braunschweig, Germany) and dried on aheating plate at 72°C. The sections were oxidized for 30 minutes with1% periodic acid, washed and stained for another 30 minutes withSchiff’s reagent (Sigma-Aldrich). The sections were embedded inEntellan New Mounting Media (Electron Microscopy Sciences, Hatfield,USA) for microscopy.

Seeds were harvested at the indicated time points and stained to detectGUS activity as described (Köhler et al., 2003b). The seeds weredehydrated and embedded as described above. Clearing analysis of seedswas performed as described (Köhler et al., 2003b).

Microscopy imaging was performed using a Leica DM 2500 microscopewith DIC optics. Images were captured using a Leica DFC300 FX digitalcamera, exported using Leica Application Suite version 2.4.0.R1 andprocessed using Photoshop CS5 (Adobe Systems, San Jose, USA).

RESULTSEndosperm cellularization failure correlates withembryo arrest in fis2 and ede1 mutantsLoss of FIS function causes an endosperm cellularization failureand arrest of embryo development, leading to seed abortion ~8days 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 whetherarrest of embryo development upon loss of FIS function is a directconsequence of endosperm cellularization failure or rather anindirect consequence of dysregulated gene expression impactingon embryo development. If embryo arrest is a direct consequenceof endosperm cellularization failure, mutants defective inendosperm cellularization are expected to have a fis-like embryoarrest phenotype. We tested this hypothesis by analyzing the ede1mutant, which has a mutation in a plant-specific microtubule-associated protein (Pignocchi et al., 2009). Loss of EDE1 functioncauses endosperm cellularization failure, resulting in seeds thatcontain uncellularized endosperm and in embryo arrest at lateheart stage (Pignocchi et al., 2009). Under our conditions, at 8DAP 11.4% of the seeds derived from homozygous ede1-1mutants had a fis-like phenotype, with uncellularized endospermand embryos arrested at heart stage (n340; Fig. 1A-C), whereas88.6% of the seeds had cellularized endosperm and embryosprogressed in their development beyond heart stage (Fig. 1E).Development of those ede1 embryos that progressed beyond heartstage was still delayed compared with wild-type seeds; at 8 DAP,ede1 mutant embryos had only reached the early torpedo stage ofdevelopment, whereas wild-type embryos had already entered thebent cotyledon stage (Fig. 1E,D). Delayed embryo developmentwas accompanied by a delay in endosperm cellularization;whereas cellularization of the peripheral endosperm was clearlyvisible in wild-type endosperm at 6 DAP, ede1-1 seeds reached asimilar cellularization state only at ~8 DAP (Fig. 1A,E). Embryo

2033RESEARCH ARTICLEEndosperm cellularization

Fig. 1. Endosperm cellularization failurecorrelates with embryo arrest in fis2 andede1 mutants. Schiff-stained Technovit sectionsof wild-type, fis2-5 and ede1-1 Arabidopsisseeds. (A)Wild-type seed with embryo attorpedo stage and cellularizing endosperm at 6DAP. (B)fis2-5 seed with embryo arresting atheart stage and uncellularized endosperm at 8DAP. (C)ede1-1 seed with embryo arresting atheart stage and uncellularized endosperm at 8DAP. (D)Wild-type seed with embryo at bentcotyledon stage and fully cellularized endospermat 8 DAP. (E)ede1-1 seed with embryo attorpedo stage and cellularizing endosperm at 8DAP. (F)ede1-1 seed with embryo at bentcotyledon stage and fully cellularized endospermat 10 DAP. CV, central vacuole. Scale bar:100m.

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development remained delayed and ede1 embryos reached thebent cotyledon stage 2 days later than wild-type embryos, at 10DAP (Fig. 1F).

Together, their close correlation in ede1 mutant seeds supportsthe idea that embryo arrest is a consequence of endospermcellularization failure. It is however possible that a fraction of ede1mutant embryos arrest development due to a direct requirement ofEDE1 function in the embryo.

Cellularization failure is connected with increasedhexose content in the endospermWe considered the possible consequences of cellularization failureimpacting on embryo development. Endosperm cellularizationcauses the central vacuole to become smaller, until it becomescompletely invaded by endosperm cells (Olsen, 2001). The centralvacuole of the endosperm is the main storage pool for hexoses inthe seed; therefore, a decrease in vacuole size in response tocellularization is likely to cause a decrease in hexose content(Morley-Smith et al., 2008). Indeed, the time of endospermcellularization 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 toundergo endosperm cellularization.

We tested this hypothesis by measuring glucose, fructose(referred to as hexoses) and sucrose levels in fis2 mutant seeds at6, 7 and 8 DAP, whereby the fis2 phenotype allowed discriminationand separate measurement of developing and arresting seeds infis2/+ siliques from 7 DAP onwards. Before 7 DAP, discriminationbetween developing and arresting fis2 seeds was not possible,

therefore we measured a mixture of 50% wild-type and 50% fis2seeds. At 6 DAP, hexose levels in wild type and fis2 werecomparable (Fig. 2A), correlating with the presence of a largecentral vacuole in wild-type seeds (Fig. 1A). At 7 DAP, the centralvacuole in wild-type seeds had strongly decreased in size(supplementary material Fig. S1), correlating with decreasedhexose levels (Fig. 2A). Consistent with the predictions, hexoselevels in fis2 mutant seeds remained high at 7 and 8 DAP, whereaswild-type hexose levels strongly decreased to ~25% of thosepresent at 6 DAP. At 8 DAP, arresting fis2 seeds contained hexoselevels that were more than five times higher than those in wild-typeseeds at the same time point (Fig. 2A). At 8 DAP, sucrose levels inwild-type seeds decreased to about half the amount at 6 DAP,whereas sucrose levels in fis2 remained unchanged over theinvestigated time period (Fig. 2A).

The increased levels of hexoses and sucrose in fis2 seeds at 8DAP correlated with increased expression of the APL3 gene, whichencodes one of the large subunits of the ADP-glucosepyrophosphorylase (AGPase), a key enzyme of starch biosynthesis(Zeeman et al., 2010) (Fig. 2B-E). Expression of APL3 is highlyresponsive to sucrose and glucose (Crevillen et al., 2005), makingthe APL3 promoter fused to the -GLUCURONIDASE (GUS) asuitable reporter to monitor the tissue-specific localization ofcarbohydrates. Whereas in fis2 seeds at 8 DAP a strong GUS signalcould be observed in the seed coat, wild-type seeds from the samesilique had only very weak GUS staining in the seed coat (Fig. 2B-E). By contrast, wild-type seeds at 5 DAP had similarly strongGUS 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 endospermcellularization mutants. (A)Enzymatic measurementsof soluble carbohydrates (glucose, fructose and sucrose)in wild-type and isolated fis2-1/+ Arabidopsis seeds at6, 7 and 8 DAP. Error bars indicate s.e.m. (n3biological replicates) (B-H) Analysis of APL3::GUSexpression in fis2 seeds. Clearings (B,C) and Technovitsections (D-H) of GUS-stained seeds expressingAPL3::GUS. (B,D)Wild-type seed at 7 DAP; (C,E) fis2-1seed 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 solublecarbohydrates in isolated ede1-1/– seeds at 8 DAP. Errorbars indicate s.e.m. (n3 biological replicates). Gluc,glucose; Fruc, fructose; Suc, sucrose; wt, wild type.Scale bar: 100m.

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However, whereas wild-type embryos had a pronounced GUSstaining zone surrounding the embryo (Fig. 2H), this zone wasabsent in fis2 embryos (Fig. 2G). This staining is likely to originatefrom the endosperm surrounding the embryo, suggesting that theembryo receives carbohydrates from the neighboring endosperm inwild-type seeds, whereas the embryo-surrounding endosperm infis2 fails to perform this function.

If increased hexose levels are a consequence of cellularizationfailure, uncellularized ede1 seeds should have similarly increasedhexose levels as fis2 seeds. To test this hypothesis, we isolated ede1seeds that failed to undergo endosperm cellularization at 8 DAP.

These seeds were phenotypically clearly distinguishable fromcellularized wild-type seeds by their white and glossy appearance.Uncellularized ede1 seeds had similarly increased hexose levels asuncellularized fis2 seeds (Fig. 2I), supporting the hypothesis thatincreased hexose levels are a consequence of cellularization failurein the endosperm.

To conclude, endosperm cellularization failure correlates withelevated hexose levels in the developing seeds, suggesting thatincoming sucrose is not properly delivered to the developingembryo, causing embryo arrest.

Restoration of cellularization by maternal loss ofAGL62 normalizes fis2 seed developmentIf endosperm cellularization failure is the cause of embryodevelopmental arrest, restoration of endosperm cellularization infis2 mutants should promote embryo development. Loss of theMADS-box transcription factor AGL62 causes precociousendosperm cellularization, implicating AGL62 as a major regulatorof endosperm cellularization in Arabidopsis (Kang et al., 2008). Inagreement with previous studies revealing prolonged expression ofan AGL62 reporter construct in fis mutant endosperm (Kang et al.,2008), AGL62 transcript levels were strongly increased andprolonged in fis2 seeds compared with wild-type transcript levels,which were undetectable at 5 DAP (Fig. 3A). Reduced expressionof AGL62 correlates with the onset of endosperm cellularization inArabidopsis seeds (Kang et al., 2008), suggesting repression ofendosperm cellularization in fis2 seeds by extended and increasedAGL62 expression.

To test whether reduced dosage of AGL62 can suppress the fis2mutant phenotype we generated fis2/+; agl62/+ double mutants.When double-heterozygous mutants were pollinated with wild-typepollen a new seed class was observed that was clearlydistinguishable from arresting fis2 seeds (Fig. 3B). At 14 DAP,~23% of the seeds were not collapsed but instead phenotypicallydistinguishable from wild-type seeds by their enlarged size (Fig.3C; 25.8% aborted seeds, 23.2% non-aborted enlarged green seeds,51% normal seeds; n635), suggesting that agl62 could partiallysuppress the fis2 mutation. Although we also observed a fractionof non-collapsed seeds in fis2/+ single mutants at 14 DAP (12.2%,n433 total seeds), none of these seeds progressed beyond the heartstage of development. By contrast, ~20% of fis2 agl62 embryosdeveloped up to the torpedo stage (n147 total fis2 agl62 seeds).Sections of the enlarged green fis2 agl62 seeds at 14 DAP revealedeither progressing or full cellularization of the endosperm (Fig.3D), whereby advanced levels of cellularization correlated withadvanced embryo development. Development of fis2 agl62embryos was delayed compared with wild-type embryos: whereasmost wild-type embryos were in the bent cotyledon stage at 9 DAP,fis2 agl62 embryos still had a torpedo-like shape (Fig. 3D). Thedelayed embryo development in fis2 agl62 seeds correlated with adelay of endosperm cellularization, which occurred 4-5 days laterthan in wild-type seeds (Fig. 3D), suggesting that AGL62 isimportant but might not be the only dysregulated FIS targetimpacting endosperm development. Alternatively, it is possible thatthe paternally contributed wild-type AGL62 allele remainedexpressed at higher levels in fis2 seeds.

To further test the hypothesis that the agl62 mutation cansuppress the fis2 mutant phenotype, we examined whether theconcomitant presence of the agl62 and fis2 mutations would allowtransmission of fis2 through the maternal gametophyte. fis2/+agl62/+ mutants were pollinated with wild-type pollen and F1seedlings derived from this cross were tested for the presence of


Fig. 3. Reduced maternal genome dosage in the fis2-5/+ agl62-2/+ double mutant suppresses the fis2 phenotype. (A)QuantitativeRT-PCR analysis of AGL62 expression in wild-type and fis2-5Arabidopsis seeds from 2-5 DAP. Error bars indicate s.e.m. (B)Seedsfrom wild type (left), from the cross fis2-5 � wild type (center) andfrom the cross fis2-5/+ agl62-2/+ � wild type (right) at 14 DAP. Insetsshow isolated embryos from seeds of the same developmental stageand genotype. (C)Distribution of seed phenotypes from the cross fis2-5� wild type and fis2-5/+ agl62-2/+ � wild type at 14 DAP, includingmaternal transmission of the fis2-5 mutation in the respective crosses.Numbers above bars indicate seeds analyzed per genotype. Numbersfor 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 stageembryos (9 and 14 DAP). fis2-5 agl62-2 seeds contain early torpedo (9DAP) and bent cotyledon (14 DAP) stage embryos. Scale bars: 100m.DEVELO

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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 agl62mutation. By contrast, out of 48 seedlings derived from the controlcross of fis2/+ pollinated with wild-type pollen, only one seedlingcontained the fis2 mutation (Fig. 3C). However, this seedling wasnot viable and arrested development immediately after germination.We conclude that agl62 allows transmission of the fis2 allelethrough the female gametophyte, albeit at reduced frequency.

Loss of maternal AGL62 normalizes triploid seeddevelopmentInterploidy crosses of diploid maternal and tetraploid paternalplants cause increased expression of AGL62, which correlateswith endosperm cellularization failure and seed abortion (Erilovaet al., 2009). We tested whether maternal loss of AGL62 couldsuppress triploid seed failure by pollinating agl62/+ plants withdiploid pollen from the omission in second division 1 (osd1)mutant. Pollination of wild-type plants with osd1 pollen leads tothe formation of 100% triploid progeny (d’Erfurth et al., 2009),similar to interploidy crosses of diploid maternal and tetraploidpaternal plants. Triploid seeds in the Columbia (Col) accessionhave similar phenotypic abnormalities as fis mutant seeds andabort containing embryos arrested at heart stage surrounded bylargely uncellularized endosperm (Dilkes et al., 2008). Uponpollination of wild-type plants with diploid osd1 pollen, 88% ofthe seeds were dark brownish and collapsed, whereas 12% werea mixture of phenotypically normal seeds (3%) and enlargedabnormally shaped seeds that nonetheless contained developedembryos (9%) (n461 total seeds; Fig. 4A). Pollination ofagl62/+ with osd1 pollen more than doubled the number of

phenotypically normal seeds and enlarged seeds (7% and 20%,respectively; n522 total seeds; Fig. 4A-C), supporting the viewthat reduced levels of AGL62 can restore viable triploid seedformation. We tested the germination capacity of triploid seedsand, consistent with increased numbers of phenotypically normaland enlarged triploid seeds derived from pollinated agl62 mutantplants, the germination rate of those seeds was almost twice ashigh as that of wild-type triploid seeds (19% versus 36%, n155and 85 total seeds, respectively; Fig. 4A). Together, thesefindings support the hypothesis that maternal loss of AGL62partially restores triploid seed viability.

Restoration of endosperm cellularization isconnected with decreased hexose levelsIf cellularization failure is causally connected with increasedhexose levels, restoration of endosperm cellularization should beconnected with decreased hexose levels. To test this hypothesis, weperformed carbohydrate measurements in fis2 agl62 and fis2 seedsat 8 DAP. At this time point, fis2/+; agl62/+ double-mutant seedscannot be distinguished from the fis2/+ seeds by eye, resulting ina mixture of 50% double-mutant and 50% fis2 seeds in the double-mutant sample. Nonetheless, in the double-mutant sample asignificant reduction in soluble carbohydrate levels was observed,supporting the idea that endosperm cellularization is connectedwith a decrease in hexose levels (Fig. 5A). Decreased hexose levelswere connected with decreased sucrose levels (Fig. 5A), suggestingthat endosperm cellularization caused decreasing sink strength, asreflected by reduced sucrose levels.

Together, our data reveal that restoration of endospermcellularization and the resulting decrease in hexose levels allowprogression of embryo development, suggesting that, as aconsequence of endosperm cellularization, incoming sucrose canbe channeled to the embryo to support its growth. If so, weexpected that fis2 embryos should survive when cultured in vitroon optimal sucrose concentrations (340 mM) (Eastmond et al.,2002). Indeed, isolated fis2 embryos had high survival rates whencultured in vitro on plates containing 340 mM sucrose (16developing fis2 embryos out of 40 isolated embryos; Fig. 5B).Genotyping of ten surviving embryos revealed that five of themwere homozygous for the fis2 mutation, four were heterozygousand one embryo was wild type, confirming that fis2 mutantembryos can indeed be rescued when cultured under appropriateconditions. By contrast, fis2 embryos did not progress in theirdevelopment when cultured on low-sucrose medium (30 mM; nodeveloping fis2 embryos out of 12 isolated embryos), similar to afailure of wild-type embryos to progress in their development whencultured on low-sucrose medium (Eastmond et al., 2002).

AGL62 is a direct target of the FIS PRC2 complexIncreased and prolonged expression of AGL62 in fis2 seedssuggests that AGL62 could be directly regulated by the FIS PRC2complex. Although AGL62 is not a target of PRC2 complexesduring vegetative development (Zhang et al., 2007), recent datafrom our laboratory revealed that the FIS PRC2 complex targets adifferent set of genes to PRC2 complexes during vegetativedevelopment (Weinhofer et al., 2010). Microarray profiling data ofH3K27me3 localization in the endosperm revealed substantiallevels of H3K27me3 at the AGL62 locus, suggesting that AGL62is a specific FIS PRC2 target in the endosperm (Fig. 6A). Weexperimentally tested the presence of H3K27me3 at the AGL62locus by ChIP experiments using purified endosperm chromatin.These experiments confirmed that AGL62 is substantially marked


Fig. 4. Triploid seed failure is suppressed by agl62. (A)Distributionof mature seed phenotypes from the cross wild type � osd1-1 andagl62-2/+ � osd1-1 including the germination rate of the respectivecrosses. 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 imagesof seeds from cross agl62-2/+ � osd1-1 at 12 DAP corresponding tonormal (left), aborted (middle) and non-aborted (right) phenotypes. D

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by H3K27me3 in the endosperm, whereas AGL62 does not containH3K27me3 marks in seedlings, thus rendering AGL62 anendosperm-specific PcG target gene (Fig. 6B).

If AGL62 acts directly downstream of the FIS complex, agl62 isexpected to be epistatic over fis2. To test this hypothesis, weanalyzed seed development in self-fertilized fis2/+; agl62/+ mutants.As in the agl62/+ single mutant, ~25% of the seeds in self-fertilizedfis2/+; agl62/+ siliques were phenotypically indistinguishable fromagl62/– seeds (23.7% early arresting seeds; n654;c20.59<c0.05[1]

23.84; Fig. 6C), revealing that agl62 is epistatic overfis2. Together, these data strongly support the hypothesis that AGL62is a downstream target gene of the FIS PRC2 complex.

DISCUSSIONOur study has revealed that endosperm cellularization failure in fis2as well as ede1 mutants is closely correlated with an arrest ofembryo development at the heart stage. Conversely, endosperm

cellularization in a fraction of ede1 seeds correlated with generallynormal, but slightly delayed, embryo development, leading toviable seeds. Endosperm cellularization failure in response tointerploidy crosses also causes an arrest of embryo development atthe heart stage (Scott et al., 1998), supporting the idea thatcellularization of the endosperm is crucial for the embryo toproceed beyond heart stage. Endosperm cellularization in wild-typeseeds was associated with reduced hexose content, which is likelyto be caused by a decrease in the size of the central vacuole.Vacuolar hexoses are likely to serve as substrates for cell wallbiosynthesis or, alternatively, to be channeled to the growingembryo. Embryo arrest in fis2 and ede1 mutants was accompaniedby strongly increased hexose levels, correlating with a failure ofthe endosperm to cellularize.

The mechanism of endosperm cellularization failure is likely todiffer in fis2 and ede1. Loss of FIS functions causes strongoverexpression of glycosyl hydrolase-encoding genes (Weinhoferet al., 2010). Glycosyl hydrolases preferentially hydrolyse themajor component of endosperm cell walls, callose (Minic andJouanin, 2006), suggesting that repression of cell wall-degradingenzymes is a requirement for successful endosperm cellularization.This furthermore implies that endosperm cellularization failure infis2 is caused by a failure to repress genes that encode cell wall-degrading enzymes. By contrast, EDE1 is required for microtubuleorganization (Pignocchi et al., 2009). Microtubules are part of the


Fig. 5. Restoration of endosperm cellularization is connectedwith decreased hexose levels, and fis2 embryos can be rescuedon high-sucrose medium. (A)Enzymatic measurements of solublecarbohydrates (glucose, fructose and sucrose) in wild-type seeds,isolated fis2-5 mutant seeds, isolated wild-type sibling seeds in a fis2-5silique, and isolated non-aborting fis2-5/+ agl62-2/+ seeds derived fromthe cross fis2-5/+ agl62-2/+ � wild type at 8 DAP. *P<0.05 for glucoseand fructose, *P0.05 for sucrose, versus arresting fis2-5 seeds(unpaired t-test). Error bars indicate s.e.m. (n3 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:200m.

Fig. 6. AGL62 is a specific target of the FIS PRC2 complex in theendosperm. (A)H3K27me3 profiles of AGL62 (At5g60440) invegetative tissues or endosperm based on published data (Weinhofer etal., 2010; Zhang et al., 2007). The gray bar represents the annotatedgene body from transcription start (left) to transcription end (right).(B)ChIP analysis of H3K27me3 levels in isolated endosperm nuclei andseedlings. Non-specific IgG antibodies served as a negative control.Recovery of chromatin was determined by quantitative real-time PCRand is shown as percentage of input. Error bars indicate s.e.m.(C)Clearings of progeny of self-fertilized fis2-5/+ agl62-2/+. fis2 seedswere clearly phenotypically distinguishable from fis2 agl62/+ seeds at14 DAP (left and right panels). fis2 agl62/– seeds were phenotypicallyindistinguishable from agl62/– seeds and arrested development at 3DAP (center panel). Scale bars: 100m for left and right; 50m forcentral.

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cell wall-forming phragmoplast, suggesting that disturbedphragmoplast formation is the cause for endosperm cellularizationfailure in ede1.

Despite the distinct causes of endosperm cellularization failurein each mutant, the effect on embryo development is similar,strengthening the idea that endosperm cellularization is essentialfor embryo development. Endosperm cellularization causes thecentral vacuole to decrease in size. As a corollary, incomingsucrose will no longer be predominantly channeled to the centralvacuole and can instead be partitioned to the embryo, which willform the major sink in the developing seed (Morley-Smith et al.,2008). Failure of endosperm cellularization will cause the centralvacuole to remain the major sink in the seed, consistent withhigh hexose-to-sucrose ratios in fis2 and ede1 mutant seeds. Asa consequence, it is possible that sucrose is not sufficientlychanneled to the embryo, but remains to be transported to thevacuole and is cleaved into hexoses. In agreement with this view,delayed endosperm cellularization in the apetala 2 mutant issimilarly connected with increased hexose-to-sucrose ratios andto a delay in embryo development (Ohto et al., 2005; Ohto et al.,2009).

The tissue-specific localization of carbohydrates using the APL3promoter supports the idea that, as a consequence of endospermcellularization, sucrose is channeled to the embryo through thesurrounding endosperm in wild-type seeds, in agreement withsucrose tracer experiments in oilseed rape (Morley-Smith et al.,2008). Consistently, APL3 expression could not be detected in theregion surrounding the fis2 embryo, suggesting that owing to thelack of endosperm cellularization in fis2 the incoming sucroseremains to be channeled to the central vacuole and does not reachthe embryo. Alternatively, it is possible that the embryo-surrounding endosperm in fis2 and ede1 mutants fails toappropriately differentiate to deliver incoming sucrose to theembryo. The sucrose transporter AtSUC5 is expressed in themicropylar region of the endosperm (Baud et al., 2005), indicatingthat this region has to achieve a specific differentiation status todeliver sucrose to the embryo. Together, we hypothesize thatreduced provision of the embryo with sucrose is the likely causefor embryo arrest in mutants that fail to undergo endospermcellularization. In line with this view is the fact that the patterningof heart stage embryos lacking FIS activity is undistinguishablefrom that of wild-type embryos (Leroy et al., 2007), making adefect in establishing organ identity rather unlikely as the cause forembryo arrest.

Our data further reveal that the type I MADS-box transcriptionfactor 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 (Kanget al., 2008) and our data suggest that the endosperm cellularizationfailure in fis2 is largely a consequence of increased AGL62expression. The agl62 mutant is epistatic over fis2, in agreementwith the view that AGL62 is acting downstream of FIS2. The fis2phenotype could partially be reversed by maternal loss of AGL62function. Restoration of endosperm cellularization in fis2/+agl62/+ double-mutant seeds occurred concomitantly with embryodevelopment, leading to the formation of viable fis2 embryos.Delayed endosperm cellularization in fis2/+ agl62/+ mutant seedswas accompanied by delayed embryo development, implying thatprogression of embryo development is strictly coupled toendosperm cellularization. As AGL62 is exclusively expressed inthe endosperm (Kang et al., 2008), it is most likely that thenormalization of fis2 embryo development in fis2/+ agl62/+

mutant seeds is a consequence of restored endospermcellularization. Similarly, triploid seed failure could be suppressedby maternal loss of AGL62 function, in agreement with the viewthat endosperm cellularization failure is largely responsible fortriploid seed failure (Scott et al., 1998). Loss of maternal AGL62function has previously been shown to cause partial rescue ofhybrid seeds derived from crosses of Arabidopsis thaliana andArabidopsis arenosa (Walia et al., 2009), suggesting that increasedAGL62 expression and consequently endosperm cellularizationfailure are common mechanisms underlying interploidy andinterspecies seed failure.

AcknowledgementsWe thank Sabrina Huber for excellent technical support; Wilhelm Gruissem forsharing laboratory facilities; Samuel Zeeman and Sebastian Streb for sharingmaterials and intellectual support; Abed Chaudhury and Max Bush forproviding fis2-1 and ede1-1 seeds, respectively; and Lars Hennig and membersof the C.K. laboratory for critical comments on this manuscript.

FundingThis research was supported by Swiss National Science Foundation grants[PP00P3_123362 and CRSI33_127506/1 to C.K.].

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077057/-/DC1

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