endosperm and imprinting, inextricably linked1[open] · in maize, endosperm is de-ﬁned...

Update on Plant Gene Imprinting

Endosperm and Imprinting, Inextricably Linked1[OPEN]

Mary Gehring* and P.R. Satyaki

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 (M.G., P.R.S.); and Departmentof Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (M.G.)

ORCID IDs: 0000-0003-2280-1522 (M.G.); 0000-0002-5035-7652 (P.R.S.).

The endosperm is often viewed as a complicated andrather strange tissue: its genome is triploid, it is formedfrom a second fertilization event specific to floweringplants, and it is a frequent source of incompatibilitybetween plants, yet does not contribute geneticmaterialto the next generation. Additionally, recent investiga-tions have demonstrated that the endosperm is perhapsthe most epigenetically divergent plant tissue, withunique DNA methylation and chromatin structurefeatures. The endosperm is also the site of imprintedgene expression, an epigenetic phenomenon intimatelylinked to the endosperm’s unusual properties. Imprin-ted genes are expressed predominantly from either thematernally or paternally inherited allele. This Updatefocuses on progress over the last several years in un-derstanding imprinted gene expression, from molecu-lar control to physiological significance, within thecontext of endosperm formation and function.

Seeds are a biological marvel—they house the nextgeneration of plant and, in the right conditions, canremain viable for thousands of years (Sallon et al.,2008). The first seed plants appeared approximately360 million years ago. About 200 million years later,flowering plants (angiosperms) arose and rapidly be-came the dominant form of plant life (Doyle, 2012). Oneof the key innovations in flowering plant seed devel-opment was the addition of a second fertilizationevent (Fig. 1). While both flowering and nonflowering(gymnosperms) seed plants produce two sperm fromeach meiotic event, only one sperm participates in fer-tilization in most nonflowering seed plants. Resourcesare provisioned to the developing gymnosperm em-bryo through the female gametophyte, which exten-sively proliferates prior to fertilization (Fig. 1). Bycontrast, in flowering plants the second sperm fertilizesa second gametic cell, the central cell, leading to for-mation of the endosperm (Drews and Koltunow, 2011).Whilemany fascinating variations on female gametophyte

development and the second fertilization event exist, inmost flowering plants endosperm is triploid, consisting oftwo maternally inherited and one paternally inheritedgenome (Friedman et al., 2008). Although the idea remainsunproven, the acquisition of a second fertilization eventhas been credited as one key to angiosperm dominancebecause the maternal parent devotes resources only tothose seeds that have been successfully fertilized (Barouxet al., 2002).

The endosperm is the site of imprinted gene expres-sion. In each plant species that has been examined inmolecular detail, dozen to hundreds of genes that areeither expressed from the maternally (maternally ex-pressed imprinted genes or MEGs) or paternally (pa-ternally expressed imprinted genes or PEGs) inheritedalleles have been identified (Fig. 2). The embryo lacksimprinting (Gehring et al., 2011; Hsieh et al., 2011; Luoet al., 2011; Waters et al., 2011), except perhaps at thevery earliest stages of development (Jahnke andScholten, 2009; Nodine and Bartel, 2012; Raissig et al.,2013), as do other plant tissues (Zhang and Borevitz,2009). It is thought that imprinting is restricted to the

1 This work was supported by National Science Foundation GrantMCB 1453459 to M.G.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Mary Gehring ([email protected]).

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endosperm because the formation of a sexual embryo-nourishing tissue may have created the right conditionsfor the evolution of genomic imprinting (Wolf, 2009;Haig, 2013; Patten et al., 2014; Spencer and Clark, 2014;see Box 1). Major unanswered or only partially an-swered questions about imprinting include: (1) How isimprinted expression controlled from the level of DNAsequence to three-dimensional chromatin structure? (2)

What is the function of imprinted genes? (3) When andwhy did imprinted expression in endosperm evolve?Reflecting the current state of knowledge in the field,this Update focuses on the first two questions, with asmattering of the third, and primarily relies on insightsgained from experiments performed in maize (Zeamays), rice (Oryza sativa), and Arabidopsis (Arabidopsisthaliana).

A BRIEF PRIMER ON ENDOSPERM DEVELOPMENTAND FUNCTION

Seed formation requires extensive intergenerationaland intertissue communication and coordination (Garciaet al., 2005; Ingouff et al., 2006; Ingram, 2010; Li et al.,2013; Xu et al., 2016). Angiosperm seeds consist of threegenetically distinct entities: the diploid embryo, withone paternal and one maternal genome; the triploidendosperm, with two maternal and one paternal ge-nome; and the protective integuments/seed coat, con-sisting of diploid maternal tissue from the previousgeneration (Fig. 1). After fertilization, the primary en-dosperm nucleus (in the fertilized central cell) rapidly

Figure 1. Endosperm is an important angiosperm innovation. In gymnosperms, a haploid sperm fertilizes one of the haploid eggs.The embryo develops surrounded and nourished by female gametophyte tissue. In angiosperms, one haploid sperm fertilizes theegg and the other fertilizes the diploid central cell. The central cell is diploid because two haploid nuclei, the polar nuclei, migratetoward one and fuse either before (Arabidopsis) or at the time of (maize) fertilization. The diploid embryo is nourished by thetriploid endosperm. A single layer of endosperm remains in mature Arabidopsis seeds; in maize the endosperm supports thegerminating seedling.

Figure 2. Imprinted genes can be eitherMEGs or PEGs. Imprinted genesare expressed in a parent-of-origin-dependent manner. Although thediagram depicts complete silencing of one allele, in practice manyimprinted genes are differentially expressed between maternal andpaternal alleles, not monoallelically expressed.

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divides before the onset of zygotic division. In cereals,Arabidopsis, andmany other species, initial endospermdivisions are synchronous and syncytial, meaning thatthere is nuclear division without concomitant cellula-rization. The surrounding maternal tissue also dividesto accommodate the growing seed (Garcia et al., 2005;Li et al., 2013). After a defined period of nuclear divi-sion, endosperm cellularization in Arabidopsis coin-cides with reduced rates of mitotic division and theonset of endosperm differentiation (Mansfield and

Briarty, 1990a, 1990b; Boisnard-Lorig et al., 2001; Berger,2003). Cellularization proceeds from the micropylarpole to the chalazal pole. Arabidopsis endosperm isdistinguished into three tissue types: micropylar,which surrounds the embryo; peripheral, which linesthe seed cavity, eventually displacing the large cen-tral vacuole; and chalazal, which abuts the termina-tion of maternal vascular tissue (Mansfield and Briarty,1990b; Boisnard-Lorig et al., 2001; Sørensen et al., 2002).Each endosperm tissue has its own gene expression

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program, with the chalazal endosperm being quitedistinct from the micropylar and peripheral endosperm(Belmonte et al., 2013). The chalazal endosperm is char-acterized by larger nuclei, the result of nuclear fusions orendoreduplication (Boisnard-Lorig et al., 2001; Brownet al., 2003; Baroux et al., 2004), and is also the site ofmaternal nutrient transfer. In maize, endosperm is de-fined as ESR (embryo surrounding region, analogous tomicropylar), starchy, aleurone, or BETL (basal endo-sperm transfer layer), which mediates nutrient transferto the endosperm from the maternal parent (Sabelli andLarkins, 2009). Gene expression programs have alsobeen defined for maize endosperm types at multiplestages of development (Li et al., 2014). In maize, celldivision continues for several days after cellularization.Cessation of mitotic division is coupled to the onset ofendoreduplication and the accumulation of storageproteins and starch, which is followed by programmedcell death (Sabelli and Larkins, 2009). In Arabidopsis,the endosperm begins to break down as the cotyledonsbecome the primary storage organs. At maturity, anArabidopsis seed has one layer of remaining endospermcells. In maize and other cereals, a large portion of en-dosperm is persistent and is subsequently consumedby the germinating seedling (Fig. 1). Endosperm pro-liferation affects final seed size—a greater number ofendosperm cells is generally correlated with biggermature seeds, even in species like Arabidopsis wherethe endosperm is ephemeral (Scott et al., 1998; Garciaet al., 2005; Sundaresan, 2005). Several excellent reviewson endosperm development provide further details(Berger, 2003; Sabelli and Larkins, 2009; Li and Berger,2012).

The endosperm is a food supply for the embryo/seedling (and other creatures). Recent excitingwork hasshown that the endosperm is not just a passive bag ofnutrients but also a source of molecules that are re-quired for proper embryo patterning. For example, theArabidopsis and maize endosperm-specific basic-helix-loop-helix ZHOUP transcription factors are expressedin the ESR and are required for two genetically sepa-rable functions: embryo epidermis patterning and en-dosperm breakdown (Xing et al., 2013; Grimault et al.,2015). Short Cys-rich peptides initially expressed in thecentral cell before fertilization and in the endospermESR after fertilization are required for elongation of theembryonic suspensor, a structure that connects theembryo proper to a nutrient supply during early seeddevelopment (Costa et al., 2014). Additional functionsfor endosperm and more cell nonautonomous actingmolecules will likely be discovered over the next sev-eral years.

IMPRINTING MECHANISMS AND THE UNIQUEENDOSPERM EPIGENOMIC LANDSCAPE

Imprinted genes offer a fascinating gene regulationpuzzle: How do alleles of a gene, perhaps with identicalsequence, living together in the same nucleus, existin two different transcriptional states (Fig. 2)? One

possibility is that epigenetic differences are establishedbefore fertilization in the male and female gameto-phytes, and are faithfully propagated in the endospermafter fertilization; another is that differences are estab-lished postfertilization. Several lines of evidence indi-cate that the embryo and endosperm exist in distincttranscriptional and chromatin states immediately afterfertilization, and that these states are established duringspecification of the egg cell and central cell in the femalegametophyte (Gutiérrez-Marcos et al., 2006; Barouxet al., 2007b; Garcia-Aguilar et al., 2010; Pillot et al.,2010). Because sperm cells are capable of fertilizing ei-ther the egg cell or central cell (Frank and Johnson,2009), differences between the zygote and the endo-sperm and between maternal and paternal DNA in theendosperm are likely established in the mature femalegametophyte before fertilization. This is a fairly narrowtimeframe to establish dimorphism; the nuclei that willcontribute to the egg and central cell are not distin-guished until the last mitotic division of the femalegametophyte and occupy a common cytoplasm untilcellularization.

EndospermDNA is hypomethylated and has a looserchromatin structure compared with the embryo orother somatic tissues. Most observations of gameticand embryo and endosperm chromatin have beenmicroscopic, and because of their inaccessible nature,there is a paucity of molecular details on the organi-zation or epigenetic state of egg and central cellgenomes. Development of cell or nuclei taggingapproaches combined with decreasing amounts ofmaterial needed for chromatin, gene expression, orDNA methylation analysis is anticipated to rectifythis within the next few years. At present, it is knownthat the central cell has low levels of H3K9me2, ahistone modification usually associated with tran-scriptional repression, and lacks cytological chromo-centers, in contrast to the egg cell or other somaticnuclei (Garcia-Aguilar et al., 2010; Pillot et al., 2010).The central cell also lacks a histone variant, H2A.W,required for heterochromatin formation (Yelagandulaet al., 2014).

DNA methylation is generally associated with tran-scriptional silencing, although many exceptions tothis exist, particularly in endosperm (Makarevichet al., 2008; Gehring et al., 2009a). The central cell isthe site ofDEMETER (DME) expression, which encodesa 5-methylcytosine DNA glycosylase that removesmethylcytosine from DNA by base excision repair(Choi et al., 2002; Gehring et al., 2006). DME is an es-sential gene, and seeds that inherit a mutant maternaldme allele undergo a prolonged period of endospermdivision and then abort about a week after fertilization(Choi et al., 2002). Because base excision repair disruptsnucleosomes, DNA demethylation probably both di-rectly and indirectly leads to changes in chromatinmodifications (Gehring et al., 2009b), although this hasnot been shown. The mechanism of targeting 5-meCDNA glycosylases to particular sites in the genome re-mains unknown.


Gehring and Satyaki



The expression of the DNAmethylation machinery isalso low in the central cell (Jullien et al., 2012). In somatictissues, DNA methylation is deposited by multiplepathways and is positively reinforced by chromatinmodifications (Law and Jacobsen, 2010). The de novoDNA methyltransferases DRM2 and CMT2, whichfunction in different but overlapping genomic contexts,establish methylation (Stroud et al., 2013, 2014; Zemachet al., 2013). Most methylation in plant genomes is foundin transposable elements (TEs) or other repetitive DNA.DRM2 is guided to its targets by 24-nucleotide smallRNAs, in a process termed RNA-directed DNA meth-ylation (RdDM; Matzke and Mosher, 2014). Somewhatcounter intuitively, RdDM requires accessible chromatinin order to act, explaining its activity in more euchro-matic regions (Schoft et al., 2009; Gent et al., 2014). DNAmethylation often co-occurs with H3K9me2, and to-gether these are important for repressing TE transcrip-tion.DNAmethylation ismaintained by themaintenancemethyltransferase enzyme MET1 (CG methylation) andby the chromomethyltransferase CMT3 (CHG methyla-tion). CMT3 interacts with H3K9me2, leading to positivereinforcement between histone methylation and DNAmethylation. Asymmetric methylation (CHH) must beconstantly established de novo after DNA replication(Law and Jacobsen, 2010). Reporter gene fusions showthat MET1 is not expressed in the central cell, and DRM2expression is detectable but low (Jullien et al., 2012).Epigenetic differences established in the central cell

persist in the endosperm after fertilization. In the pe-ripheral endosperm of young Arabidopsis seeds, anendosperm-specific form of chromatin has been de-scribed, characterized by an absence of chromocentersand interspersed, diminished regions of heterochro-matin (Baroux et al., 2007a). The decondensed chro-matin appears to be driven by the maternal endospermgenome (Baroux et al., 2007a). Decondensed hetero-chromatin is associated with increased RdDM activityin other tissues (Schoft et al., 2009). Thus, looser chro-matin structure in the endosperm might be responsiblefor the abundance of small RNAs observed in seeds(Mosher et al., 2009). Most studies on DNAmethylationin seeds have been performed between 6 and 8 d afterpollination. At this stage, wild-type endosperm ishypomethylated at TEs and related sequences, partic-ularly at short TEs (i.e. fragments or remnants of TEs)that reside near genes (Gehring et al., 2009; Hsieh et al.,2009; Ibarra et al., 2012). Hypomethylation is specific tomaternal alleles and depends on DME (Gehring et al.,2006; Ibarra et al., 2012). Thus, expression of DME in thecentral cell creates epigenetic asymmetry between ma-ternal alleles in the embryo and endosperm, and be-tween maternal and paternal alleles in endosperm(Gehring et al., 2006; Ibarra et al., 2012). Interestingly,endosperm from Arabidopsis dme mutants exhibits acomplex methylation phenotype. Regions proximal togenes and/or corresponding to short TEs are hyper-methylated, as expected, but longer TEs are hypo-methylated, contrary to expectations for an enzymethat removes 5-methylcytosine. These may be indirect

effects but suggest a requirement for DNA demethyl-ation activity to maintain methylation in heterochro-matin (Ibarra et al., 2012). Maize and rice endospermDNA is also hypomethylated, and it is presumed that aDME homolog is responsible (Lauria et al., 2004;Zemach et al., 2010; Waters et al., 2011; Zhang et al.,2014).

By tagging and isolating nuclei of specific cell types ininterspecific crosses, Moreno-Romero et al. (2016) wererecently able to examine allele-specific DNA methyla-tion and histone modifications in Arabidopsis seedsmuch earlier than previous studies, at 4 d after polli-nation. Their findings suggest that DNA methylationis dynamic during endosperm development. CHHmethylation is absent from endosperm at 4 d afterpollination (Moreno-Romero et al., 2016). This agreeswith gene expression profiling of laser capture dis-sected endosperm tissue that indicates low expressionof the RdDM components in the endosperm until theheart stage (Belmonte et al., 2013) and with observa-tions on the expression of methyltransferase reportergenes (Jullien et al., 2012). By 6 d after pollinationCHH methylation is well established in the endosperm(Pignatta et al., 2014), indicating an increase in denovo DNA methylation activity. Moreno-Romero et al.(2016) also demonstrated that maternal allele-specifichistone H3 trimethylation at Lys-27 (H3K27me3) is lo-cated at sites demethylated by DME. H3K27me3 isestablished by PRC2 (Polycomb repressive complex 2)and represses the expression of developmental stage-specific genes (Holec and Berger, 2012). PRC2 com-ponents are essential genes required for endospermdevelopment, and prc2mutants share some aspects ofendosperm developmental (Choi et al., 2002) andDNA methylation (Ibarra et al., 2012) defects withdme. These data provide further impetus to fully de-scribe and understand epigenome dynamics fromgamete specification to seed maturity.

How do these unique endosperm features relate togene imprinting? As of yet there is no clear under-standing of how the cytological observation of decon-densed endosperm chromatin relate to endosperm geneexpression or imprinting, although it is thought thatdecondensation primarily affects the maternal genome(Baroux et al., 2007a). Techniques that would assayoverall chromatin structure, like variations of chromo-some conformation capture (Denker and deLaat, 2016),have not yet been applied to endosperm because of thelarge amounts of material required for these experi-ments. The relationship of DNA methylation to geneimprinting is better understood. Imprinted genes areoften associated with differentially methylated regions(DMRs) between the maternal and paternal alleles, al-though there are many more DMRs in endosperm thanthere are imprinted genes (Gehring et al., 2009). SomeMEGs have hypermethylated promoters in somatictissues, like the Arabidopsis genes FWA and SDC, anddemethylation of the maternal allele by DME promotestranscription in the central cell and endosperm. Si-lencing of the paternal allele depends on MET1 and





RdDM (Kinoshita et al., 2004; Vu et al., 2013). However,this relatively simple regulatory scenario describes aminority of imprinted genes. DNA hypomethylationalso appears to influence transcriptional start sitechoice, leading to the production of different transcriptsfrom maternal and paternal alleles (Luo et al., 2011; Duet al., 2014). The association between DMRs andimprinted genes in maize, rice, and Arabidopsis ismuch stronger for PEGs than for MEGs. Maternal al-leles of PEGs are hypomethylated (Rodrigues et al.,2013; Pignatta et al., 2014; Zhang et al., 2014). This wasinitially surprising: Why is the hypomethylated allelethe silent allele? Why is methylation required for ex-pression of the active allele? It is now clear that thehypomethylated maternal alleles of most PEGs are alsopreferentially modified by H3K27me3 (Du et al., 2014;Zhang et al., 2014; Moreno-Romero et al., 2016), whichis generally correlated with repression. This suggeststhat DNA methylation might “protect” the paternalallele from gaining H3K27me3 (Weinhofer et al., 2010).In line with these observations, mutations in PRC2components cause biallelic expression of PEGs (Hsiehet al., 2011). Interestingly, the epigenetic profile of PEGsis altered in Arabidopsis lyrata, an outcrossing speciesclosely related to Arabidopsis (Klosinska et al., 2016).This species is also characterized by maternal genomehypomethylation, and many PEGs are associated with59 or 39 DMRs. However, unlike Arabidopsis, the silentmaternal alleles of PEGs become CHG hypermethylatedin the gene body. This phenotype has not been observedin wild-type Arabidopsis endosperm but suggests thatimprinting mechanisms may be flexible on relativelyshort evolutionary time scales.

Imprinting can be variable (also referred to as allelespecific) within species (Kermicle, 1978; Alleman andDoctor, 2000; Waters et al., 2013; Pignatta et al., 2014),and this may be related to variability in fidelity of epi-genetic modifications. The sequences demethylated byDME and hypomethylated in Arabidopsis endospermare enriched for TE remnants, often of the Helitron andMutator types (Gehring et al., 2009; Ibarra et al., 2012;Pignatta et al., 2014, Moreno-Romero et al., 2016). Thepresence of TEs or their methylation status can be var-iable within a species (Cao et al., 2011; Schmitz et al.,2013), which has implications for imprinting stability.Pignatta et al. (2014) examined intraspecific variationin imprinting and found several examples of genesimprinted in one strain of Arabidopsis but not another,which was correlated with variation in methylation of anearby TE. These data suggest that gene expression pro-grams in the endospermmight be particularly susceptibleto natural epigenetic variation (Schmitz et al., 2013) be-cause it is in endosperm that methylation state is relevantto the expression of some genes (Pignatta et al., 2014).

IDENTITY AND FUNCTIONS OF IMPRINTED GENES

Transcriptomic studies have identified imprintedgenes on a genome-wide scale in the cereals rice, maize,and sorghum (Sorghum bicolor) and in the dicots

Arabidopsis, A. lyrata, Capsella rubella, and castor bean(Ricinus communis) (Gehring et al., 2011; Hsieh et al.,2011; Luo et al., 2011; Waters et al., 2011; Wolff et al.,2011; Zhang et al., 2011; Xin et al., 2013; Xu et al., 2014;Hatorangan et al., 2016; Klosinska et al., 2016;M. Zhanget al., 2016). Typically, F1 endosperm from crosses be-tween two different strains is isolated at specific stagesof seed development, mRNA-seq performed, andimprinted genes identified by a deviation from the ex-pected 2:1 ratio of maternal to paternal transcripts.Maternal and paternal transcripts are distinguishedby sequence polymorphisms. Although the speciesthat have been examined are vastly different in termsof genome complexity, organization, and epigeneticmodification, and the criteria to define imprinted genesvary somewhat between groups, common themes haveemerged.

The list of imprinted genes identified in any givenexperiment is significantly impacted by sequencingdepth and the availability of sequence polymorphismsbetween the two parents (if a gene does not have apolymorphism in the coding sequence, parent-of-origin-specific transcript information cannot be collected). Po-tential contamination from maternal integument/seedcoat tissue remains a concern. Nevertheless, in eachspecies approximately 75 to 200 imprinted genes havebeen identified. Protein coding genes, noncoding RNAs,and TEs are all subject to imprinting. Different sets ofgenes can be imprinted at different stages of seed de-velopment (Xin et al., 2013). This is perhaps not surpris-ing given that each stage of seed development is typifiedby its own gene expression program, and understandingthe imprinted gene universe for a species will requireanalysis at multiple stages of seed development.

Importantly, not all imprinted genes are mono-allelicly expressed, meaning that expression betweenthe parental alleles is differential but not binary. (Onecould instead strictly define imprinted genes as thosethat are monoallelic, although this would mean therewere hardly any imprinted genes and would excludesome genes identified as imprinted before the onset ofgenomics.) Partial imprinting could arise from twoscenarios: a mixture of imprinted and nonimprintedexpression among individual endosperm cells or tissuetypes, or partial imprinting within each cell. Thesescenarios have bearing on understanding the molecularmechanisms of imprinting establishment and mainte-nance and imprinting function. This question could beaddressed in greater detail by RNA-FISH or single celltranscriptomic studies.

As noted above, imprinting can also be variablewithin species, meaning that only some alleles of a geneare MEGs or PEGs. The first imprinted gene described,the maize R gene, exhibits allele-specific imprinting(Alleman and Doctor, 2000). Imprinting studies inmaize and Arabidopsis that employed reciprocalcrosses between multiple inbred lines concluded thatabout 10% of imprinted genes are variably imprinted(Waters et al., 2013; Pignatta et al., 2014). The functionalsignificance of this variation remains unclear. Some of


Gehring and Satyaki



the imprinting variation could be caused by rapidturnover of imprinting machinery as a result of conflict(see Box 1). Or, variable imprinting could reflect intra-specific differences in endosperm gene expressionprograms, as the timing of various aspects of seed de-velopment can be variable even within species. Anotherpossibility is that allele-specific imprinting is the result ofrelaxed selection on the imprinted state of a gene, sug-gesting that imprinting of that gene is not functionallyrelevant per se. Additional experimentation and under-standing of gene functionwill be required to resolve thesedisparate possibilities for individual genes.Analysis for enriched functional categories of imprin-

ted genes have returned fairly modest results (Gehringet al., 2011; Waters et al., 2011; Wolff et al., 2011;Pignatta et al., 2014). In Arabidopsis and maize, PEGsare somewhat enriched for putative chromatin modi-fying functions or epigenetic regulators (Gehring et al.,2011; Waters et al., 2013; Pignatta et al., 2014;). This isparticularly intriguing because regulators of imprint-ing may themselves become part of an imprintingnetwork under a conflict model of imprinting (see Box1; Patten et al., 2016). Many of these PEGs are predictedto promote DNA methylation or repressive histonemodifications, including putative histone methyl-transferases, components of the RdDM pathway, andgenes required to maintain CG methylation, includingVIM genes and MET1 homologs (Gehring et al., 2011;Hsieh et al., 2011; Klosinska et al., 2016). By contrast,the PRC2 complex genes MEA and FIS2, which arerequired to maintain imprinted expression during en-dosperm development, are MEGs. In addition to pro-teins, imprinting regulators can include cis-regulatorynoncoding RNA as well as trans-regulatory small RNA.In maize, four noncoding RNAs, speculated to haveregulatory functions, are maternally expressed fromwithin protein-coding loci that are paternally expressed(Zhang et al., 2011). In rice, small RNAs of the type thatparticipate in RdDM and have the potential to regulateimprinting are themselves imprinted (Rodrigues et al.,2013). However, it should be noted that apart from thePRC2 encoded proteins, the ability of these other factorsto regulate imprinting remains to be demonstrated. Thisshould be a fruitful area of future research.Whether imprinting is functionally important for the

plant or an inconsequential side effect of a relaxed en-dosperm epigenome has been debated (Berger et al.,2012). Mutations in several Arabidopsis PEGs have noobvious effect on seed development (Wolff et al., 2015).Yet the list of imprinted genes with clear functions inendosperm is growing. The imprinted gene Meg1 isrequired for normal transfer cell development in maizeBETL; altering the dosage of Meg1 alters kernel size(Gutiérrez-Marcos et al., 2004; Costa et al., 2012). Thefirst imprinted genes discovered in Arabidopsis, MEAand FIS2, prohibit endosperm proliferation and areessential for normal endosperm development, althoughthey also have important functions in the female ga-metophyte (Chaudhury et al., 1997; Grossniklaus et al.,1998; Kiyosue et al., 1999). One finding that could point

to imprinting being “important” or functional is if ho-mologous genes or genes that function in the samepathways were commonly imprinted among species.Comparisons of imprinting between maize and Arabi-dopsis or rice and maize report limited, but statisticallysignificant, overlap among imprinted genes (Waterset al., 2013). FIE, another member of the PRC2 complex,is imprinted in rice and maize (Danilevskaya et al.,2003; Luo et al., 2009). Other conserved imprinted genesincluded auxin biosynthesis genes and epigenetic reg-ulators, like the VIM maintenance methylation genes.Interestingly, VIM103 is positively correlated with in-creased seedweight in a long-term selection experimentin maize (X. Zhang et al., 2016). Endosperm-specificYUCCA genes, required for auxin biosynthesis, arePEGs in rice, maize, Arabidopsis, and related species(Gehring et al., 2011; Hsieh et al., 2011; Luo et al., 2011;Waters et al., 2011; Klosinska et al., 2016). Auxin isrequired for normal endosperm proliferation in Arabi-dopsis (Figueiredo et al., 2015), and in maize YUC1positively regulates endosperm proliferation; plantswith mutations in the gene produce lighter kernels withreduced endosperm cell number (Bernardi et al., 2012).

Because the absolute number of instances of con-served or convergent imprinting is few, the perceptionis that imprinting is rapidly changing among plantspecies. In practice, comparing imprinting betweenspecies is not trivial. Genes identified as imprinted inone species might not be called imprinted in anotherspecies for several technical reasons. It may be chal-lenging to identify orthologs between distantly relatedspecies. Or, genes of interest might not have a sequencepolymorphism in all species, preventing discriminationbetween maternal and paternal alleles. Genes that arelowly expressed, as are some imprinted genes, are lesslikely to be represented in RNA-seq libraries. Addi-tionally, the developmental stages of the seeds beingused to assay imprinting may not be comparable be-tween species. Thus, it is important to only compareimprinting among genes that can be confidentlyassessed for imprinting in both species (Klosinska et al.,2016). Two recent studies have compared imprintingbetween the closely related species Arabidopsis and C.rubella, and Arabidopsis and A. lyrata, which divergedfrom one another around 10 to 15 million years ago(Hatorangan et al., 2016; Klosinska et al., 2016). Whileone study characterized imprinting as rapid and theother characterized it as fairly conserved, in bothcomparisons imprinting of around 50% of genes wasshared. PEGs appear to be more conserved than MEGs.Gene-focused comparisons of imprinting betweenspecies do not take into account the possibility thatother genes working in the same pathway could beimprinted, if the function of the pathway, rather thanthe function of an individual gene, is under selection.For example, in A. lyrata and Arabidopsis, differentcomponents of the RdDM pathway are PEGs, and inmaize and rice a different PRC2 component is imprin-ted than in Arabidopsis. On the whole, based on bothcomparisons of imprinting between species and on





known function of imprinted genes, there appears to beample evidence that genes subject to imprinting areimportant for endosperm proliferation and seed de-velopment, as will be further detailed below.

IMPRINTING AND INTERSPECIES OR INTERPLOIDYHYBRID SEED PHENOTYPES

Perturbation of endosperm development is a majorsource of seed lethality between different species andbetween plants of the same species but differentploidies. Although both embryo and endosperm de-velopment are defective in hybrid seeds, in many casesF1 embryos can be rescued, suggesting that defects inendosperm development are causal for seed death andapparent effects on embryo growth are secondary(Ishikawa et al., 2011; Rebernig et al., 2015). Historicalandmore recentmolecular evidence suggests a possiblerole for imprinting, or perturbation of imprinted geneexpression, in both interploidy and interspecific hybridincompatibility. A key observation linking imprintingto hybrid seed endosperm failure is that reciprocal en-dosperm phenotypes are generated depending onwhich species or ploidy is the male parent and which isthe female in the cross. This is termed a parent-of-origineffect and is classically associated with imprinting inplants and animals. It is important to note that parent-of-origin effects can arise for reasons other than imprinting,including cytonuclear interactions and gametophytic ef-fects, and additional experiments must be undertaken toexclude these other possibilities before fingering im-printing as the culprit (Kermicle, 1970).

A key developmental decision point during seeddevelopment is the timing of endosperm cellulariza-tion (Hehenberger et al., 2012; Fig. 3). Early cellula-rization leads to reduced endosperm size, whereaslater cellularization or the absence of cellularizationleads to larger endosperm. Both conditions can belethal. For example, C. rubella and Capsella grandifloradiverged from each other only 100,000 years ago. When

C. grandiflora is the female in interspecific crosses, hy-brid seed lethality is associated with lack of endospermcellularization. Some viable seeds (60%) are producedin the reciprocal cross, but these are small due to earlyendosperm cellularization (Rebernig et al., 2015).C. rubella is a self-fertilizing species and C. grandiflora isan outcrossing species, which may be related to the lackof completely reciprocal phenotypes (Brandvain andHaig, 2005; Rebernig et al., 2015). Similar phenotypeshave been observed in rice interspecific crosses(Ishikawa et al., 2011), although in these instances theresultant seeds are larger or smaller than seeds fromintraspecific crosses, rather than aborted. Monkey-flower (Mimulus), a genus with many recently divergedmembers that have overlapping ranges, is emerging asan interesting system for the study of hybrid endo-sperm effects. Pollination and fertilization occur equallywell between Mimulus guttatus and Mimulus nudatus asin self-fertilizations, but the resultant interspecific hy-brid seeds are flat or shriveled because of the absence ofor delayed endospermproliferation (O’Neal et al., 2016).Using a clever breeding strategy, Garner et al. recentlymapped quantitative trait loci responsible for hybridseed lethality between M. guttatus and Mimulus tilingii(Garner et al., 2016). Although the underlying geneshave yet to be identified, many of the quantitative traitloci effects were dependent on the parent-of-origin,further supporting the idea that imprinted genes mightbe involved. Several new mutants with parent-of-origineffects on maize endosperm development, albeit withincomplete penetrance, have also recently been identified(Bai et al., 2016), and it will be informative to determinethe molecular identity and imprinting status of the un-derlying genes.

Seed lethality is also a frequent outcome of inter-ploidy crosses within species. In maize, maternal andpaternal genomes must be in a 2:1 ratio in the endo-sperm for the production of viable seeds (Lin, 1984;Pennington et al., 2008), and altering that dosage dis-rupts the transition from endospermmitotic division toendoreduplication (Leblanc et al., 2002). Other species,like Arabidopsis, are somewhat more tolerant of devi-ation from the 2:1 ratio (Scott et al., 1998). However,even when interploidy crosses are not lethal, endo-sperm syncytial division is either prolonged or attenu-ated based on whether excess genomes are inheritedfrom the male or female parent. Maternal genomic ex-cess is associatedwith early cellularization and paternalgenomic excess with delayed (or in cases were seedsabort, the absence of) cellularization (Fig. 3; Scott et al.,1998; Pennington et al., 2008). The resemblance of re-ciprocal endosperm phenotypes produced in inter-ploidy and interspecific crosses has led to the idea thatthese are functionally similar phenomenon, both pos-sibly related to gene imprinting (Lin, 1984; Bushell et al.,2003; Lafon-Placette and Köhler, 2016). Howmight thisbe so? In the framework of the popular kinship theoryof imprinting (Box 1), MEGs are predicted to attenuateendosperm proliferation while PEGs promote it, andthis is somewhat borne out by the known functions of

Figure 3. Timing of endosperm cellularization is a key developmentaldecision point. Interspecies and interploidy crosses alter when or ifendosperm cellularizes, which can lead to smaller or larger seeds, oreven seed abortion. Mutations in some epigenetic regulators can par-tially ameliorate cellularization defects. WT, Wild type.


Gehring and Satyaki



imprinted genes (see section above). In maternal ge-nomic excess the dosage of active alleles for MEGs in-creases while the relative dosage of PEGs decreases,with the reverse true in instances of paternal genomicexcess. Additionally, different genes might be imprin-ted in different species, leading to an altered gene ex-pression program in F1 endosperm depending onwhich species serves as which parent.Most molecular characterization of the gene expres-

sion changes that accompany interspecific and/orinterploidy crosses have been performed in Arabi-dopsis and related species. In crosses between diploidArabidopsis females and diploid Arabidopsis arenosamales, endosperm overproliferates and 95% of seedsabort. Seed lethality is significantly reduced if theArabidopsis female is tetraploid (Josefsson et al., 2006),suggesting, in congruence with the endosperm balancenumber hypothesis (Johnston et al., 1980; Kinoshita,2007), that A. arenosa males are functionally tetraploidin relation to Arabidopsis females. Expression of sev-eral PEGs, including the AGAMOUS-LIKE (AGL)MADS box transcription factor PHERES1 (PHE1), isup-regulated from the normally silent maternal allele inthe lethal cross, and mutation of phe1 in Arabidopsispartially ameliorates the hybrid incompatibility phe-notype (Josefsson et al., 2006; Burkart-Waco et al., 2015).Failure of endosperm cellularization has been associ-ated with increased expression of AGL MADS boxtranscription factors, in particular AGL62 (Kang et al.,2008). OtherAGL genes also contribute to seed lethality,some of which are imprinted (Walia et al., 2009;Shirzadi et al., 2011). Many of the same genes are mis-regulated in interploidy crosses within Arabidopsis(Erilova et al., 2009; Tiwari et al., 2010), further high-lighting the molecular similarities of interploidy andinterspecific seed lethality. Expression of theAGL genesis regulated by the PRC2 complex (Hehenberger et al.,2012), which is active in the female gametophyte anddeveloping seeds. Seeds that inherit mutations in meaand fis2 from the female parent phenocopy interploidycrosses with paternal genomic excess (Fig. 3), under-going seed abortion after an abnormally prolongedperiod of endosperm syncytial division. Consistentwith this, the prc2 mutant phenotype is suppressed ifthe maternal parent contributes two extra genomes(producing triploid embryos and pentaploid endo-sperms), presumably by restoring effective, rather thanactual, genomic balance of maternal to paternal ge-nomes in the endosperm (Kradolfer et al., 2013a).A causal relationship between specific imprinted

genes and seed abortion induced by interploidy crosseshas recently been genetically demonstrated (Kradolferet al., 2013b; Wolff et al., 2015). Taking a genetic ap-proach, Kradolfer et al. (2013b) identified mutationsthat suppressed the seed abortion produced whenwild-type Arabidopsis females are pollinated by unre-duced 2n pollen (a cross that normally generates inviableseeds with triploid embryo and tetraploid endosperm).Paternal inheritance of mutations in the ADMETOS(ADM) gene, a PEG, causes partial normalization of

endosperm cellularization timing and AGL expression,suppressing seed abortion (Kradolfer et al., 2013b). Mu-tations in ADM have no effect on seed development intraditional diploid seeds. ADM encodes a putative mo-lecular chaperone whose expression is up-regulated intriploid seeds destined for death. Interestingly, the in-crease inADM expression is all from the paternal allele—imprinting of ADM remains intact. ADM is also a targetof the FIS-PRC2 complex, and mutations in adm weaklysuppress the mea seed abortion phenotype (Kradolferet al., 2013b). Paternally inherited mutations in threeadditional PEGs, the putative histone methyltransferaseSUVH7, PEG2, and PEG9, also normalize endospermcellularization timing andpartially suppress triploid seedabortion (Wolff et al., 2015). Like ADM, SUVH7 andPEG2 imprinting is maintained in triploid seeds, but ex-pression levels are higher. Together, these data have ledto the suggestion that imprinted genes establish inter-ploidy hybridization barriers, ultimately promotingspeciation (Gutierrez-Marcos et al., 2003; Lafon-Placetteand Köhler, 2016). Although it is clear that imprintedgenes can impact this process, it is less clear that parent-of-origin-specific expression is the relevant feature withregard to hybrid seed failure. In many instances it is notloss of imprinting—i.e. both alleles being transcription-ally active (biallelic) or both alleles being transcriptionallysilent—that is the outcome of hybrid crosses. Rather, asdocumented for the paternal allele ofADM, it is increasedexpression of the allele that is active in nonhybrid crosses.It has been argued that these effects are more about genedosage than the specific parent-of-origin from which agene is expressed (Dilkes and Comai, 2004; Birchler,2014). However, because imprinting is itself a type ofdosage effect and imprinted genes are predicted to bedosage sensitive (Patten et al., 2014), it is difficult to dis-tinguish these possibilities. If imprinting is insteadviewed as a process required for expression of a gene(going from zero active copies to one [PEGs] or two[MEGs] active copies), then increased expression of theexpressed allele of imprinted genes in triploid seedscould be viewed as an imprinting effect.

CONCLUDING REMARKS

The past 5 years have seen an immense expansion ofthe plant imprinting field. Imprinted genes have beenidentified genome-wide in multiple species, and therelative ease of these experiments ensures that morewill follow. The exploration of seed epigenome dy-namics from fertilization to seed maturity is in its in-fancy, as is the understanding of what those dynamicsmeanwith regard to imprinted gene expression ormorebroadly. The recent findings that imprinted genes cancause postzygotic lethality nicely complement work onhybrid incompatibility stretching back to the first half ofthe 20th century. Many questions (see OutstandingQuestions) remain on the horizon for this field. Advanc-ing understanding will depend on a range of approaches,from deploying the latest genomic technologies to





access and assess parent-of-origin-specific endo-sperm chromatin structure and determine the rela-tionship to active DNA demethylation, to detailedmolecular genetic studies of regulation and function ofindividual imprinted genes.Received August 25, 2016; accepted November 22, 2016; published November28, 2016.

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Gehring and Satyaki



endosperm and imprinting, inextricably linked1[open] · in maize, endosperm is de-ﬁned...

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