Epigenetic regulation of gene expression plays a central role in eukaryotic development and takes place through modulation of chromatin structure. Highly organized chromatin consisting of DNA wrapped around histone proteins in nucleosomal structures can switch between relaxed and condensed states associated with transcriptional activity and transcriptional repression, respectively [1]. This is accomplished though cytosine methylation of DNA and post-translational modifications of nucleosomal histone tails. Histone modifications include changes in methylation, acetylation, and phosphorylation on specific lysines and arginines residues. Generally methylation of histone 3 on lysine 9 and 27 (H3K9, H3K27) leads to a repressive chromatin state correlating with gene silencing whereas methylation of histone 3 on lysine 4 (H3K4) leads to a permissive chromatin state correlating with gene activation [2]. Histone acetylation and deacetylation result in active and silent chromatin forms, respectively [3]. This kind of epigenetic regulation operates on various aspects of plant growth and development, including seed formation and stress responses (reviewed in [4, 5]).

In flowering plants, the seed is formed through the process of double fertilization. Fertilization of the egg cell by a sperm cell from the male gametophyte generates the diploid embryo from which the organs, tissues and shoot meristems of the plant will be generated. Fertilization of the adjacent central cell by a second sperm cell forms a triploid endosperm, in angiosperms, which supports embryo growth and development by producing storage proteins, lipids, and starch [6].

Bypass of double fertilization is observed in natural apomicts or after mutagenesis of specific gene loci. In Arabidopsis mutations of the FIS (Fertilization Independent Seed) class of genes: FERTILIZATION INDEPENDENT SEED (FIE), MEDEA (MEA), FERTILIZATION INDEPENDENT SEED2 (FIS2), MULTICOPY SUPRESSOR OF IRA1 (MSI1) and BORGIA (BGA), result in the formation of seed-like structures in the absence of double fertilization that eventually collapse [7–9]. The phenotype of fie, mea, fis2 and msi1 mutants is accompanied by another impressive characteristic, that is, endosperm overproliferation [9, 10]. Endosperm overproduction and seed size increases are also observed in crosses where extra paternal genomes or hypomethylated maternal genomes are used [11]. Another feature of the fis phenotype is that when the female gametophyte carrying a mutation in a fis locus is fertilized by a wild type plant, the seed finally collapses indicative of a lethal maternal effect of the fis mutation and a gametophytic maternal control over seed development by FIS genes [7, 8]. This is partially attributed to parental imprinting of FIE, MEA and FIS2 where only the maternal allele is expressed during the early stages of seed development [12–14].

The common phenotypic effects of the fis mutants are explained by the shared function of the FIS proteins in a PcG complex. The participation of these proteins in a PcG complex was first proposed by [10] and it is now well established that FIE, MEA, FIS2 and MSI1 form the so called FIE/MEA complex, one of the three PRC2 (Polycomb Repression Complex 2) homologues in Arabidopsis, regulating seed formation [15].

The PRC2 complex also known as EXTRA SEX COMBS-ENHANCER OF ZESTE (ESC-E(Z)) complex is one of three PcG complexes in Drosophila. PRC2 has been shown to play a role in the epigenetic silencing of target genes involved in cell growth and proliferation and in early development both in Drosophila and mammals. Its function involves H3K27 methyltransferase activity [16]. Methylation of lysines 9 and 27 of histone 3 serve as epigenetic marks of transcriptional silent loci. The PcG/PRC2 complex in Drosophila contains four essential subunits: two WD40 proteins, named EXTRA SEX COMBS (ESC), and P55 containing the WD40 domain involved in protein-protein interactions, a zinc finger protein, SUPRESSOR OF ZESTE 12 [Su(z)12], and a SET-domain protein responsible for methyltransferase activity, ENHANCER OF ZESTE E(Z) [E(Z)] [17, 18].

Different variants of the PRC2 complex have been identified in plants depending on developmental stage, the best studied one being the FIE/MEA complex which regulates the initiation of seed development [18]. It consists of FIE, MEA, FIS2 and MSI1, which are homologous to the Drosophila ESC, E(Z), Su(z)12, and P55, respectively [19, 20, 13, 10]. In Arabidopsis all PcG complexes are predicted to contain the FIE and MSI1 subunits. Depending on cell type and function the different PcG complexes contain one of the three homologues of the Drosophila EZ protein, MEA, CURLY LEAF (CLF) or SWINGER (SWN), and one of the three homologues of the Drosophila Su(z)12 protein, EMBRYONIC FLOWER 2 (EMF2), FERTILIZATION INDEPENDENT SEED 2 (FIS2), and VERNALIZATION2 (VRN2), respectively. The EMF2-CLF/SWN-FIE-MSI1 complex has been suggested to play a role in suppressing the transition from vegetative development to flowering and flower organ formation [15, 21] and the VRN2-CLF/SWN-FIE-MSI1 has been implicated in the regulation of the process of vernalization [15, 22, 23]. EMF2, FIS2 and VRN2, the three Su(z)12-like proteins in Arabidopsis, contain a classical C₂H₂ zinc finger similar to the fingers found in sequence-specific DNA-binding proteins and another stretch of amino acids, located C-terminal to the zinc finger, that is conserved between DsSu(z)12, HsSu(z)12, EMF2, VRN2 and FIS2 and has been termed VEFS box (VRN2-EMF2-FIS2-Su(z)12 box) [24].

Homologues of the PcG proteins of the FIE-E(Z) complex have been identified in other plant species including monocots, indicating their involvement in a conserved regulatory mechanism among all higher plants [17].

Three target genes for the Arabidopsis FIS2-FIE-MEA-MSI1 complex operating in seed development have been identified so far. The first one is PHERES1(PHE1), a type I MADS box gene [25] which is parentally imprinted in the developing seed, with the maternal allele being silenced and the paternal allele expressed in specialized endosperm cells during the first two days after fertilization [26, 27]. The second target is the MEA gene itself, demonstrating an autoregulating mechanism for the imprinting of a PcG epigenetic silencer [28, 29]. The third one is FUS3, a transcription factor belonging to the plant-exclusive B3 domain TF family, specifically expressed in seeds during the seed filling phase [30].

Seed development, dormancy and germination are under the control of plant hormones, in particular ABA and gibberellic acid (GA). ABA inhibits germination and is required for the acquisition of dessication tolerance and entry into seed dormancy acting antagonistically to GA during these processes [31, 32]. It is also involved in various aspects of plant growth and development including abiotic stress tolerance. Histone modifications have been suggested to play a major role in ABA-mediated processes such as seed development, germination and abiotic stress adaptation [4]. For example, in leaves of Phaseolus vulgaris, externally supplied ABA has been shown to induce methylation of H3K4 and acetylation of H3K14 and H4K5 (indicative of gene activation) of a chromatin region around the phaseolin promoter, and ectopic expression of the otherwise silent in leaves phaseolin gene (encoding a major seed storage protein) [33]. Likewise, ABA-treated seeds of an Arabidopsis mutantof PICKLE (a chromatin remodelling factor mediating suppression of embryonic identity upon germination) showed high expression levels of the ABA-induced transcription factors, ABI3 and ABI5, involved in seed filling. This correlated with reduced H3K9 and H3K27 methylation at ABI3 and ABI5 promoters, in the ABA treated mutant seeds than in wild type seeds [34]. In another study, ABA treatment caused severe reduction in expression of the Arabidopsis histone deacetylase gene, AtHD2C, whereas overexpression of AtHD2C resulted in enhanced abiotic stress tolerance and both repression or induction of several ABA-responsive genes [35]. Recently, ABA was suggested to be a positive regulator of SWI3B, a subunit of the chromatin remodelling complex SWI/SNF, as in Arabidopsis swi3b mutant seedlings exposed to external ABA there was reduced expression of ABA-responsive genes [36]. Furthermore, ABA or an ABA signal was suggested to affect epigenetic states, the DNA replication machinery and chromatin mediated gene expression in Arabidopsis seedlings [37].

Despite the extensive studies on epigenetic regulation of seed development in Arabidopsis, knowledge on epigenetic regulation through DNA and chromatin modulation in cereal seed development is only now starting to emerge. The expression of a large number of genes encoding structural proteins, metabolic enzymes, transcription factors and DNA and chromatin modulators during seed development and germination has been investigated in barley, through large-scale transcriptome analysis [38]. Very recently, as part of our efforts to study epigenetic control mechanisms during seed development in barley, we isolated members of the two families of histone deacetylases, the plant specific HD2 family and the RPD3/HDA1 family as well as members of the histone acetyltransferase families, GCN5, MYST, ELP3, respectively, and investigated their expression at different seed developmental stages, in different grain-size cultivars and in response to external hormonal stimuli [39–41].

Considering the economic importance of cereal crops such as barley, rice, wheat and maize, we report here the characterization of the barley PcG gene homologues. More specifically, a FIE homologue, termed HvFIE, an E(Z) homologue, termed HvE(Z), and two Su(z)12-like homologues, termed HvSu(z)12a and HvSu(z)12b, respectively, were characterized. The mapping position of HvFIE, HvE(Z), HvSu(z)12a, and HvSu(z)12b on different barley chromosomes was assigned, and their expression was examined in different tissues and seed developmental stages and in barley cultivars which differ in seed size and weight. Furthermore, the expression of HvFIE, HvE(Z) and HvSu(z)12b was investigated after treatment with the plant hormone ABA, a modulator of gene expression during developmental and environmental changes, operating often through epigenetic modifications of target genes.

In the current study we present the identification, mapping and expression profiles of the genes encoding PcG chromatin modifiers from barley. This work serves as a first step in understanding the role played by PcG genes in the epigenetic mechanisms that control seed formation and stress response in a monocot cereal plant. Unlike dicots, where the endosperm is consumed by the embryo during seed maturation, in monocots such as cereals, the endosperm persists after embryo development is completed and constitutes the major portion of the mature kernel [6, 7]. The endosperm of cereal crops like barley, wheat, rice, maize, stores reserves such as proteins, starch, and fatty acids, and represents 60% of the world's food and feed supply. Consequently, understanding and manipulating the molecular mechanisms governing endosperm development for increasing seed yield is of utmost interest for agriculture.

Four barley PcG gene homologues encoding the putative PcG proteins HvFIE, HvEZ, HvSu(z)12a and HvSu(z)12b were identified and analyzed. In Arabidopsis the respective PcG counterparts are [AtFIE], [AtMEA or AtSWN or AtCLF] and [AtFIS2 or AtEMF2 or AtVRN2]. Three different PcG complex variants can be formed depending on the different combinations of these subunits. The changeable subunits are the MEA and FIS2 homologues whereas FIE is likely to be common in all three complexes. The FIE, MEA, FIS2 proteins form a complex essential for viable seed formation. MEA, CLF, and SWN, constitute the three Arabidopsis E(Z) homologues, which cluster into three separate clades, and are members of different PcG complexes with distinct functions. MEA is important for proper seed development and SWN acts redundantly with MEA having a role in preventing endosperm development as well [56]. However according to another report by Spillane et al. 2007 [57], swn/clf double mutants in Arabidopsis produce normal seeds suggesting that neither SWN nor CLF has a role in seed formation. In addition, SWN and CLF are involved in the vernalization process [23]. It was proposed that these homologues have arisen through an old duplication and separation of CLF from the MEA/SWN lineage, which occurred before the divergence of monocots and dicots, whereas a more recent duplication produced the separation between MEA and SWN in only a few species [57]. No direct orthologues of MEA have been found in any other species than Arabidopsis. E(Z) homologues identified in maize and rice [58–60], two monocots with sequenced genomes, cluster in two clades: ZmMEZ2, ZmMEZ3 and OsiEZ1 form the so-called SWN-like clade, and ZmMEZ1 and OsjEZ (also named OsCLF) form the CLF-like clade. Phylogenetic analysis showed that the barley homologue HvEZ is a close relative of the wheat, rice and maize E(Z) homologues belonging in the SWN-like clade. Unlike Arabidopsis, which has at least three E(Z) homologues functioning at different stages of the plant's life cycle, a second HvEZ homologue has not been identified in barley as yet. It might be possible that HvEZ is a common member of the different PcG complex variants that may be operating at different stages of barley development. However, this hypothesis awaits sequencing of the barley genome and further experimentation. In the absence of a MEA counterpart it is possible that the role of MEA is played by one of the putative SWN or CLF homologues both in barley and in the other species lacking MEA.

HvFIE maps on chromosome 7Hcen which is colinear with rice chromosome 8, where the rice OsnFIE2 homologue (Os08g04270) has been also localized. HvE(Z) maps on chromosome 4Hcen which is colinear with rice chromosome 3, where the rice OsiEZ1 homologue (Os03g19480) is found [60]. Similarly, HvSu(z)12a maps on chromosome 2Hshort which is colinear with rice chromosome 4, where the OsEMF (Os04g08034) homologue is localized, and HvSu(z)12b maps on chromosome 5Hlong which is colinear with rice chromosome 9 where the rice homologue OsVEF (Os09g13630)is localized [60]. These observations are in agreement with the extensive synteny between barley and rice chromosomes.

HvFIE and Hv(E)Z are located in the centromeric region of chromosome 7H and 4H repectively, where important clusters of malting quality QTLs have been identified by several authors and reviewed by [61]. Noteworthy, using the Oregon Wolfe Barley map [53] that integrates 2383 loci and a comprehensive summary of 154 QTLs of malting quality http://wheat.pw.usda.gov/ggpages/maps/OWB/, it is possibile to relate the high resolution genomic location of the two PcG genes with historical QTL data. Hv(E)Z location is coincident with scind03751 and 1.1 cM proximal to Tef2, the significant marker for the beta-Glucanase activity QTL (QBgsg.StMo-4H) identified in the Steptoe x Morex population. HvFIE maps to a cluster of OPA and DArT markers that includes MWG808 and is 10 cM proximal to MWG2031, the significant marker for both Grain protein content (QGpc.HaMo-7H) and Kernel plumpness (QKp.HaMo-7H) in the Harrington x Morex population.

Likewise the map position of the two Su(z)12 homologues (HvSu(z)12a on the short arm of chromosome 2H and HvSu(z)12b close to the centromere of chromosome 5H), is in proximity to genomic regions historically involved in the genetic control of malting quality traits [61]. However, all these gene-QTL relationships cannot be unequivocally established and thus further research is needed to determine the role of PcG in endosperm-related traits.

Qualitative RT-PCR analysis demonstrated that the barley HvFIE shows a tissue expression profile similar to that of AtFIE, maize ZmFIE2 and rice OsFIE2 [13, 62, 58, 63, 60] in that it is expressed in both vegetative and reproductive tissues. Similarly, the barley HvE(Z) is expressed throughout development like the maize homologues ZmMEZ1, ZmMEZ2 and ZmMEZ3 [58] and the rice E(Z) homologues, OsiEZ1 and OsCLF (same as OsjEZ) [60], whereas AtMEA is not expressed in leaves [19, 12, 64]. The expression pattern of HvSu(z)12b is similar to the rice OsEMF homologues, being expressed both in vegetative and reproductive tissues [60]. Conversely, AtFIS2 is expressed only in the central cell and endosperm [13]. No expression studies on the maize Su(z)12 homologues, ZmEMF-like and ZmEMF2-like, have been reported so far. The differential expression of AtMEA and AtFIS2 compared to E(Z) and Su(z)12 homologues from monocots and other dicots, together with the fact that the protein sequences of AtMEA and AtFIS2, are distantly related to the E(Z) and Su(z)12 homologues from cereals and other dicots supports the view proposed by Luo et al. 2009 [60], that the MEA and FIS2 genes arose only within the Brassicaceae.

In Arabidopsis, mutations in the PcG genes resulting in downregulation of AtMEA, AtFIE and AtFIS2, cause seed-like structure formation in the absence of fertilization and the production of non-viable seeds after fertilization. In both cases endosperm overproliferation has been observed. Endosperm overproliferation could be a desirable feature of viable and qualitatively acceptable seeds in crops where the seed and in particular the endosperm is the agronomically important product. In this study we attempted to analyze quantitatively the expression of the PcG gene homologues in different seed developmental stages and in two barley cultivars differing in seed size, in order to unravel any association between the expression of these genes with seed development and the size of seed. Quantitative real time PCR expression analysis revealed a differential expression pattern during seed development and significant differences between two barley cultivars with varying seed size for the PcG genes. HvFIE exhibits a pronounced increase after fertilization for both cultivars, which declines in Caresse after 3-5 DAF, but increases remarkably in Ippolytos (approximately 7 fold) at 5-10 and 10-15 DAF. Interestingly, HvFIE transcript levels are markedly higher in Ippolytos at 5-10 and 10-15 DAF than in Caresse by approximately a factor of 1.5 and 3.5, respectively. HvE(Z) expression shows a decline in Caresse seeds 3-15 days after fertilization as compared to flowers prior to fertilization, with a marked 2 fold decrease at 3-5 DAF. Conversely, such a reduction is not observed in Ippolytos, where HvE(Z) transcript levels do not show any significant changes among seed stages. In barley, endosperm cellularization begins at approximately 4 DAF and ends at 6-8 DAF, when the seed maturation process begins [6]. It might be possible that the differences in HvE(Z) and HvFIE expression between the two cultivars during these critical stages of endosperm development are associated with the processes of cellularization and seed filling and ultimately with the size of seed. For HvSu(z)12b, transcript levels show an induction after fertilization at 5-10 and 10-15 DAF, implying a role for HvSu(z)12b in the cellularization and seed maturation process for both cultivars. Further experiments will be needed to elucidate the precise roles of these PcG genes in barley seed development.

The plant hormone ABA, is involved in various plant developmental processes, including seed development, germination and abiotic stress tolerance. In Arabidopsis, ABA plays a central signalling role during seed filling, interacting with master regulator genes such as ABI3 and FUS3, in a gene activation manner [reviewed in [65]]. Interestingly, FUS3 is one of the targets of both the AtCLF and AtMEA genes (acting in vegetative and reproductive stages, respectively) and it is silenced in leaves and closed flowers [30] where it was also shown to contain the repressive mark H3K27me3. In this work we examined the response of PcG genes to ABA after exogenous ABA application on seedlings and observed that both HvE(Z) and HvFIE genes are induced by ABA. The observed increase in HvE(Z) and HvFIE expression upon ABA treatment suggests that these genes might play a role as epigenetic regulators of ABA-mediated processes in seedlings. Chromatin modulators, such as histone deacetylase genes, have been shown previously to be affected by exogenous ABA in Arabidopsis, rice and barley [35, 66, 39]. This is the first time that an induction of PcG genes upon external ABA exposure has been demonstrated. It is possible that the expression of PcG genes is also affected by ABA during seed development, to activate or suppress key regulators, as the cell would require.

In this work we have presented the identification and characterization of four barley PcG genes, HvFIE, HvE(Z), HvSu(z)12a, and HvSu(z)12b. All genes were mapped on different barley chromosomes and co-localized with known chromosomal regions responsible for malting quality related traits. Should this be confirmed PcG genes might be used as molecular markers for marker-assisted selection in breeding programmes. Differential expression of HvFIE, HvE(Z), and HvSu(z)12b in different seed developmental stages and in two barley cultivars differing in seed size, suggested a role for these PcG genes in seed development. In addition HvFIE and HvE(Z) were found to be induced by the hormone ABA involved in seed development and stress response. The present work provides a basis for future studies aiming at unraveling the function of barley PcG genes and understanding seed development and stress-response epigenetic regulation in barley and other cereals.