Epigenetics in Plants—Vernalisation and Hybrid Vigour☆

Biochimica et Biophysica Acta 1809 (2011) 427–437

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagrm

Review Epigenetics in plants—vernalisation and hybrid vigour☆

Michael Groszmann a,b, Ian K. Greaves a,c, Nicolas Albert a, Ryo Fujimoto a, Chris A. Helliwell a, Elizabeth S. Dennis a,b, W. James Peacock a,⁎ a Commonwealth Scientiﬁc and Industrial Research Organisation, Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia b NSW Agricultural Genomics Centre, PMB, Wagga Wagga, NSW 2650, Australia c Department of Genome Biology, John Curtin School of Medical Research, Australian National University, ACT 0200, Australia article info abstract

Article history: In this review we have analysed two major biological systems involving epigenetic control of gene activity. In Received 3 March 2011 the first system we demonstrate the interplay between genetic and epigenetic controls over the Received in revised form 24 March 2011 transcriptional activity of FLC, a major repressor of flowering in Arabidopsis. FLC is down-regulated by low Accepted 25 March 2011 temperature treatment (vernalisation) releasing the repressor effect on flowering. We discuss the Available online 1 April 2011 mechanisms of the reduced transcription and the memory of the vernalisation treatment through vegetative

Keywords: development. We also discuss the resetting of the repressed activity level of the FLC gene, following Arabidopsis vernalisation, to the default high activity level and show it occurs during both male and female gametogenesis Heterosis but with different timing in each. Flowering In the second part of the review discussed the complex multigenic system which is responsible for the Methylation patterns of gene activity which bring about hybrid vigour in crosses between genetically similar but Paramutation epigenetically distinct parents. The epigenetic systems that we have identiﬁed as contributing to the heterotic Epialleles phenotype are the 24nt siRNAs and their effects on RNA dependent DNA methylation (RdDM) at the target loci leading to changed expression levels. We conclude that it is likely that epigenetic controls are involved in expression systems in many aspects of plant development and plant function. © 2011 Elsevier B.V. All rights reserved.

1. Introduction states directly through targeted effects on DNA methylation, or on the production and/or destruction of transcripts from the gene. All these Thirty years ago, in probing the molecular basis for control of gene components of chromatin contribute to epigenetic controls of gene activity, emphasis was on the coded sequences in the DNA molecule. activity. In both plants and animals, a number of sequence motifs important for transcriptional activation were identified. More recently, it has been 2. Chromatin packaging realised that it is important to look at both the DNA and the proteins with which it is associated in chromatin. We now know the histone The primary unit of chromatin packaging is the nucleosome. The proteins of chromatin have modifications such as acetylation and nucleosome core particle is comprised of an octamer of histones methylation of particular amino acid residues which are associated H2A, H2B, H3 and H4 with an associated 147 bp of DNA. Chromatin with the control of gene activity. Methylation of cytosine residues in can undergo modifications to either DNA or histones that can have DNA can also influence the transcriptional activity of a gene. The consequences for gene expression by affecting access of RNA poly- makeup of the suite of these different epigenetic marks for any gene is merases or the interaction of transcription factors with specific DNA critical in determining the transcriptional state of that gene, probably sequences. through effects on localised chromatin architecture. Protein com- Until recently, determining the location of nucleosomes in the plexes which bind to chromatin are responsible for the modification genome was a laborious process, however the development of high of histones and small RNA molecules also influence transcriptional throughput sequencing technologies has made genome-wide mapping of nucleosome occupancy straightforward [1]. Some general trends have emerged from these studies, most notably that eukaryotic genes, including those of Arabidopsis, generally have a nucleosome- ☆ This article is part of a Special Issue entitled: Epigenetic Control of cellular and free region at the beginning and end of the transcription unit which developmental processes in plants. could facilitate access by RNA polymerase although is not sufficient for ⁎ Corresponding author. CSIRO Plant Industry (Black Mountain Laboratories) Acton, ACT 2601, Australia. Tel.: +61 2 6246 5250. high transcriptional activity as this feature is present at many genes E-mail address: [email protected] (W.J. Peacock). that are expressed at a low level.

The extent to which the position of a nucleosome is determined by pseudogenes and intragenic regions [7,8]. Approximately one third of the underlying DNA sequence appears to be somewhat variable. Arabidopsis genes contain CG methylation in their gene body [7,8]. Alignment of nucleosome core DNA sequences shows a 10 bp peri- These are predominantly constitutively expressed genes. Promoter odicity of AT and GC rich sequences. Nucleosomes are often aligned to methylation appears important for conferring specific tissue expres- the same position, particularly the nucleosome closest to the trans- sion [7]. The higher frequency of CHG and CHH methylation in the cription start site. Other nucleosomes do not have as well defined flanking regions of genes correlates with siRNA distribution across positions. Data from comparisons of expression states suggest that genic regions, consistent with RdDM establishing and maintaining positions of key nucleosomes near transcription starts can change, these two methylation contexts. but that the bulk of nucleosomes do not show expression-related Recently an important contribution was made by Lister et al. [9] differences in position. comparing the methylomes in human DNA samples extracted from Nucleosomes are enriched in coding regions and often mark intron- embryonic stem cells and from foetal fibroblast cells. They found that exon boundaries. This suggests that the nucleosome landscape may CG methylation is present in both cell types but differed in its dis- have an important role in intron splicing events as well as in trans- tribution across the genomes. Significantly, other contexts of cytosine cription. The nucleosomes also appear to be sites of DNA methylation, methylation (CHH and CHG) were present in the embryonic stem cells with a strong correlation between DNA methylation and the presence and absent from the fibroblast cells implying that these other contexts of nucleosomes [2]. of cytosine methylation are important in determining the pluripotent nature of the embryonic stem cells. It is likely that in the early paths of 3. Epigenetic marks on chromatin cell differentiation there are different patterns of alteration of the methyl cytosine residues in the genome. The CG context is the only Epigenetic controls are ubiquitous in flowering plants and are context to survive into fully differentiated cells. operative not only for the control of processes involved in develop- CG methylation in the genome has some important properties. It is ment but also for the plant's responses in its growth environment to inherited through cell divisions and is closely associated with DNA ensure its survival and function. The interaction of epigenetic me- replication. Changes in the methyl C residue distribution can occur in chanisms with environmental cues is important in many gene activity different tissues through development but ultimately the original responses. parental embryonic pattern is re-established (in animals) after meiosis In this review we discuss the role of epigenetic controls in two key in the new embryo. In flowering plants, methylation of DNA is com- biological activities of plants. One is the initiation of flowering fol- mon. The first indication that methylation is associated with regulation lowing exposure to extended periods of cold temperature—vernalisa- of gene activity came from studies on transposable elements, parti- tion. In the second part we examine the evidence that epigenetic cularly the Ac and Spm systems in maize [10,11]. The correlation that controls are significant in the patterns and levels of gene expression was evident was that the presence of methylated cytosine residues which give rise to hybrid vigour or heterosis in many groups of plants. was associated with non-active transposons and the absence of me- Arabidopsis and the knowledge of its genome sequence and the many thylation characterised active transposons, particularly in their tools that exist for detailed analysis of particular genomic regions and transcriptional activity and genome mobility. their functions have contributed to an understanding of the balance of genetics and epigenetics in the control of gene activity in biological 6. Vernalisation—an epigenetic control system processes. In plants the biological phenomenon of cold temperature-induced 4. DNA methylation and gene regulation induction of flowering, vernalisation, has properties paralleling DNA methylation. Vernalisation-induced states of gene activity instigated In the mid 1970s two key papers were published on the occurrence in the germinating seed are maintained through vegetative develop- of methylation of cytosine residues in DNA in animals [3,4]. This work ment of the plant, to finally operate in the transition of the growing focussed on the methylation of cytosine residues in CG dinucleotides. apex from a vegetative meristem to a reproductive meristem. Each The suggestion was that this epigenetic modification to the genomic generation of plants requires the exposure to a low temperature DNA was likely to be of importance in modifying the transcriptional period in order to flower. activity of genes. This was noted to occur both at a whole chromo- Treatment of germinating seeds of both Arabidopsis and wheat with somal level as evidenced by the X inactivation phenomenon in mam- 5-azacytidine, which prevents DNA methylation when incorporated mals and for individual genes operating in many metabolic pathways into DNA, caused early flowering without vernalisation. The results in cells. mimicked the accelerated initiation of flowering by vernalisation In plants, DNA methylation is found in three different contexts, CG, [12,13]. In Arabidopsis the reduction of CG methylation by inhibiting CHG and CHH (H = A, C or T). The methyl groups are added by three the activity of the methyl transferase 1 (MET1) enzyme responsible for different methyltransferase enzymes; METHYLTRANSFERASE 1 (MET1) CG methylation also accelerated flowering [14]. methylates CG sites, CHROMOMETHYLASE 3 (CMT3)methylatesCHG Genetic analysis of the vernalisation response identified two genes sites and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) estab- which had major effects in the response, FLOWERING LOCUS C (FLC)and lishes de novo methylation of all three contexts and maintains CHH sites FRIGIDA (FRI) [15–17]. FRI ensures a high level activity of FLC through a [5]. promotive transcription complex. FLC is a repressor of flowering and the The RNA-dependent DNA methylation (RdDM) pathway uses a vernalisation treatment reduces FLC transcriptional activity. When the series of plant-specific polymerases and processing enzymes to gene- FLC gene was cloned we were able to determine that there was only one rate short interfering RNAs (siRNAs). These siRNAs are then used as methyl C in intron one and no other methyl Cs in the gene body or in the targeting sequences for gene specific transcriptional silencing through flanking promoter region [18,19]. We found there was no change in the establishment of de novo DNA methylation of all three contexts and methylation pattern following vernalisation and concluded that the maintenance of CHH methylation [5,6]. reduction of CG methylation across the genome must result in reduction of FLC activity not through a direct action on the FLC gene but through 5. Distribution of DNA methylation across the genome interactions with products from at least one other gene which had changed activity levels as a result of the demethylation treatment. DNA methylation is predominately found in transposons and The vernalisation treatment represses the transcription of FLC and repetitive sequences and in promoter regions, intergenic regions, poses the question as to how the down regulation of FLC occurs. A M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 429 complex of proteins binding to FLC chromatin modifies the histones to in H3K27me3 [21,31]. Time course studies show only a small change in repress FLC activity [20,21]. These marks are retained after vernalisa- H3K27me3 at FLC in the cold at the transcription/translation start region tion, determining the state of suppression of FLC activity through contrasting with a progressive increase along the gene once plants are development of the plant. Since the requirement of vernalisation for returned to higher temperatures [29]. The changes in H3K27me3 at FLC flowering initiation is found in many groups of plants we have asked lag behind the changes in FLC transcription suggesting they may be a whether FLC is the key response gene in species other than Ara- consequence of the decreased transcription [33]. bidopsis. FLC occurs in other members of the Brassicaceae [22], the family to which Arabidopsis belongs, and it has been reported in 8. Two phases in the vernalisation response sugarbeet [23] but no details of its epigenetic behaviour have been established in these species. We were not able to identify a homo- A number of lines of evidence suggest that there are two phases to logue of FLC in the temperate cereals, wheat or barley, despite these the vernalisation response—the initial down-regulation of FLC with species being strongly dependent on vernalisation for flowering in repression of transcription and the subsequent phase of maintenance their winter varieties. It has been possible to define an equivalent of the repressed state. The regions of the gene responsible for the two epigenetic system in the cereals [24,25]. phases are different [34]. The maintenance phase is better understood than the initial repression. Deletion studies with an FLC:GUS fusion 7. Vernalisation causes epigenetic changes to FLC chromatin construct have indicated that the large first intron of FLC is required for stable down-regulation. In constructs where the intron was largely The repression of Arabidopsis FLC expression by vernalisation is deleted, down-regulation of the transcript occurred but the repressed one of the best-studied examples of the epigenetic repression of a state was not maintained through plant development. The stable gene in plants [18,19]. The process of vernalisation has long been maintenance of the repressed state but not the initial repression suspected of having an epigenetic basis as there can be a considerable requires the proteins of the polycomb complex, CURLY LEAF/SWINGER temporal separation between the vernalising cold period and the (CLF/SWN), VRN2 and FIE [32,35]. These data suggest that the initial onset of reproductive development. In Arabidopsis, expression of FLC down-regulation of FLC is independent of PRC2 function. Maintenance confers a vernalisation-responsive late flowering phenotype. FLC of the inactive state requires the addition of H3K27me3 to the encodes a MADS box protein that acts to repress flowering by direct chromatin by the CLF/SWN homolog of Enhancer of Zeste. Initially interaction with the floral integrator genes SUPPRESSOR OF OVER- H3K27me3 is added to the chromatin at the transcription/translation EXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT) region. This repressive histone mark later spreads across the first [26,27]. FLC expression is down-regulated during vernalisation and intron and beyond to the 3′ terminus of the gene. DNA replication is maintained at a low level after plants are returned to warm con- required for the maintenance events to occur. In the absence of DNA ditions. This regulation of FLC is the basis of the epigenetic memory of replication in older leaves FLC is down-regulated during vernalisation the vernalisation treatment. and H3K27me3 is added at the transcription/translation start region. The arrangement of nucleosomes across the FLC locus appears to be With further growth of the plant in normal temperatures, the gene typical of an expressed gene with a distinct nucleosome-free region returns to the default active state and the repressive histone marks are around the transcription start site. To date no evidence has been removed [29,36]. obtained for nucleosome redistribution with vernalisation. The histone The observations that H3K27me3 is required to stably repress FLC variant H2AZ, which is associated with active genes and destabilises expression and that H3K27me3 increases in response to decreases in FLC nucleosomes, is inserted at the 5′ and 3′ end of the FLC transcription unit. transcription after vernalisation, prompted us to investigate the Loss of H2AZ incorporation leads to low FLC expression indicating that it interaction between H3K27me3 and transcription in more detail. We is a requirement for FLC expression [28]. However loss of H2AZ does not found that the FLC gene body is heavily H3K27 trimethylated in the occur when FLC is inactivated by vernalisation (there is in fact a slight absence of the FLC promoter suggesting that sequences in the gene have increase [29]). This suggests that H2AZ has a role in marking genes for intrinsic H3K27me3-recruiting properties [33]. Using a version of this transcriptional activity, but at least in the case of FLC it does not have a gene construct with an inducible promoter showed that H3K27me3 qualitative role in regulation of expression. recruitment is a consequence of stopping transcription of the gene. The A number of mutant screens have been carried out to identify kinetics of this recruitment closely match those of H3K27me3 recruit- factors involved in the vernalisation response. Among the genes ment after vernalisation. This suggests that the addition of H3K27me3 to identified are VERNALISATION 2 (VRN2), encoding a homolog of the FLC after vernalisation can be explained as a consequence of the loss of Suppressor of Zeste 12 protein that is a component of polycomb re- FLC transcription. H3K27me3 levels in ecotypes with a range of FLC pressive complex 2 (PRC2) [20]. The PRC2 complex in Drosophila is expression show a quantitative inverse relationship with transcription, composed of the core subunits Enhancer of Zeste, Suppressor of Zeste reinforcing the link between transcription and H3K27me3 occupancy. 12, Extra sex combs and P55. In Arabidopsis small gene families When PRC2 function is reduced in lines expressing either high or low encode these proteins with the exception of FERTILIZATION INDEPEN- levels of FLC, FLC expression increases showing that the presence of DENT ENDOSPERM (FIE), the EsC homolog, which is present in single H3K27me3 inhibits transcription [32]. Taken together these data suggest copy [30]. Subsequent studies have shown that other components of a feedback loop between transcription and H3K27me3 that stabilises the PRC2 complex, which adds the repressive histone3 lysine 27 expression levels of FLC [33]. trimethylation (H3K27me3) chromatin mark, are also required for This function of PRC2 in vernalisation mirrors the role of the repression of FLC expression in the ambient temperatures following polycomb system in repression of homeotic genes in Drosophila vernalisation. The VERNALISATION INDEPENDENT 3 (VIN3) and VRN5 where initial expression patterns are set in a polycomb-independent genes which encode plant homeodomain (PHD) proteins are also manner by transcription factor binding which then require polycomb required for vernalisation repression of FLC [31]. Protein studies have function to maintain repression in cells where the homeotic genes shown that these PHD proteins associate with the PRC2 complex. VIN3 were inactivated [37]. is only expressed during cold temperatures and its association with the PRC2 complex has led to the suggestion that it enhances recruit- 9. The FLC environment in the genome—coordinate epigenetic ment of the PRC2 complex to FLC during the low temperature period controls of genes [32]. Consistent with the requirement for the PRC2 complex, the re- FLC is located in a chromosomal domain that at least in some respects pression of FLC expression by vernalisation is associated with an increase shows coordinated regulation of the genes in the domain [38].The 430 M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 expression of UFC, the locus approximately 5 kb upstream of the FLC microsporogenesis. Activity then apparently ceases with no FLC activity promoter region, parallels that of FLC in a number of ecotypes, in mutants detectable in the subsequent pollen cells [40]. of the autonomous flowering pathway genes and with the presence of FRI. Induced reduction of DNA CG methylation co-ordinately down- 11. Non-coding RNAs and FLC Expression regulates both FLC and UFC. An insertion of a nos::nptII gene construct into the region between FLC and UFC also follows the vernalisation Two long non-coding RNAs have been reported that may play a induced regulation pattern of FLC and UFC. This nptII construct, inserted role in FLC repression and/or maintenance [42]. COOLAIR antisense elsewhere in the genome, does not respond to vernalisation. However transcripts are expressed from a promoter located at the 3′ end of the if the construct is engineered to include the FLC gene and inserted FLC gene and at least two differently processed transcripts are pro- into the genome both FLC and nptII respond to vernalisation treatment duced. One transcript extends past the transcription start site of FLC [39]. Over-expression of FLC driven by the 35S promoter and inserted and another is truncated and only extends into the sixth intron of the elsewhere in the genome does not alter the transcriptional level of the gene. COOLAIR has been suggested to play a role in the initial down- UFC gene showing that it is not the transcriptional or translational regulation of FLC. The COOLAIR transcript is maximally induced by product of FLC that regulates UFC in the coordinate responses. 10 days in the low temperature treatment period and precedes the In addition to the coordinate response of UFC, the gene down- independent VIN3 induction. The COOLAIR promoter confers low stream from FLC (DFC) also responds to vernalisation treatment with temperature induced down-regulation of a GFP or Luciferase reporter reduced expression [38]. Again, as with UFC, the repression is not construct. As to the mechanism of COOLAIR RNA down-regulating FLC, stably maintained in the manner that FLC is. Beyond those two loci it has been suggested that promoter interference results from the there is no indication of a vernalisation response in other neighbour- presence of the antisense transcript causing repression of the FLC ing genes so there appears to be a finite region of the genome with a gene, which sets up a cascade of events that leads to the repression of perimeter beyond UFC upstream of FLC and beyond DFC downstream sense transcription of the FLC gene. The lack of FLC product then of FLC which has this property of coordinated responses. This applies results in early flowering. to low temperature and to the action of mutants of the autonomous These findings around COOLAIR transcription are not consistent flowering pathway. The region may have defined termini but if it is with earlier reports that show that constructs with deletions of the 3′ lengthened as in the nptII insertion in flc11 which introduces 15kb end of the FLC gene, including the region of the COOLAIR promoter, adjacent to FLC, the whole region has the coordinated response. still respond to vernalisation by inducing repression of FLC transcrip- This coordinated response to low temperature and to epigenetic tion [34]. controls together with the response to reduced CG genome methyl- Recently another long non-coding RNA, COLDAIR,whichis ation, suggests that the transcription responses may be a consequence transcribed in the sense direction from within the first intron of FLC of a property of the chromatin of the region—perhaps to a special has been suggested as being required for the vernalisation repression packing regime of that region. There may be specific sequences at the of FLC [43]. COLDAIR is induced by the low temperature period and termini but we have been unable to find any conserved elements so reaches a maximum at 20 days. The transcription start site of COLDAIR this regional response property remains unexplained. is within the vernalisation responsive element (VRE), a region located in intron 1 and previously shown to be required for vernalisation- 10. Resetting of FLC mediated repression of FLC [44]. The putative promoter of COLDAIR confers cold-inducible expression of a Luciferase reporter gene cons- One of the characteristics of vernalisation is that the low tem- truct. COLDAIR transcripts become associated with the PRC2 complex, perature treatment has to be applied each generation. This implies binding to the CLF protein. RNAi knockdown lines, targeted to the that the vernalised state with inactive FLC is reset to an active state in region of the COLDAIR transcript, resulted in late flowering following the progeny [40]. FLC is active in 3-day-old germinating seeds and an vernalisation, the plants having high levels of FLC. In fact an initial examination using a FLC:GUS reporter construct of the paternal and repression of FLC transcripts does occur in the RNAi lines but the maternal derived FLC genes in the zygote shows that both alleles are repression was not maintained through plant development. The reset by the early globular stage embryo. Furthermore, if the FLC allele COLDAIR effect is on the maintenance phase and not the initiation is derived from the male gamete it is active in the single cell zygote. If phase of the vernalisation response. the FLC allele is derived from the female the active state is not reset These RNAi experiments have other possible interpretations. The until approximately three or four mitotic divisions have occurred. RNAi construct could be operating against the unprocessed FLC Resetting to an active state in the embryo is not dependent on FRI transcript itself rather than against the COLDAIR transcript. RNAi tar- activity or any of the autonomous pathway genes, which are im- geting an intron has been successful in other systems in disrupting portant for FLC activity in later development of the plant. transcription. The suggestion that the COLDAIR RNA may be involved in The resetting in the developing seed is restricted to the embryo targeting the polycomb complex to FLC chromatin is similar to that and FLC is not operative in the endosperm. The differential resetting of proposed for the long intergenic non-coding RNA, HOTAIR, in mam- FLC activity in the male and female FLC alleles is evidence of the mals. HOTAIR RNA targets the HOX gene cluster for PRC2, H3K27me3 intracellular environment interacting with FLC chromatin. For and stable transcription repression [45]. Even if COLDAIR RNA targets example the two sperm, both delivered by mitosis of the generative the complex to the first intron by base homology there still needs to be nucleus have different FLC futures depending on whether they are an explanation why the first H3K27me3 is placed at the transcription/ involved in fertilisation of the central cell to produce endosperm or in translation start site. the formation of the zygote by fusion with the egg cell. Another example of an epigenetic contribution to resetting is the role 12. Autonomous pathway genes are involved in control of FLC of the FVE gene which codes for a retinoblastoma-associated protein, activity and prevents the resetting of the expression of FLC in the endosperm; in an fve mutant FLC is reset to an active state in the cells of the endosperm There are a number of pathways involving different loci that can at the torpedo stage of embryo development [41].Incontrasttothe affect the activity state of the FLC gene. Genes operating in the so-called situation in animals where DNA methylation patterns are reset in the autonomous pathway affect expression and repress FLC without new generation embryo, the resetting of FLC follows a more complex exposure to low temperature. The autonomous pathway regulatory course. FLC is reset in the anthers of the vernalised plant, in the maternal genes were first identified genetically by Koornneef [16] by their wall tissue of the anther prior to meiosis, and at the earliest stages of effects in delaying flowering. Subsequently a number of studies have M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 431 shown that these autonomous pathway genes participate in control of A vernalisation response element (VRE) in the FLC gene has been the activity state of FLC. For example, the RNA binding protein pro- located in the first intron, roughly 1300 bp from the ATG and probably duced from the FCA gene interacts with the polyadenylation signalling corresponds to the tenth nucleosome of the locus. Before VIN3 trans- protein FLOWERING LOCUS Y (FY) controlling FLC expression. The cription ceases, COLDAIR transcripts are produced from a promoter control is possibly through the regulation of the expression of FLC within intron 1 in the 5′ region of the VRE. The COLDAIR transcript binds antisense RNA (COOLAIR) by alteration of the 3′ terminus of the to the CLF/SWN proteins of the PRC2 complex and may be involved in transcript [46]. FPA is another gene operating in the autonomous recruiting the VIN3/VRN5 heterodimer, triggering the spreading of the pathway which also affects 3′ processing of FLC antisense RNA. Two H3K27me3 which subsequently maintains the absence of FLC transcrip- other genes, FLOWERING LOCUS D (FLD), a lysine specific demethylase tion. Subsequent to the vernalisation period, these events have acted so [47], and FVE, a retinoblastoma associated protein homologue, affect as to ensure the stabilization of FLC down-regulation. Another protein, the structure of chromatin at the FLC region. These autonomous LHP1, which binds to H3K27me3 marks appears to be necessary for this pathway genes have similar effects on other targets as shown by their stabilization of the inactive state [44]. role in silencing transgenes and controlling targets of RNA-mediated DNA methylation [48]. Alternative 3′ polyadenylation of COOLAIR 14. Epigenetic control of vernalisation in temperate cereals antisense FLC transcripts are altered in an fpa mutant where there is relatively more of the long transcript, and correspondingly when FPA is Since the beginning of the 20th Century plant breeders have overexpressed the long COOLAIR transcripts are reduced in frequency worked with winter and spring varieties of wheat, barley and rye. [46]. When the long antisense transcript is reduced there is also a Winter lines of these temperate cereals require exposure to a pro- reduced expression of FLC, a result contrary to the suggestion that longed period of low temperature, as experienced by the crops in the COOLAIR transcripts may interfere with FLC transcription. ground during winter, to ensure flowering and grain development in the spring and early summer. Spring varieties flower and develop grain 13. Multiplicity of epigenetic controls—a summary without the need for the low temperature exposure. Genetic analyses in wheat and barley have identified a small Prior to vernalisation, FLC is transcriptionally active in accessions number of key genes involved in the response to the low temperature where there is an active FRI gene. The FRI transcription complex (FRI- vernalisation period [50,51]. As in Arabidopsis, the environmental cue C) recruits general transcription and chromatin modification factors brings about changes in the activity states of the key genes. These ensuring FLC chromatin is in a transcriptionally active state and is activity states are retained through the development of the plant until highly expressed. However in the earliest stages of embryo develop- the plant initiates reproductive development of the terminal me- ment, FLC activity occurs without the requirement of an active FRI ristem, producing the inflorescence and the harvestable head of grain. gene in the genotype. This FRI independent activity remains until the In these winter cereals, plants of the next generation require a ver- late torpedo stage of embryo development and then decays. Before nalisation treatment identical to that needed by the parent crop. This vernalisation, the FLC gene has a low level of H3K27me3 marks. How mirrors the resetting of FLC in Arabidopsis. The implication is that the transcriptional activity relates to the low level of H3K27me3 is cold-induced activity states of the vernalisation response genes are unclear; it may be that a certain percentage of cells do not have the reset to the default state during the sexual reproduction process. This H3K27me3 marks and these are the cells in which FLC is active, or it genome/environment interaction cycle implies a reversible epigenetic may be that a particular density of H3K27me3 is needed in order to control of transcription state of the key response genes whose pro- prevent transcription. A negative correlation between the level of ducts are involved in the initiation of flowering. H3K27me3 and the level of transcriptional activity is displayed in The properties of the vernalisation-induction flowering cycle in different accessions. the monocot cereals parallel those of the dicot Arabidopsis [52]. The We have no knowledge of the mechanism that initiates the key vernalisation response gene of the dicot is FLC and it could be reduction of FLC activity in the early stages of the cold treatment conserved across flowering plants, operating in monocots with the period. Mutant searches have not identified any loci concerned with same group of epigenetic controls as found in Arabidopsis. This is not this process, so the possibilities are redundant gene mechanisms or, the case. In the cereals the cold-induced epigenetic cycle involves the the process may involve a localised change in chromatin structure, induction of a key promotive gene VERNALISATION 1 (VRN1) stimu- rather than the interaction with a gene product. The earliest gene- lating the vegetative/reproductive transition [24,25]. Vernalisation based activity is the transcription of COOLAIR RNA, reaching maximal causes the production of leaf primordia at the shoot apex to be activity by 10 days into the cold period. The suggestion has been made replaced by the appearance of floral primordia. Following vernalisa- that the COOLAIR antisense RNA interferes with the sense transcription in the winter, long day lengths in the spring are required for the tion of the FLC gene thus down-regulating FLC. The proposed COOLAIR floral primordia to develop into inflorescence branches bearing the interference effect may operate together with other controls florets. Long days are a required cue but cause rapid flowering only generating the down-regulation. The other epigenetic controls are after plants have been vernalised. Vernalisation of the germinating concerned with maintenance of the FLC down-regulation through seed changes the transcription activity state of the genetically mapped plant development. major response gene, VRN1, the induced state being maintained VIN3 induction is another prominent response to the low tem- through development of the plant, returning to the pre-vernalisation perature treatment, its activity building to a maximum by 40 days. state in the next generation plants. The cellular (mitotic) memory of VIN3 transcriptional activity is reduced quickly following the ces- the vernalisation-induced state is demonstrated by the observation sation of the cold period. The VIN3 protein can interact with VRN5 that plants regenerated from tissue culture cells of a winter variety do protein to form a heterodimer which associates with the PRC2 com- not require a vernalisation treatment for early flowering in long days plex positioned at an FLC chromatin region proximal to and extending if the tissue culture cells themselves had been vernalised [53]. into the first intron [49]. This probably represents the region of the Prior to vernalisation exposure, VRN1, a MADS box gene, as is FLC second and third nucleosome from the ATG of FLC. The binding of the in Arabidopsis, has only a low level of transcription. Vernalisation VIN3 heterodimer with PRC2 induces CLF/SWN to increase H3K27me3, induces high transcript levels in leaf and apical tissue. In the same initiating the spreading of H3K27me3 along the gene, through the cells and tissues, two other genes VRN2 and VRN3 (FT1) [54,55] show intron and extending to the 3′ terminus. Even though the VIN3 pro- marked changes in transcription activity levels. Both of these genes duct is not present after the cold period, H3K27me3 spreading may have been identified genetically as being involved in the vernalisa- continue. tion/long day flowering response which reduces VRN2 transcripts and 432 M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 increases VRN3 transcripts. VRN2, encoding a protein with a zinc chanisms involved have shown that the large number of genes have finger and a CCT domain, delays flowering by inhibiting expression of changed levels and patterns of activity. Recent results are beginning FT1. VRN2 down-regulation by vernalisation is likely to be mediated to disclose the involvement of epigenetic mechanisms. One of the by the low temperature induction of VRN1, thus removing the re- puzzles has been that significant heterosis can be obtained even when pression of FT1 activity which results from the action of VRN2 [56]. FT1 the parents are genetically very similar. These early experimental data transcription is up-regulated in long days and is directly involved in indicate that parents with highly similar genome sequences may have the transformation of the terminal apex into a reproductive meristem. epigenomes which are sufficiently different from each other to be of Plants lacking VRN2 do not require vernalisation for early flowering. significance in the generation of the heterotic phenotypes. So the genetic identification and biological behaviour of the key vernalisation response genes in cereals provide a strong case for an 16. Epigenetics influences development epigenetic control of flowering equivalent to the Arabidopsis story. There are only limited data on the molecular basis of the elements of Epigenetic regulation plays a vital role during embryogenesis and the epigenetic control cycle [57]. Pre-vernalisation barley VRN1 chro- development in animals [59–61]. Mutations in epigenetic regulators matin has high levels of H3K27me3. The vernalisation period of low such as Polycomb or Trithorax in Drosophila cause altered homeotic temperature results in increased levels of H3K4me3 and removal of gene expression leading to developmental defects in organ position H3K27me3 at the locus. H3K27me3 is a histone mark indicating lack and number [59]. Despite the many differences between plant and of transcriptional activity whereas H3K4me3 is associated with animal development, it is becoming clear that epigenetic regulation is transcriptionally active genes. also required for normal plant development. Initial indications were The vernalisation induced reduction of H3K27me3 and increase of the pleiotropic phenotypes of mutants for genes involved in the H3K4me3 at VRN1 are maintained through plant development, a similar biosynthesis of epigenetic regulators. For example, genome-wide loss mitotic memory of induced histone marks as seen in Arabidopsis. of CG methylation through mutation in met1 results in plants with The same two histone modifications were not altered at VRN2 or curled leaves, altered branching patterns, delayed phase-change, FT1 during the low temperature period, pointing to transcript pro- altered flowering times, variable floral morphological abnormalities duction at VRN1 being the major response in the vernalisation induc- and decreased fertility [14,62–64]. Mutants of the polycomb complex tion of flowering. In cereals the epigenetic response is the activation genes CLF/SWN, and EMBRYONIC FLOWER 2 (EMF2), produce many of the key response gene VRN1 whereas in Arabidopsis the response is developmental defects including premature flowering immediately repression of transcriptional activity of the key response gene FLC. after germination [65,66]. In Arabidopsis, addition and maintenance of the H3K27me3 marks Further evidence that epigenetics plays an important role in plant are achieved through the action of the PRC2 protein complex. The development comes from epigenetic Recombinant Inbred Lines (epi- component proteins of the PRC2 complex have been identified in RILs). These epi-RILs are generated between lines of the same genetic cereals [58] so it is likely that the complex is involved in the addition background except that one parent has an altered epigenome due to a of H3K27me3 marks at VRN1 prior to vernalisation, and recently a mutation in a gene affecting epigenetic marks. Epi-RILs generated from histone demethylase, specific to the H3K27me3 mark, has been DECREASE IN DNA METHYLATION (ddm1)ormet1 mutant lines display a identified. It should be possible to check for the induced activity of this range of developmental phenotypes, including altered flowering time, gene during the vernalisation period in barley. The VRN1 gene of plant stature, biomass, salinity tolerance, and pathogen resistance barley varieties that flower without vernalisation (spring barleys) has [67,68]. Some of these traits varied phenotypically across the col- high levels of H3K4me3 and low levels of H3K27me3. lection of epi-RILs, indicating a polygenic basis [67]. There are unknowns in this pathway of vernalisation-induced Epi-alleles with DNA hypermethylation in the promoters of genes flowering in the cereals. Studies of transcript levels in mutants and at affect a diverse range of developmental processes. These include colourless different developmental stages of the plant point to a VRN2 repression non-ripening, a SPB box transcription factor which affects fruit ripening in of FT1 activity but the mechanism is unknown. Similarly how VRN1 tomato [69], a naturally occurring epi-allele of CYCLOIDIA in Linaria represses the transcriptional activity of VRN2 is unknown. In vulgaris that causes peloric conversion of floral organs [70] and the clark Arabidopsis, FT in concert with FD, induces APETALA 1 (AP1) activity kent and floral organ number1 epi-alleles of SUPERMAN which result in which triggers floral development but in cereals the action of the abnormal anther and carpel development in Arabidopsis [71,72].Such homologous FT1 gene has not been determined. epi-alleles corresponding to transcription factor genes and other regulatory loci, regulate developmental processes and metabolic 15. Hybrid vigour—epigenetic contributions? pathways.

The results from many laboratories studying the FLC locus have 17. Natural variation of the epigenome shown that the relationship between the gene sequence and the phenotype is not dependent solely on the DNA sequence itself. The Closely related species having differences in gene expression in data have shown that FLC chromatin, which includes the DNA, the response to environmental stresses show differences in their epige- histones and other protein complexes and RNA molecules, are all netic systems. These epigenetic alterations also have the advantage of involved. FLC is probably one of the best studied plant genes in terms being heritable, but also reversible, without any changes being made of the epigenetic controls involved in the determination of its activity to the underlying genetic sequence [73]. There are large differences in state, and it is clear that the path from the coded message in the DNA the epigenetic architecture in both distantly and closely related to the phenotypes (there are more than one) is complex with many species [74–83]. One reason for these differences in the epigenomes is interactions between the control mechanisms. Given the understand- that they could relate to adaptation to different environmental niches. ing of the way in which the genome and epigenome interact in For example, the mangrove Laguncularia racemosa, contains multiple determining the transcriptional activity of this single gene, we expect DNA methylation polymorphisms between sub-populations depend- that the phenomenon of hybrid vigour will have significant and ing on whether they are growing by a river or within a salt marsh [84]. complex interactions between the genome and the epigenome. Genome-wide studies of the epigenomes of Arabidopsis thaliana Heterosis has been studied over the last century and has been used accessions revealed that transposable elements are methylated with extensively in agricultural production systems where the increased little variation between the different accessions, but there are harvestable yield has been of importance to farmers. Attempts at a substantially different methylation patterns within gene bodies and genome level to analyse the causes of hybrid vigour and the me- promoters [76,77,82]. Similarly, siRNA-associated loci show M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 433 significant variation between Arabidopsis accessions [78,82]. Among to a “balancing out” of the siRNAs contributed by each parental locus. ~15,300 siRNA clusters indentified between the Arabidopsis acces- Decreases in the high parent level were greater than the increases in sions C24 and Landsberg erecta, only ~38% had similar levels of siRNA the low parent level, resulting in a net reduction in the siRNA levels. It in both accessions, ~45% had large differences in the levels of siRNA is possible that the trans-effects at loci exhibiting large decreases in between corresponding clusters, and 17% occurred in only one of the siRNA levels is an extreme example of the ‘balancing out’ seen at loci two accessions [82]. with less disparity in parental siRNA levels. Limitations in the parental The abundance and localisation of DNA methylation, siRNA contribution analysis prevented examination of loci with large production, and active and repressive histone marks also differ differences in siRNA levels between parents. between the closely related rice sub-species, japonica and indica [80]. In the rice hybrids, a substantial number of loci show non-additive The 4,538 genes that are differentially expressed between these two DNA methylation [80]. The reciprocal rice hybrids differ in the number sub-species mostly show differences in DNA methylation, in the active of loci showing non-additive methylation; however, in both hybrids H3K4me3 marks and in the repressive H3K27me3 marks [80]. approximately 75% of the non-additively methylated loci have in- It is now apparent that there can be large differences in the creased levels of methylation [80]. Loci with altered levels of me- epigenetic make-up of related species, even between ecotypes which thylation are also prevalent in Arabidopsis hybrids and show a general have genomes that are very similar. This epigenetic variation may trend of increased methylation [82, Greaves et al., unpublished]. As in profoundly influence the development of heterosis of the F1 hybrids, the siRNA loci, non-additive DNA methylation in the hybrids occurs at particularly in genetically close parents. loci that are differentially methylated between parents [82, Greaves et al., unpublished]. In both the rice and Arabidopsis hybrids, locus- 18. Epigenomes of Hybrids Differ from the Parental Epigenomes specific increases in DNA methylation above MPV suggest Trans Chromosomal Methylation (TCM), where the presence of a hyper- The molecular basis for hybrid vigour is being investigated in a methylated parental locus confers methylation onto the hypomethy- number of plant species, including the important crops wheat, rice, lated locus, creating two similarly methylated epi-loci. Although in barley and canola, along with laboratory plants such as Arabidopsis. both the rice and Arabidopsis hybrids the general trend is towards The majority of these investigations have revealed non-additive gene increased methylation at non-additively methylated loci, a significant expression in hybrids [reviewed in [85–87]. The focus is now shifting proportion show reduction in methylation towards the low-parent to the mechanisms causing this non-additive gene expression. An indicating Trans Chromosomal de-Methylation (TCdM). epigenetic contribution is of particular interest in intra-species Epi-alleles influence development and may contribute to the hybrids derived from genetically close parents where allelic diversity likelihood of obtaining heterosis in an F1 hybrid. The locus-specific producing heterosis may be provided by epi-alleles rather than by changes to siRNA and methylation levels through trans effects cor- differences in DNA sequences in genetic alleles. relate with altered gene expression in the hybrids [82,Greaves et al., Early studies showed the inheritance of DNA methylation was unpublished]. Epigenetic changes can affect expression levels by non-additive, providing the initial indicator that hybrids had an either reducing or increasing allelic expression bias. Differences in altered epigenome [79,88–93]. The development and availability of siRNA and DNA methylation levels between parents and in hybrids deep sequencing technologies has enabled more extensive large-scale occur most frequently in genes and their flanking regions [82,Greaves analyses on all components of the epigenome [80–82]. et al., unpublished]. We suggest these epi-allelic variants result from In rice intra-species hybrids as well as in Arabidopsis inter-species selection pressures in their native habitats and may influence de- hybrids, a small number of miRNAs are non-additively expressed, velopmental or functional properties and contribute to hybrid vigor. affecting transcript levels of their targeted genes [80,81]. In contrast, Loci with altered levels of siRNAs in the Arabidopsis hybrids are fre- miRNAs and their target genes appeared to be additively expressed in quently associated with genes with higher order molecular functions intra-species Arabidopsis hybrids [82]. However, by far the most (i.e. transcription, protein binding, and signal transduction) [82]. This significant epigenetic alterations were associated with DNA methyl- suggests that the altered activity of relatively few genes affected by an ation and 24-nt siRNA levels [80,82]. initial epigenetic change can lead to cascading effects on a larger On a genome-wide scale there is no difference in the frequency number of genes contributing to heterosis. and distribution of siRNAs and DNA methylation between parents and Changes to the epigenome of hybrids have been seen in several hybrids [80,82, Greaves et al., unpublished]. This has led to a con- systems. Whether these changes alter development and contribute to clusion that the hybrid epigenome is no different to those of the heterosis depends on the particular epi-alleles present in the parents parents when less sensitive analytical techniques are used [94]. In fact (Fig. 1). The change in epi-alleles in the hybrids, through altered the hybrid epigenome shows extensive locus specific differences from siRNA levels, TCM, or TCdM, could fit the classic genetic heterosis the average of the additive contributions of the two parents—Mid models of dominance and over dominance indicating that epi-alleles Parent Value (MPV). Both the rice and Arabidopsis hybrids exhibit need to be considered as part of the mechanism of hybrid vigor. What locus specific variation in 24-nt siRNA accumulation, with the vast is clear is the hybrid epigenome differs significantly from the additive majority of these loci showing decreased levels compared to the MPV product of the two parents. [80,82]. This is consistent with data from Arabidopsis inter-species hybrids which show a reduction in total siRNA levels [81]. The Ara- bidopsis intra-specific hybrids show that the locus-specific decreases 19. Increased methylation levels in hybrids through trans in siRNAs correlate with differences in siRNA levels between the chromosomal methylation parents [82]; dramatically reduced siRNA levels in the hybrids occur exclusively at loci with large differences in siRNA levels between We have suggested that in the hybrids locus-specific Trans Chro- parents. This implies that locus-specific siRNA decreases in the mosomal Methylation (TCM) results in increased levels of methyla- hybrids are mediated through trans-effects between the two parental tion above the mid parent value. This has similarities to the situation alleles. In the rice hybrids [80], although not specifically analysed, the in paramutation where a dominant paramutagenic allele, equivalent results indicate a similar relationship at loci showing non-additive to the high parent methylated locus, induces changes in the ex- siRNA levels. pression state of a paramutable allele, equivalent to the low parent Trans-effects are also evident in approximately one-fifth of the methylated locus [95–97]. Parallels between TCM and paramutation siRNA loci in Arabidopsis hybrids having moderate to low differences may aid in understanding possible mechanisms causing the altered between parental siRNA levels [82]. At these loci the trans effects lead epigenome of the hybrids. 434 M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437

Fig. 1. Model for inheritance of locus-specific siRNA and DNA methylation levels in the F1 hybrid. A schematic representation of four hypothetical loci and associated epi-alleles with variations in chromatin states, DNA methylation and/or siRNA levels between parental lines [Note: (i) parental lines are homozygous for their respective alleles (ii) only one chromatid from each chromosome is depicted (iii) the specific contexts of DNA methylation not differentiated]. The transcriptional activity of each allele is dictated through the associated chromatin state which is influenced by siRNA and DNA methylation. In turn, the chromatin state can influence the levels of siRNA required to establish DNA methylation in the region. Several possible interactions may account for the hybrid epigenome differing from the expected average of the two parental lines (i.e. mid-parent value—MPV). Locus 1—Additive inheritance of the parental alleles resulting in transcription levels being at MPV. The presence of siRNA is a requirement for trans effects to occur. The lack of siRNA associated with the hypermethylated P1 allele results in additive inheritance in the hybrid. Locus 2—In this example, siRNA are associated with the methylation of the P2 allele. In the hybrids the P2 derived siRNA molecules associate across both alleles resulting in levels sufficient to retain methylation at the P2 allele but not enough to exceed the threshold set by the active chromatin state of the P1 allele. Therefore, the parental alleles retain their native methylation states resulting in additive inheritance and MPV transcription levels. Locus 3—The P1 derived siRNA molecules associate with both alleles, remaining above threshold levels to not only maintain methylation at the P1 allele, but to also establish de novo methylation of the hypomethylated P2 allele. This process of Trans Chromosomal Methylation (TCM) results in both parental alleles being methylated and silenced in the hybrid leading to below MPV transcription levels. In addition, the MPV siRNA levels are now being equally contributed by both parental alleles possibly as a consequence of the TCM process and the reinforcing interaction that exists between DNA methylation and siRNA production. Locus 4—In the hybrid, the P2 derived siRNA initially become associated across both parental alleles. This co- association results in siRNA levels at each allele falling below the required threshold to establish or even maintain DNA methylation causing the hypomethylation of the P2 allele. The loss of DNA methylation may also interrupt the process maintaining siRNA levels resulting in the subsequent loss of siRNAs and further reduction of DNA methylation. This process of Trans Chromosomal de-Methylation (TCdM) results in both parental alleles being active in the hybrid, leading to above MPV transcription levels. M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 435

A well characterised example of paramutation is the Booster 1 (b1) Localised non-additive decreases in DNA methylation levels are also locus in maize encoding a transcription factor involved in anthocyanin observed in the met1-derived epi-RILs [68], indicating that the TCdM biosynthesis [95–97]. Generation of a plant heterozygous for the B’ and phenomenon is dependent on the interactions between the two B-I alleles, results in the lower expressed B’ allele conferring a B’-like parental epi-alleles and not due to differences in the nucleotide state on the more highly expressed B-I allele essentially creating a B’ sequences. homozygote. Paramutation is exerted through methylation of an up- Loci exhibiting TCdM in both the Arabidopsis hybrids and the stream repeat sequence enhancer element causing a repressive chro- met1-derived epi-RILs are frequently associated with decreased siRNA matin state and therefore low expression of the b1 gene [98,99]. The levels [68,82] and in Arabidopsis hybrids is predominantly associated increased methylation at B-I under the influence of B’ requires 24-nt with decreased CHH methylation which requires 24-nt siRNA for siRNAs [100]. Similarly, 24-nt siRNAs are required for Trans Chromo- maintenance [82]. This implies that the localised reductions in 24-nt somal Methylation (TCM) in hybrids. In intra-species Arabidopsis siRNA at these loci are the primary cause of methylation reduction, hybrids TCM occurred in all three methylation contexts at loci with emphasising the role of 24-nt siRNA in shaping the altered hybrid large differences in siRNA and methylation levels between parental methylome. Although methylation contexts were not specified for the alleles and where hybrid siRNA production is ≥ MPV. However, at loci rice hybrids, it is likely that non-additive decreases in methylation are with similar differences between the parents but with decreased also frequently associated with CHH methylation given the large siRNA production in the hybrids, TCM is absent or severely reduced in number of loci showing decreased siRNA levels [80]. the CHG and CHH contexts and there is only a low level in the CG It is possible that TCM and TCdM are extremes of the same process context [82]. This suggests that siRNAs are involved directly. where siRNA produced from the hypermethylated parent associate Trans chromosomal methylation in the hybrids is reminiscent of with both parental loci in the hybrids. In TCM, siRNAs levels across the re-methylation of hypomethylated loci in ddm1 and met1 both alleles remain above the threshold levels required to maintain recombinant inbred lines (RILs) [67,68,101]. Within the ddm1-derived and initiate methylation, and could initiate siRNA production through epi-RILs, the hypermethylated wild type and hypomethylated ddm1 a feed-forward loop. In TCdM, the siRNAs levels across both alleles fall epi-loci resemble differentially methylated parental alleles which below threshold levels causing decreased methylation of the hyper- frequently exhibit TCM in the hybrids [82, Greaves et al., unpub- methylated parental loci, affecting the feed-forward loop needed to lished]. Two categories of loci were identified within the ddm1-derived maintain siRNA production at the once hypermethylated locus (Fig. 1) epi-RILSs: (i) “non-remethylatable” loci, which are similar to loci in [8,68,82,Greaves et al., unpublished]. hybrids showing additive inheritance of methylation. (ii) “remethylatable loci” which show non-additive siRNA-dependent increased levels 21. Loss of hybrid vigour beyond the F1 generation of methylation (i.e. TCM). In the met1-derived epi-RILs changes do occur in the F1 hybrids as well as over subsequent generations [68], paralleling The degree of heterosis in the F1 is decreased in subsequent ge- the situation in the hybrids. nerations, possibly by segregation of the heterosis promoting allelic The presence of siRNA alone is not enough to ensure TCM as not all combinations. However, if epi-alleles are involved, then the decrease hypermethylated siRNA-associated loci in the hybrids or epi-RILs in heterosis is due to more than simple Mendelian segregation. The exhibit trans-chromosomal methylation [82,101]. It is likely the dynamic nature of the epigenetic mechanisms at play in the hybrids abundance of siRNAs at the hypermethylated locus and the chromatin suggests that any heterosis promoting epi-alleles would not only state of the hypomethylated recipient locus are important factors for segregate from each other, but the epi-alleles themselves could change TCM in hybrids. Support for this comes from the well studied through the same mechanisms responsible for producing the altered FLOWERING WAGENINGEN A (FWA) gene from Arabidopsis. FWA is F1 epigenome. For instance, loci with reduced levels of 24-nt siRNAs or transcriptionally silenced by methylation of repeat elements in the DNA methylation in the F1 may show increased levels in subsequent promoter and 5′ UTR [102]. siRNAs are associated with the methylated generations as has been reported for inter-species Arabidopsis hybrids region and can confer TCM on a newly integrated transgene with no and epi-RILs, respectively [67,68,81,101]. In addition, further methyl- chromatin marks [103–105]. However, if the transgene is transcrip- ation or de-methylation may occur through trans-effects altering the tionally active before being introduced to the siRNA from the newly segregated epi-alleles, changing their heterosis promoting hypermethylated siRNA producing FWA, it will not be trans-chromo- expression. somally methylated [104]. This suggests that a highly active chromatin In the epi-RILs, little change in TCM can be detected during the state hinders siRNA directed TCM. However, silencing of highly vegetative phase [106]. It is likely that the hybrids are similar, sug- transcribed fwa epi-alleles can be forced by the production of large gesting that the epigenetic alterations are restricted to the reproduc- amounts of siRNA [102], implying that the required threshold of siRNA tive phase where large-scale re-organising of the epigenome occurs abundance for TCM may be determined by the chromatin state of the [107]. recipient locus. A similar conclusion can be drawn from recent work on the paramutagenic b1 locus which shows siRNAs are not only associated with the silenced B' allele but also with the active B-I epi- 22. Concluding remarks allele [100]. Presumably the active chromatin state of B-I impairs silencing. This can be overcome by increasing the levels of siRNA [100]. The DNA molecules of the genome are in partnership with other The implication for the hybrids is that not all loci that are dif- molecules to form the chromatin within the nucleus. Modifications of ferentially methylated between parents will show non-additive the chromatin histones alter packaging of DNA, influencing local increases in methylation through TCM. The occurrence of TCM in region architecture and providing a go or no-go state for transcription. the F1 hybrids is likely to be dependent on (i) the presence and Initially, DNA methylation and histone modification were dis- abundance of siRNA and (ii) the chromatin state of the hypomethy- cussed mainly in relation to animal genomes, but by the end of the lated parental locus (Fig. 1). 1980s it was realized that plant genomes had these same epigenomic interventions influencing gene activity. Other epigenetic controls 20. Decreased methylation in hybrids is predominantly due to soon became evident, most importantly in the advent of the small reduced siRNA levels RNA era. Small RNAs, either as micro RNAs (miRNAs), involved in the control of gene action in development, or small interfering RNAs Rice and Arabidopsis hybrids show a significant proportion of loci (siRNAs) involved in defence against virus infection, and in the con- with reductions in DNA methylation towards the low-parent level. trol of gene activity are critical epigenetic molecules. 436 M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437

When the double helix structure of DNA was described, the [20] A.R. Gendall, Y.Y. Levy, A. Wilson, C. Dean, The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis, Cell 107 (2001) 525–535. complementarity of the sequence of the two chains of nucleotides was [21] R. Bastow, J.S. Mylne, C. Lister, Z. Lippman, R.A. Martienssen, C. Dean, Verna- recognized as being critical for its replication and transcription. Se- lization requires epigenetic silencing of FLC by histone methylation, Nature 427 quence complementarity is also basic to the RNA control of the activity (2004) 164–167. [22] M. Tadege, C.C. Sheldon, C.A. Helliwell, P. Stoutjesdijk, E.S. Dennis, W.J. Peacock, state of genes. The RNA molecules specify the regions where histone Control of flowering time by FLC orthologues in Brassica napus, Plant J. 28 (2001) modifications or DNA methylation will occur by targeting protein com- 545–553. plexes to these regions to effect epigenetic control of the gene activity. [23] P.A. Reeves, Y.H. He, R.J. Schmitz, R.M. Amasino, L.W. Panella, C.M. Richards, The epigenetic control of gene activity in the development of the Evolutionary conservation of the FLOWERING LOCUS C-mediated vernalization response: evidence from the sugar beet (Beta vulgaris), Genetics 176 (2007) organism and the function of the genome are of ubiquitous importance 295–307. in biology. [24] L. Yan, A. Loukoianov, G. Tranquilli, M. Helguera, T. Fahima, J. Dubcovsky, Positional cloning of the wheat vernalization gene VRN1, Proc. Natl. Acad. Sci. USA 100 (2003) 6263–6268. Acknowledgments [25] B. Trevaskis, D.J. Bagnall, M.H. Ellis, W.J. Peacock, E.S. Dennis, MADS box genes control vernalization-induced flowering in cereals, Proc. Natl. Acad. Sci. USA 100 (2003) 13099–13104. This work was supported by National Collaborative Research [26] I. Searle, Y.H. He, F. Turck, C. Vincent, F. Fornara, S. Krober, R.A. Amasino, G. Infrastructure Strategy (NCRIS) and Science and Industry Endowment Coupland, The transcription factor FLC confers a flowering response to ver- Fund (SIEF) funded by the Australian Government, and by the Agri- nalization by repressing meristem competence and systemic signaling in Arabidopsis, Genes Dev. 20 (2006) 898–912. cultural Genomics Centre, New South Wales Government BioFirst [27] C.A. Helliwell, C.C. Wood, M. Robertson, W.J. Peacock, E.S. Dennis, The Arabidopsis initiative FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a Ryo Fujimoto was supported by a Japan Society for the Promotion high-molecular-weight protein complex, Plant J. 46 (2006) 183–192. [28] R.B. Deal, C.N. Topp, E.C. McKinney, R.B. Meagher, Repression of flowering in of Science Overseas Fellowship. Arabidopsis requires activation of FLOWERING LOCUS C expression by the We wish to thank Nerida Gibb for excellent manuscript preparation. histone variant H2A.Z, Plant Cell 19 (2007) 74–83. [29] E.J. Finnegan, E.S. Dennis, Vernalization-induced trimethylation of histone H3 lysine 27 at FLC is not maintained in mitotically quiescent cells, Curr. Biol. 17 References (2007) 1978–1983. [30] N. Ohad, L. Margossian, Y.C. Hsu, C. Williams, P. Repetti, R.L. Fischer, A mutation [1] C.Z. Jiang, B.F. Pugh, Nucleosome positioning and gene regulation: advances that allows endosperm development without fertilization, Proc. Natl. Acad. Sci. through genomics, Nat. Rev. Genet. 10 (2009) 161–172. USA 93 (1996) 5319–5324. [2] K. Chodavarapu, S.H. Feng, Y.V. Bernatavichute, P.Y. Chen, H. Stroud, Y.C. Yu, J.A. [31] S.B. Sung, R.M. Amasino, Vernalization in Arabidopsis thaliana is mediated by the Hetzel, F. Kuo, J. Kim, S.J. Cokus, D. Casero, M. Bernal, P. Huijser, A.T. Clark, U. PHD finger protein VIN3, Nature 427 (2004) 159–164. Kramer, S.S. Merchant, X.O. Zhang, S.E. Jacobsen, M. Pellegrini, Relationship [32] C.C. Wood, M. Robertson, G. Tanner, W.J. Peacock, E.S. Dennis, C.A. Helliwell, The between nucleosome positioning and DNA methylation ramakrishna, Nature Arabidopsis thaliana vernalization response requires a polycomb-like protein 466 (2010) 388–392. complex that also includes VERNALIZATION INSENSITIVE 3, Proc. Natl. Acad. Sci. [3] A.D. Riggs, X-inactivation, differentiation, and DNA methylation, Cytogenet. Cell USA 103 (2006) 14631–14636. Genet. 14 (1975) 9–25. [33] D.M. Buzas, R. M., E.J. Finnegan, C.A. Helliwel, Transcription-dependence of [4] R. Holliday, J.E. Pugh, DNA modification mechanisms and gene activity during histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC, development, Science 187 (1975) 226–232. Plant J. (2011) DOI: 10.1111/j.1365-313X.2010.04471.x. [5] J.A. Law, S.E. Jacobsen, Establishing, maintaining and modifying DNA methyla- [34] C.C. Sheldon, A.B. Conn, E.S. Dennis, W.J. Peacock, Different regulatory regions are tion patterns in plants and animals, Nat. Rev. Genet. 11 (2010) 204–220. required for the vernalization-induced repression of FLOWERING LOCUS C and [6] M. Matzke, T. Kanno, B. Huettel, L. Daxinger, A.J.M. Matzke, Targets of RNA- for the epigenetic maintenance of repression, Plant Cell 14 (2002) 2527–2537. directed DNA methylation, Curr. Opin. Plant Biol. 10 (2007) 512–519. [35] D. Schubert, L. Primavesi, A. Bishopp, G. Roberts, J. Doonan, T. Jenuwein, J. Goodrich, [7] X.Y. Zhang, J. Yazaki, A. Sundaresan, S. Cokus, S.W.L. Chan, H.M. Chen, I.R. Silencing by plant Polycomb-group genes requires dispersed trimethylation of Henderson, P. Shinn, M. Pellegrini, S.E. Jacobsen, J.R. Ecker, Genome-wide high- histone H3 at lysine 27, EMBO J. 25 (2006) 4638–4649. resolution mapping and functional analysis of DNA methylation in Arabidopsis, [36] S.J. Wellensiek, The effect of periodic dark interruptions in long photoperiods on Cell 126 (2006) 1189–1201. floral induction in the long-day plants Silene-armeria l, annual Lunaria-annua l [8] R. Lister, R.C. O'Malley, J. Tonti-Filippini, B.D. Gregory, C.C. Berry, A.H. Millar, J.R. and Samolus-parviflorus raf, J. Plant Physiol. 117 (1984) 257–265. Ecker, Highly integrated single-base resolution maps of the epigenome in [37] R. Margueron, D. Reinberg, The Polycomb complex PRC2 and its mark in life, Arabidopsis, Cell 133 (2008) 523–536. Nature 469 (2011) 343–349. [9] R. Lister, M. Pelizzola, R.H. Dowen, R.D. Hawkins, G. Hon, J. Tonti-Filippini, J.R. [38] E.J. Finnegan, C.C. Sheldon, F. Jardinaud, W.J. Peacock, W.S. Dennis, A cluster of Nery, L. Lee, Z. Ye, Q.M. Ngo, L. Edsall, J. Antosiewicz-Bourget, R. Stewart, V. Arabidlopsis genes with a coordinate response to an environmental stimulus, Ruotti, A.H. Millar, J.A. Thomson, B. Ren, J.R. Ecker, Human DNA methylomes at Curr. Biol. 14 (2004) 911–916. base resolution show widespread epigenomic differences, Nature 462 (2009) [39] C.C. Sheldon, E.J. Finnegan, W.J. Peacock, E.S. Dennis, Mechanisms of gene 315–322. repression by vernalization in Arabidopsis, Plant J. 59 (2009) 488–498. [10] D. Schwartz, E. Dennis, Transposase activity of the ac controlling element in [40] C.C. Sheldon, M.J. Hills, C. Lister, C. Dean, E.S. Dennis, W.J. Peacock, Resetting of maize is regulated by its degree of methylation, Mol. Gen. Genet. 205 (1986) FLOWERING LOCUS C expression after epigenetic repression by vernalization, 476–482. Proc. Natl. Acad. Sci. USA 105 (2008) 2214–2219. [11] J.A. Banks, P. Masson, N. Fedoroff, Molecular mechanisms in the developmental [41] J. Choi, Y. Hyun, M.J. Kang, H. Yun, J.Y. Yun, C. Lister, C. Dean, R.M. Amasino, B. Noh, Y.S. regulation of the maize suppressor-mutator transposable element, Genes Dev. 2 Noh, Y. Choi, Resetting and regulation of FLOWERING LOCUS C expression during (1988) 1364–1380. Arabidopsis reproductive development, Plant J. 57 (2009) 918–931. [12] J.E. Burn, D.J. Bagnall, J.D. Metzger, E.S. Dennis, W.J. Peacock, DNA methylation, [42] S. Swiezewski, F.Q. Liu, A. Magusin, C. Dean, Cold-induced silencing by long vernalization, and the initiation of flowering, Proc. Natl. Acad. Sci. USA 90 (1993) antisense transcripts of an Arabidopsis Polycomb target, Nature 462 (2009) 287–291. 799–802. [13] R.D. Brock, J.L. Davidson, 5-azacytidine and gamma-rays partially substitute for [43] J.B. Heo, S. Sung, Vernalization-mediated epigenetic silencing by a long intronic cold treatment in vernalizing winter-wheat, Environ. Exp. Botany 34 (1994) noncoding RNA, Science 331 (2011). 195–199. [44] S.B. Sung, Y.H. He, T.W. Eshoo, Y. Tamada, L. Johnson, K. Nakahigashi, K. Goto, S.E. [14] E.J. Finnegan, W.J. Peacock, E.S. Dennis, Reduced DNA methylation in Arabidopsis Jacobsen, R.M. Amasino, Epigenetic maintenance of the vernalized state in thaliana results in abnormal plant development, Proc. Natl. Acad. Sci. USA 93 Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1, Nat. Genet. (1996) 8449–8454. 38 (2006) 706–710. [15] K. Napp-Zinn, Vernalization—environmental and genetic regulation, Manipula- [45] J.L. Rinn, M. Kertesz, J.K. Wang, S.L. Squazzo, X. Xu, S.A. Brugmann, L.H. tion flowering (1987) 123–132. Goodnough, J.A. Helms, P.J. Farnham, E. Segal, H.Y. Chang, Functional demarca- [16] M. Koornneef, C.J. Hanhart, J.H. Vanderveen, A genetic and physiological analysis tion of active and silent chromatin domains in human HOX loci by Noncoding of late flowering mutants in Arabidopsis-thaliana, Mol. Gen. Genet. 229 (1991) RNAs, Cell 129 (2007) 1311–1323. 57–66. [46] C. Hornyik, L.C. Terzi, G.G. Simpson, The spen family protein FPA controls [17] J.E. Burn, D.R. Smyth, W.J. Peacock, E.S. Dennis, Genes conferring late flowering in alternative cleavage and polyadenylation of RNA, Dev. Cell 18 (2010) 203–213. Arabidopsis-thaliana, Genetica 90 (1993) 147–155. [47] F.Q. Liu, V. Quesada, P. Crevillen, I. Baurle, S. Swiezewski, C. Dean, The Arabidopsis [18] C.C. Sheldon, J.E. Burn, P.P. Perez, J. Metzger, J.A. Edwards, W.J. Peacock, E.S. RNA-Binding protein FCA requires a lysine-specific demethylase 1 homolog to Dennis, The FLF MADS box gene: a repressor of flowering in Arabidopsis downregulate FLC, Mol. Cell 28 (2007) 398–407. regulated by vernalization and methylation, Plant Cell 11 (1999) 445–458. [48] I. Baurle, L. Smith, D.C. Baulcombe, C. Dean, Widespread role for the flowering- [19] S.D. Michaels, R.M. Amasino, FLOWERING LOCUS C encodes a novel MADS domain time regulators FCA and FPA in RNA-mediated chromatin silencing, Science 318 protein that acts as a repressor of flowering, Plant Cell 11 (1999) 949–956. (2007) 109–112. M. Groszmann et al. / Biochimica et Biophysica Acta 1809 (2011) 427–437 437

[49] T. Greb, J.S. Mylne, P. Crevillen, N. Geraldo, H.L. An, A.R. Gendall, C. Dean, The PHD ization between Oryza sativa and O. officinalis, TAG, Theor. Appl. Genet. 117 finger protein VRN5 functions in the epigenetic silencing of Arabidopsis FLC, (2008) 1271–1279. Curr. Biol. 17 (2007) 73–78. [80] G.M. He, X.P. Zhu, A.A. Elling, L.B. Chen, X.F. Wang, L. Guo, M.Z. Liang, H. He, H.Y. [50] A.T. Pugsley, Genetic analysis of spring-winter habit of growth in wheat, Australian Zhang, F.F. Chen, Y.J. Qi, R.S. Chen, X.W. Deng, Global epigenetic and J. Agricultural Research 22 (1971) 21–33. transcriptional trends among two rice subspecies and their reciprocal hybrids, [51] R. Takahashi, S. Yasuda, Genetics of carliness and growth habit in barley, Barley Plant Cell 22 (2010) 17–33. genetics II. Proceedings of Second International Barley Genetics Symposium. [81] M. Ha, J. Lu, L. Tian, V. Ramachandran, K.D. Kasschau, E.J. Chapman, J.C. Washington State University, Pulman, 1970, (1971) 388–408 pp. Carrington, X.M. Chen, X.J. Wang, Z.J. Chen, Small RNAs serve as a genetic buffer [52] E.S. Dennis, W.J. Peacock, Epigenetic regulation of flowering, Curr. Opin. Plant against genomic shock in Arabidopsis interspecific hybrids and allopolyploids, Biol. 10 (2007) 520–527. Proc. Natl. Acad. Sci. USA 106 (2009) 17835–17840. [53] I. Marcinska, F. Dubert, J. Biesagakoscielniak, Transfer of the ability to flower in [82] M. Groszmann, I.K. Greaves, Z.I. Albertyn, G.N. Scofield, W.J. Peacock, E.S. Dennis, winter-wheat via callus-tissue regenerated from immature inflorescences, Plant Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic Cell Tissue Organ Cult. 41 (1995) 285–288. contribution to hybrid vigor, Proc. Natl. Acad. Sci. USA 108 (2011) 2617–2622. [54] L.L. Yan, A. Loukoianov, A. Blechl, G. Tranquilli, W. Ramakrishna, P. SanMiguel, J.L. [83] I. Makarevitch, R.M. Stupar, A.L. Iniguez, W.J. Haun, W.B. Barbazulk, S.M. Bennetzen, V. Echenique, J. Dubcovsky, The wheat VRN2 gene is a flowering Kaeppler, N.M. Springer, Natural variation for Alleles under epigenetic control by repressor down-regulated by vernalization, Science 303 (2004) 1640–1644. the maize chromomethylase Zmet2, Genetics 177 (2007) 749–760. [55] L. Yan, D. Fu, C. Li, A. Blechl, G. Tranquilli, M. Bonafede, A. Sanchez, M. Valarik, S. [84] C.F. Lira-Medeiros, C. Parisod, R.A. Fernandes, C.S. Mata, M.A. Cardoso, P.C.G. Yasuda, J. Dubcovsky, The wheat and barley vernalization gene VRN3 is an Ferreira, Epigenetic variation in mangrove plants occurring in contrasting natural orthologue of FT, Proc. Natl. Acad. Sci. USA 103 (2006) 19581–19586. environment, PLoS One 5 (2010). [56] M.N. Hemming, W.J. Peacock, E.S. Dennis, B. Trevaskis, Low-temperature and [85] Z.B. Lippman, D. Zamir, Heterosis: revisiting the magic, Trends Genet. 23 (2007) daylength cues are integrated to regulate FLOWERING LOCUS T in barley, Plant 60–66. Physiol. 147 (2008) 355–366. [86] F. Hochholdinger, N. Hoecker, Towards the molecular basis of heterosis, Trends [57] S.N. Oliver, E.J. Finnegan, E.S. Dennis, W.J. Peacock, B. Trevaskis, Vernalization- Plant Sci. 12 (2007) 427–432. induced flowering in cereals is associated with changes in histone methylation at [87] J.A. Birchler, H. Yao, S. Chudalayandi, D. Vaiman, R.A. Veitia, Heterosis, Plant Cell the VERNALIZATION1 gene, Proc. Natl. Acad. Sci. USA 106 (2009) 8386–8391. 22 (2010) 2105–2112. [58] M. Luo, D. Platten, A. Chaudhury, W.J. Peacock, E.S. Dennis, Expression, im- [88] T. Takamiya, S. Hosobuchi, T. Noguchi, K. Asai, E. Nakamura, Y. Habu, A.H. printing, and evolution of rice homologs of the polycomb group genes, Mol. Plant Paterson, H. Iijima, Y. Murakami, H. Okuizumi, Inheritance and alteration of 2 (2009) 711–723. genome methylation in F1 hybrid rice, Electrophoresis 29 (2008) 4088–4095. [59] D. Vasanthi, R.K. Mishra, Epigenetic regulation of genes during development: a [89] X.X. Zhao, Y. Chai, B. Liu, Epigenetic inheritance and variation of DNA conserved theme from flies to mammals, J. Genet. Genomics 35 (2008) 413–429. methylation level and pattern in maize intra-specific hybrids, Plant Science [60] S.H. Feng, S.E. Jacobsen, W. Reik, Epigenetic Reprogramming in Plant and Animal 172 (2007) 930–938. Development, Science 330 (2010) 622–627. [90] K. Sakthivel, K. Girishkumar, G. Ramkumar, V.V. Shenoy, S.T. Kajjidoni, P.M. [61] S.C. Wu, Y. Zhang, Active DNA demethylation: many roads lead to Rome, Nat. Salimath, Alterations in inheritance pattern and level of cytosine DNA methylation, Rev. Mol. Cell Biol. 11 (2010) 607–620. and their relationship with heterosis in rice, Euphytica 175 (2010) 303–314. [62] M.J. Ronemus, M. Galbiati, C. Ticknor, J.C. Chen, S.L. Dellaporta, Demethylation- [91] L.Z. Xiong, C.G. Xu, M.A.S. Maroof, Q.F. Zhang, Patterns of cytosine methylation in induced developmental pleiotropy in Arabidopsis, Science 273 (1996) 654–657. an elite rice hybrid and its parental lines, detected by a methylation-sensitive [63] M.W. Kankel, D.E. Ramsey, T.L. Stokes, S.K. Flowers, J.R. Haag, J.A. Jeddeloh, N.C. amplification polymorphism technique, Mol. Gen. Genet. 261 (1999) 439–446. Riddle, M.L. Verbsky, E.J. Richards, Arabidopsis MET1 cytosine methyltransferase [92] L.N. Lukens, J.C. Pires, E. Leon, R. Vogelzang, L. Oslach, T. Osborn, Patterns of mutants, Genetics 163 (2003) 1109–1122. sequence loss and cytosine methylation within a population of newly resyn- [64] H. Saze, O.M. Scheid, J. Paszkowski, Maintenance of CpG methylation is essential for thesized Brassica napus allopolyploids, Plant Physiol. 140 (2006) 336–348. epigenetic inheritance during plant gametogenesis, Nat. Genet. 34 (2003) 65–69. [93] M.S. Zhang, H.Y. Yan, N. Zhao, X.Y. Lin, J.S. Pang, K.Z. Xu, L.X. Liu, B. Liu, [65] Y.H. Moon, L.J. Chen, R.L. Pan, H.S. Chang, T. Zhu, D.M. Maffeo, Z.R. Sung, EMF Endosperm-specific hypomethylation, and meiotic inheritance and variation of genes maintain vegetative development by repressing the flower program in DNA methylation level and pattern in sorghum (Sorghum bicolor L.) inter-strain Arabidopsis, Plant Cell 15 (2003) 681–693. hybrids, Theor. Appl. Genet. 115 (2007) 195–207. [66] S.Y. Kim, T. Zhu, Z.R. Sung, Epigenetic regulation of gene programs by EMF1 and [94] A.M.B. Moghaddam, J. Fuchs, T. Czauderna, A. Houben, M.F. Mette, Intraspecific EMF2 in Arabidopsis, Plant Physiol. 152 (2010) 516–528. hybrids of Arabidopsis thaliana revealed no gross alterations in endopolyploidy, [67] F. Johannes, E. Porcher, F.K. Teixeira, V. Saliba-Colombani, M. Simon, N. Agier, A. DNA methylation, histone modifications and transcript levels, TAG Theoretical Bulski, J. Albuisson, F. Heredia, P. Audigier, D. Bouchez, C. Dillmann, P. Guerche, F. Applied Genetics 120 (2010) 215–226. Hospital, V. Colot, Assessing the impact of transgenerational epigenetic variation [95] M. Stam, Paramutation: a heritable change in gene expression by allelic inter- on complex traits, PLoS Genet. 5 (2009). actions in trans, Mol. Plant 2 (2009) 578–588. [68] J. Reinders, B.B.H. Wulff, M. Mirouze, A. Mari-Ordonez, M. Dapp, W. Rozhon, E. [96] V.L. Chandler, Paramutation's properties and puzzles, Science 330 (2010) 628–629. Bucher, G. Theiler, J. Paszkowski, Compromised stability of DNA methylation and [97] M.A. Arteaga-Vazquez, V.L. Chandler, Paramutation in maize: RNA mediated transposon immobilization in mosaic Arabidopsis epigenomes, Genes Dev. 23 trans-generational gene silencing, Curr. Opin. Genet. Dev. 20 (2010) 156–163. (2009) 939–950. [98] M. Stam, C. Belele, J.E. Dorweiler, V.L. Chandler, Differential chromatin structure [69] K. Manning, M. Tor, M. Poole, Y. Hong, A.J. Thompson, G.J. King, J.J. Giovannoni, G.B. within a tandem array 100 kb upstream of the maize b1 locus is associated with Seymour, A naturally occurring epigenetic mutation in a gene encoding an SBP-box paramutation, Genes Dev. 16 (2002) 1906–1918. transcription factor inhibits tomato fruit ripening, Nat. Genet. 38 (2006) 948–952. [99] M. Louwers, R. Bader, M. Haring, R. van Driel, W. de Laat, M. Stam, Tissue- and [70] P. Cubas, C. Vincent, E. Coen, An epigenetic mutation responsible for natural expression level-specific chromatin looping at Maize b1 epialleles, Plant Cell 21 variation in floral symmetry, Nature 401 (1999) 157–161. (2009) 832–842. [71] S.E. Jacobsen, E.M. Meyerowitz, Hypermethylated SUPERMAN epigenetic alleles [100] M. Arteaga-Vazquez, L. Sidorenko, F.A. Rabanal, R. Shrivistava, K. Nobuta, P.J. in Arabidopsis, Science 277 (1997) 1100–1103. Green, B.C. Meyers, V.L. Chandler, RNA-mediated trans-communication can [72] H. Huang, H. Ma, The nature of the Arabidopsis fon1 mutations, Plant Cell 10 (1998) 3–4. establish paramutation at the b1 locus in maize, Proc. Natl. Acad. Sci. USA 107 [73] C.L. Richards, O. Bossdorf, K.J.F. Verhoeven, Understanding natural epigenetic (2010) 12986–12991. variation, New Phytol. 187 (2010) 562–564. [101] F.K. Teixeira, F. Heredia, A. Sarazin, F. Roudier, M. Boccara, C. Ciaudo, C. Cruaud, J. [74] C.M. Herrera, P. Bazaga, Epigenetic differentiation and relationship to adaptive Poulain, M. Berdasco, M.F. Fraga, O. Voinnet, P. Wincker, M. Esteller, V. Colot, A genetic divergence in discrete populations of the violet Viola cazorlensis, New role for RNAi in the selective correction of DNA methylation defects, Science 323 Phytol. 187 (2010) 867–876. (2009) 1600–1604. [75] Y.D. Li, X.H. Shan, X.M. Liu, L.J. Hu, W.L. Guo, B. Liu, Utility of the methylation- [102] Y. Kinoshita, H. Saze, T. Kinoshita, A. Miura, W.J.J. Soppe, M. Koornneef, T. sensitive amplified polymorphism (MSAP) marker for detection of DNA Kakutani, Control of FWA gene silencing in Arabidopsis thaliana by SINE-related methylation polymorphism and epigenetic population structure in a wild barley direct repeats, Plant J. 49 (2007) 38–45. species (Hordeum brevisubulatum), Ecol. Res. 23 (2008) 927–930. [103] X.F. Cao, S.E. Jacobsen, Role of the Arabidopsis DRM methyltransferases in de [76] M.W. Vaughn, M. Tanurdzic, Z. Lippman, H. Jiang, R. Carrasquillo, P.D. Rabinowicz, N. novo DNA methylation and gene silencing, Curr. Biol. 12 (2002) 1138–1144. Dedhia, W.R. McCombie, N. Agier, A. Bulski, V. Colot, R.W. Doerge, R.A. Martienssen, [104] S.W.L. Chan, D. Zilberman, Z.X. Xie, L.K. Johansen, J.C. Carrington, S.E. Jacobsen, Epigenetic natural variation in Arabidopsis thaliana, PLoS Biol. 5 (2007) 1617–1629. RNA silencing genes control de novo DNA methylation, Science 303 (2004) 1336. [77] X. Zhang, S. Shiu, A. Cal, J.O. Borevitz, Global analysis of genetic, epigenetic and [105] S.W.L. Chan, X.Y. Zhang, Y.V. Bernatavichute, S.E. Jacobsen, Two-step recruitment transcriptional polymorphisms in Arabidopsis thaliana using whole genome of RNA-directed DNA methylation to tandem repeats, PLoS Biol. 4 (2006) tiling arrays, PLoS Genet. 4 (2008). 1923–1933. [78] J.X. Zhai, J. Liu, B. Liu, P.C. Li, B.C. Meyers, X.M. Chen, X.F. Cao, Small RNA-directed [106] F.K. Teixeira, V. Colot, Repeat elements and the Arabidopsis DNA methylation epigenetic natural variation in Arabidopsis thaliana, PLoS Genet. 4 (2008). landscape, Heredity 105 (2010) 14–23. [79] H. Jin, W. Hu, Z. Wei, L. Wan, G. Li, G. Tan, L. Zhu, G. He, Alterations in cytosine [107] R.A. Mosher, C.W. Melnyk, siRNAs and DNA methylation: seedy epigenetics, methylation and species-specific transcription induced by interspecific hybrid- Trends Plant Sci. 15 (2010) 204–210.