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Epigenetics: The Second Genetic Code

Nathan M. Springer* and Shawn M. Kaeppler†

Contents

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Agronomy, Volume 100 # 2008

-2113, DOI: 10.1016/S0065-2113(08)00603-2 All rig

ent of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108, USAnt of Agronomy, University of Wisconsin, Madison, Wisconsin 53706, USA

Else

hts

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2. M
olecular Mechanisms of Epigenetic Inheritance 60
2
.1. D NA methylation 61
2
.2. H istone modifications 62
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.3. C hromatin structure 63
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.4. R ole of RNA in heritable silencing 64
2
.5. In teractions among DNA methylation, histone modifications,
and chromatin structure
65
3. E
pigenetic Phenomena in Plants 65
3
.1. P henotypic examples of epigenetic inheritance 66
3
.2. G enomic and molecular genetic examples
of epigenetic variation
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4. E
pigenetic Inheritance and Crop Improvement 71
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.1. E pigenetics in quantitative inheritance and selection response 72
4
.2. E pialleles and gene discovery 73
Refe
rences 73
Abstract

Plant breeders utilize directed selection and transgenics to produce novel

cultivars of diploid and polyploid species. DNA sequence is clearly important

in these processes, but growing evidence implicates epigenetics as an impor-

tant factor in controlling genetic variation and gene/transgene expression. In

this article, we focus on epigenetic variation defined as mitotically and meioti-

cally heritable but reversible states of gene expression that are not conditioned

by differences in DNA sequence. We summarize mechanisms underlying epige-

netic states of expression, and discuss implications of epigenetics in cultivar

development.

vier Inc.

reserved.

59

60 Nathan M. Springer and Shawn M. Kaeppler

1. Introduction

Plant breeders have made tremendous progress towards altering plantphenotypes to provide more productive crops. The majority of these gainshave been made through selection of variation that is present within aspecies. While it is clear that phenotypic selection has resulted in improvedplant characteristics, the molecular basis of the selected variation remainslargely unknown. Traditionally, it was assumed that the majority of herita-ble variation was due to genetic sequence differences. However, a growingbody of evidence suggests that intraspecific expression differences amonggenotypes can also be caused by epigenetic variation. To some researchers,epigenetics is used as a catchall term to describe any variation that does notseem to follow Mendelian inheritance patterns. In this article, we use theterm epigenetics to specifically describe meiotically or mitotically heritabledifferences in gene expression that are not caused by sequence differences.We will begin by describing some of the molecular changes that areassociated with epigenetic variation and then proceed to discussing someof the well characterized examples of epigenetic variation. Finally, we willdiscuss the potential impact of epigenetic inheritance in crop improvement.

2. Molecular Mechanisms of Epigenetic

Inheritance

Epigenetics involves changes in heritable phenotypes without changesin DNA sequence. With the exception of protein-based inheritance such asprions, epigenetic differences are the result of altered gene expression levels.Research on the molecular mechanisms of epigenetic inheritance has iden-tified several complementary pathways for the stable regulation of geneexpression without sequence changes.

The basis of epigenetic inheritance is the manner in which DNA ispackaged and modified. DNA in plant cells contains four standard bases,cytosine, guanine, thymine, and adenine, but can also contain the modifiedbase 5-methylcytosine. Most often, presence of 5-methylcytosine is asso-ciated with repressed gene expression. DNA in the cell is packaged byvarious sets of proteins. The first level of packaging involves wrappingDNA around cores of histone octamers containing two each of the proteins:Histone 2A (H2A), Histone 2B (H2B), Histone 3 (H3), and Histone 4 (H4).The histone proteins in the octamer can contain various modifications. Themost relevant modifications for epigenetic expression are acetylation andmethylation, which occur on the tails of H3 and H4. The tails of thesehistones extend outside of the DNA/protein core, and the modificationsaffect how those cores are packaged into a higher order structure.

Epigenetics: The Second Genetic Code 61

Various proteins are involved in methylation of the DNA, modificationof the histones, recognition of chromatin state, and in energy-dependentremodeling of one chromatin state to another. Following is a brief summaryof some of the most important players in this process, chosen largely becauseone or more members have proven effects on gene expression in plants.

2.1. DNA methylation

DNA methylation is found in the genomes of many plant and animalspecies. In eukaryotic genomes, DNA methylation refers primarily to5-methylcytosine although some evidence for methylated adenines hasbeen reported. The methyl moiety is added to cytosine residues present indouble stranded DNA by a group of enzymes referred to as DNA methyl-transferases. The majority of DNA methylation is found in CpG dinucleo-tide (plants and animals) and CpHpG trinucleotide (plants only) sequencecontexts. Plant genomes encode several different DNA methyltransferaseenzymes that fall into three different functional categories (reviewed byChan et al., 2005). The Domains Rearranged Methyltransferases (DRM)encode de novo methyltransferases. These enzymes are capable of addingmethyl groups to DNA that is unmethylated. The other two categories,DNA methyltransferases (MET) and chromomethylases (CMT), encodemaintenance methyltransferases. Following DNA replication, the parentstrand retains 5-methylcytosine but all cytosines within the daughter strandare unmethylated. This hemimethylated substrate is the target of the main-tenance methyltransferases. Hemimethylated CpGs are the target of theMET class of enzymes while hemimethylated CpHpGs are methylated byCMTs. The targeting of a DRM protein to a specific genomic location willresult in methylation of all cytosines within the region. If the targeting signalis no longer present then only the CpG and CpHpG methylation will bemaintained by maintenance activities. These are likely oversimplifications ofthe activities and preferences for these enzymes as there appears to be someredundancy and locus-specific activities for these classes (reviewed by Chanet al., 2005).

In general, DNA methylation is associated with transcriptional silencingof a locus. DNA methylation can result in silencing by directly interferingwith the binding of transcriptional activators or by recruiting proteins thatbind to methylated DNA and recruit transcriptional repressors (Bird andWolffe, 1999; Klose and Bird, 2006). DNA methylation is often associatedwith centromeres and repetitive elements (Zhang et al., 2006). Recently,the application of microarrays and high-throughput sequencing approacheshave provided a view of genome-wide DNA methylation patterns in Arabi-dopsis and the relationship of DNA methylation and gene expression (Cokuset al., 2008; Lister et al., 2008; Zhang et al., 2006; Zilberman et al., 2007).DNA methylation is quite high in transposon sequences and loss of CpG


methylation often results in transcriptional activation of these sequences. Twodifferent types of genic methylation were noted (Lister et al., 2008; Zhanget al., 2006; Zilberman et al., 2007). A significant proportion of genes (�33%)exhibit methylation within the coding region and a much smaller proportionof genes (�5%) exhibit promoter methylation. However, a relatively smallnumber of genes (�500) exhibit altered expressionwhenDNAmethylation isreduced (Zhang et al., 2006). The majority of genes which are sensitive toDNA methylation exhibit promoter methylation. Interestingly, most of thegenes controlled by CpG methylation are pseudogenes located within peri-centromeric heterochromatin while the genes regulated by CpHpGmethyla-tion are spread throughout euchromatic portions of the genome (Zhang et al.,2006). There is also evidence for altered expression of antisense and nc RNAsin plants with reduced DNAmethylation levels suggesting that DNAmethyl-ation may be required to reduce transcriptional ‘‘noise.’’

2.2. Histone modifications

The histone proteins exhibit remarkable sequence conservation in eukaryoteswithin the globular head domain that interacts with other histones and DNAas well as within the ‘‘tail’’ domain that protrudes from the central octomer–DNA complex. Recent research has identified a number of posttranslationalmodifications that occur to the histone tails including acetylation,methylation,SUMOlation, ubiquitination, and others (reviewed by Kouzarides, 2007;Pfluger andWagner, 2007). These histonemodifications can provide a varietyof functions including transcriptional activation, transcriptional repression,efficient assembly into chromatin, and DNA replication. The theory of ahistone code, in which each modification indicates a specific meaning andthe combinations of modifications result in interpretations of chromatin state,was proposed ( Jenuwein and Allis, 2001). However, most current researchsuggests that there are actually a limited number of chromatin states and thatthe many of the modifications can act in a redundant manner (Kouzarides,2007; Peterson and Laniel, 2004).

Histone modifications can have both direct and indirect effects upontranscription. The presence of modifications, such as acetylation, may affectthe ability of adjacent nucleosomes to interact. Histone modifications canalso provide binding sites for other proteins which in turn may act ascorepressors or coactivators (Jenuwein and Allis, 2001; Kouzarides, 2007;Peterson and Laniel, 2004). The epigenetic information of histone mod-ifications is generally thought to be less stable than that of DNA methyla-tion. The mechanisms for maintenance of DNA methylation patternsfollowing replication are well understood. However, the mechanisms formaintaining histone modification patterns following dispersive replicationof chromatin are unclear. In addition, most histone modifications are


reversible and the equilibrium for a particular locus is controlled by theaccess of the modifiers and demodifiers.

Recent studies have provided a genomic view of the distribution ofcertain histone modifications. Cytogenetic studies have documented thechromosomal distribution for several histone modifications (Baroux et al.,2007; Fuchs et al., 2006; Houben et al., 2003; Jackson et al., 2004;Jasencakova et al., 2003; Soppe et al., 2002). A higher level of resolutionhas been provided by studies that combine chromatin immunoprecipitationand microarray hybridization (Bernatavichute et al., 2008; Gendrel et al.,2005; Turck et al., 2007; Zhang et al., 2007). Trimethylation of lysine 27 ofhistone H3 (H3K27me3) is present at �18% of genes and is enriched attranscription factors and developmental regulators that are not expressed inthe tissues being studied (Zhang et al., 2007). H3K27me3was often enrichednear the promoter of genes. Interestingly, another histone modification thatis associated with silencing, H3K9me3, does not tend to colocalize withH3K27me3 (Turck et al., 2007). In general, H3K9me3 is more prevalent atconstitutive heterochromatin such as transposons while H3K27me3 isfound at loci with more dynamic tissue-specific regulation. The mutuallyexclusive presence of these modifications suggests two different types ofsilencing that can be conditioned by histone methylation.

2.3. Chromatin structure

Alterations to chromatin structure are also important in epigenetic regula-tion. Chromatin structure alterations can include chromatin structurechanges caused by histone variants (reviewed by Henikoff and Ahmad,2005; Williams and Tyler, 2007) or the physical remodeling of chromatinstructure. Histone variants are critical for the epigenetic definition ofcentromeres in plants (Dalal et al., 2007; Dawe and Henikoff, 2006;Zhang et al., 2008). In addition, histone variants are also involved inepigenetic processes such as vernalization and plant immunity (Deal et al.,2007;March-Diaz et al., 2008). Relatively little is known about the genomicdistribution of histone variants in plant cells. Studies on animal genomeshave revealed that the H3.3 variant histone is deposited in a replicationindependent manner at regions with active transcription (Mito et al., 2005).A recent study found differences in H3.3 distributions within the developingembryo and endosperm of Arabidopsis (Ingouff et al., 2007).

Chromatin structure can be altered by a family of enzymes that utilizeATP to physically alter chromatin (reviewed by Jerzmanowski, 2007; Kwonand Wagner, 2007). Arabidopsis encodes a large family of ATP-dependantchromatin remodeling enzymes ( Jerzmanowski, 2007; Verbsky andRichards, 2001). Several of these genes have been identified in geneticscreens for epigenetic or developmental regulators ( Jerzmanowski, 2007;Kwon and Wagner, 2007). Very little is known about how these chromatin


remodeling enzymes are targeted to specific genomic regions. However, itis clear that these chromatin remodeling activities are critical for epigeneticregulation. In most cases, the chromatin remodeling activities appear tobe important for gene regulation in response to environmental cues, notlong-term changes in expression (Kwon and Wagner, 2007). A model thatlinks histone modifications, histone variants and chromatin structure tonucleasome stability was recently proposed by Henikoff (2008).

2.4. Role of RNA in heritable silencing

Posttranscriptional gene silencing (PTGS) or RNAi is a well-characterizedprocess by which RNA is transcribed, but then degraded before translation.While PTGS is important in processes including development, transgenesilencing, and plant responses to the environment, it does not fit thedefinition of epigenetics that we have used in this review, and will thereforenot be addressed.

However, recent results provide evidence that RNA can also play acritical role in establishing and maintaining heritable chromatin states.Evidence for the role of RNA in establishing and maintaining heritablechromatin states come from several types of studies. Research utilizingmutants in RNAi genes found that plants mutant for genes in this pathwaydisplayed altered heritable DNA methylation patterns (Chan et al., 2004,2006; Lippman et al., 2003; Tran et al., 2005; Zilberman et al., 2003). Themop1mutant in maize is an example of the role of RNA in multiple types ofheritable silencing. The mop1 gene encodes an RNA-dependent RNApolymerase 2-like protein that is involved in paramutation (Alleman et al.,2006), heritable transgene silencing (McGinnis et al., 2006), and transposonsilencing (Lisch et al., 2002).

While the complete mechanism of RNA-directed heritable silencing hasnot been completely elucidated, several important players in this pathwayhave been identified. Plants contain a unique family of RNA polymerase,PolIV. These polymerases contain component proteins of the NRPD1 andNRPD2 classes, and play an important role in RNA directed DNA meth-ylation via the production of 24 nt siRNAs (Pikaard et al., 2008). Produc-tion of these siRNAs involves RNA-dependent RNA polymerase 2 typeproteins which produce dsRNA that is processed by RISC complexescontaining Argonaute 4 or Argonaute 6 (Vaucheret, 2008) homologs.Also involved in the process is SGS3, a protein of currently unknownfunction. An excellent summary of the various roles of RNA pathways intranscriptional and posttranscriptional silencing is provided by Shiba andTakayama (2007).


2.5. Interactions among DNA methylation, histonemodifications, and chromatin structure

The general descriptions of the molecular mechanisms of epigenetic inheri-tance provided above suggest discrete pathways for epigenetic regulation.However, it is clear that there is significant cross-talk and redundancybetween these molecular mechanisms. The first plant mutant with reducedDNA methylation levels was Arabidopsis ddm1 (Vongs et al., 1993). TheDDM1 gene actually encodes an ATP-dependent chromatin remodelingprotein that is required for proper DNA methylation patterns ( Jeddelohet al., 1999). A similar relationship between DNA methylation and chro-matin remodeling has been noted in mammals (Dennis et al., 2001). Thissuggests that chromatin remodeling activities are required for proper DNAmethylation patterns. There is also evidence for a strong relationshipbetween histone methylation and DNA methylation in plants. Mutationsin the H3K9 histone methyltransferase KRYPTONITE affect CpHpGmethylation ( Jackson et al., 2002; Malagnac et al., 2002). There is evidencethat alterations in DNAmethylation patterns affects the histone methylationpatterns (Gendrel et al., 2002; Johnson et al., 2002; Lawrence et al., 2004;Lindroth et al., 2004; Soppe et al., 2002; Tariq et al., 2003). The CMTDNAmethyltransferase exhibit preferential binding to chromatin containing bothH3K9 and H3K27 methylation (Lindroth et al., 2004). Several of the plantH3K9 methyltransferases contain an SRA domain which exhibit methyl-DNA specific binding activities ( Johnson et al., 2007; Woo et al., 2007,2008). These molecular properties of the DNA and histone methyltrans-ferases lead to complementary reinforcement of CpHpG DNA methylationand H3K9 histone methylation. Proper histone acetylation patterns are alsorequired for maintenance of DNA methylation (Aufsatz et al., 2002; Probstet al., 2004). Mutations in chromatin remodeling genes can also affecthistone modification patterns (Gendrel et al., 2002; Kanno et al., 2004,2005; Lippman et al., 2003). The epigenetic mechanisms for gene regulationexhibit a high level of redundancy and interdependence. It is likely thatthese relationships strengthen the heritability and help to preserve thisepigenetic information following DNA replication and cell division.

3. Epigenetic Phenomena in Plants

As discussed above there are a number of different molecular mechan-isms for storing epigenetic information. Similarly, epigenetic inheritanceexhibits a variety of different types of inheritance. In some cases, an epige-netic difference can be quite stable and can appear to follow Mendeliansegregation patterns. In other cases epigenetic states can be programmed by


the parent of origin or exposure to other alleles. We will begin by discussingseveral examples of epigenetic inheritance in plants. This discussion will belimited to examples of epigenetic inheritance that can be meiotically trans-mitted. There are examples of epigenetic gene regulation during develop-ment, such as vernalization (Schmitz and Amasino, 2007), but these do notaffect phenotypes in the off-spring. A survey of studies on epigeneticvariation within plant species will also be presented.

3.1. Phenotypic examples of epigenetic inheritance

3.1.1. ImprintingImprinting is a form of epigenetic regulation in which the maternal andpaternal alleles exhibit differential expression following fertilization. At animprinted locus, there are two alleles with identical, or nearly identical,sequences in the same nucleus, yet these two alleles exhibit differentialexpression. To date, all examples of imprinting in plants occur in endo-sperm tissue (Huh et al., 2007). The endosperm exhibits unique epigeneticstates relative to other plant tissues (Baroux et al., 2007; Lauria et al., 2004).Most imprinted genes exhibit expression only from the maternal allele butseveral examples with paternal expression have also been identified(reviewed by Huh et al., 2007). Imprinting can result in phenotypic differ-ences between reciprocal hybrids. For example, the phenotype conditionedby some r1 locus haplotypes differs depending upon maternal or paternaltransmission. The maternally transmitted haplotype provides solid kernelcoloration while the paternally transmitted allele conditions a mottledpattern (Kermicle, 1970, 1978; Kermicle and Alleman, 1990). Interestingly,while some r1 haplotypes are subject to imprinting, the majority do notexhibit any evidence of imprinting (Kermicle and Alleman, 1990). Thenumber of plant genes that exhibit complete, or binary, imprinting isrelatively small. Many of the imprinted plants genes including Medea,Fis2, ZmFie1, and Mez1, have sequence homology to proteins in thePolycomb repressive complex2 (PRC2) (Kohler and Makarevich, 2006).

There is evidence that the imprinting mechanism involves DNA meth-ylation and histone modifications (reviewed by Huh et al., 2008). In plants,the mechanism for imprinting at the Arabidopsis Medea (MEA) locus isunderstood in the greatest detail. MEA encodes a SET domain proteinthat can perform methylation of H3K27 (Baroux et al., 2006; Gehringet al., 2006; Grossniklaus et al., 1998; Jullien et al., 2006). DNA methylationestablishes a default silenced state at theMEA locus and maintenance of thissilent state requires MET1 (Xiao et al., 2003). A DNA glycosylase enzyme,DEMETER (DME ) is expressed in the central cell and removes DNAmethylation from the maternal allele of MEA (Choi et al., 2002; Gehringet al., 2006; Kinoshita et al., 2004). Upon fertilization the maternal allele is


unmethylated and active while the paternal allele is methylated. Duringendosperm growth and development the MEA protein expressed from thematernal allele is required for maintenance of silencing of the paternal allele(Baroux et al., 2006; Gehring et al., 2006; Jullien et al., 2006). The mater-nally produced MEA protein is recruited to the promoter of the paternalMEA allele and catalyzes H3K27me3. There is evidence for differentialDNA methylation of the maternal and paternal alleles of FWA (Kinoshitaet al., 2004), Fis2 ( Jullien et al., 2006), ZmFie1 (Gutierrez-Marcos et al.,2006; Hermon et al., 2007), andMez1 (Haun et al., 2007). Three imprintedmaize genes,Mez1, ZmFie1, andNrp1 also exhibit evidence for H3K27me3enrichment at the promoter of the silenced paternal allele and H3 acetyla-tion and H4 acetylation enrichment within the coding region of thematernal allele (Haun and Springer, 2008).

3.1.2. ParamutationParamutation is a form of epigenetic inheritance that involves the commu-nication of two alleles. Paramutable alleles can be heritably altered by beingexposed to a paramutagenic allele in a heterozygote (reviewed by Chandler,2007; Chandler and Stam, 2004; Hollick et al., 1997). Paramutation hasbeen well-characterized at the r1, b1, p1, and pl1 loci in maize (Brink, 1956;Coe, 1966; Hollick et al., 1995; Sidorenko and Peterson, 2001). The b1locus of maize provides the best characterized example of paramutation.The b gene encodes a transcription factor that regulates the production ofanthcyanin. The B-I allele provides dark pigmentation of several vegetativetissues while the B’ allele provides very light pigmentation of these tissues.The B-I allele is dominant over loss-of-function b alleles (Coe, 1966;Patterson et al., 1993, 1995). However, plants that are heterozygous forB-I/B’ exhibit light pigmentation similar to B’/B’ homozygotes. Self-pollination of B-I/B’ heterozygotes produces only off-spring with light-pigmentation. The paramutable B-I allele is affected by the paramutagenicB’ allele in the heterozygote such that the B-I allele is transformed into a B’allele. The transition of B-I to B’ can happen spontaneously at low rates butis 100% when exposed to B’ in a heterozygote. This transition of B-I to B’does not involve any sequence changes at the B locus. A series of elegantgenetic experiments have identified a series of direct repeats 100 kb 50 of theB gene that are required for paramutation (Stam et al., 2002a,b). Character-ization of mutants that are impaired in paramutation reveals that RNAi andchromatin remodeling are important components of paramutation(Alleman et al., 2006; Dorweiler et al., 2000; Hale et al., 2007). There is noevidence that cytosine DNAmethylation is required for paramutation at theB locus. However, altered DNA methylation patterns have been associatedwith paramutation at the R and P1 loci (Sidorenko and Peterson, 2001;Walker et al., 1998). There is genetic evidence that a common mechanism


might underlie paramutation at these distinct loci (Dorweiler et al., 2000;Hollick and Chandler, 2001; Hollick et al., 2005). There is some evidencefor paramutation-like interactions between transgenes in other plant andanimal species (reviewed by Chandler and Stam, 2004). It is unclear whetherparamutation is limited to a small number of loci or might be acting at manygenomic locations.

3.1.3. Naturally occurring epiallelesStable epigenetic alleles have also been identified by studies on intraspecificvariation. In the process of analyzing floral development mutants, Jacobsenand Meyerowitz (1997) identified several alleles of the SUP locus. Thesealleles were mapped to the SUP locus and were confirmed by complemen-tation tests. However, no sequence differences were identified at the SUPlocus. These clark kent alleles exhibit higher methylation levels than thewild-type SUP allele and reduced expression. While the clark kent allelesgenerally exhibit stable inheritance, 1–3% of progeny reverted to wild-typephenotype and these revertents had reduced, wild-type, methylation levels( Jacobsen and Meyerowitz, 1997). The epigenetic silencing of SUPrequires CpHpG DNA methylation (Lindroth et al., 2001), histone meth-ylation ( Jackson et al., 2002) and RNAi components (Zilberman et al.,2003). The PAI gene family of Arabidopsis also exhibits epigenetic variation(Bender and Fink, 1995). The four copies of PAI present in the WS ecotypeare methylated and transcriptionally silenced while the three PAI genes inCol are unmethylated and expressed (Bender and Fink, 1995). This silenc-ing in WS is triggered by the inverted repeat arrangement of the PAI1 andPAI4 genes (Melquist and Bender, 2004; Melquist et al., 1999) and requiresCpHpG methylation (Bartee et al., 2001) and histone methylation (Ebbset al., 2006; Malagnac et al., 2002). There is also evidence that a diseaseresistance gene cluster exhibits naturally occurring epigenetic variation (Yiand Richards, 2007).

There are several examples of naturally occurring epigenetic variants inother plant species as well. Linneus described a variant of Linaria vulgaris thathad altered floral morphology (Cubas et al., 1999). This natural variant hasbeen stably maintained for over 250 years and exhibits relatively stablegenetic transmission. Molecular characterization revealed that the alteredfloral morphology is due to increased cytosine methylation at the Lcyc gene(Cubas et al., 1999). There is a mutation that affects tomato fruit develop-ment and ripening that is caused by epigenetic changes at the Cnr locus(Manning et al., 2006). There are naturally occurring alleles of several maizegenes affecting seed or plant pigmentation levels, including Pl1 (DellaVedova et al., 2005; Hoekenga et al., 2000); P1 (Chopra et al., 2003;Sekhon et al., 2007; Sidorenko and Peterson, 2001) and R (Kermicleet al., 1995; Ronchi et al., 1995; Walker and Panavas, 2001), that exhibitepigenetic regulation.


3.1.4. Polyploid formationEpigenetic regulation may also play an important role in the success ofpolyploids (Hegarty and Hiscock, 2008; Liu andWendel, 2003). Polyploidyinvolves an alteration in gene dosage (autopolyploidy) or a fusion of twocomplete genomes (allopolyploidy). In both instances, a situation is createdin which the majority of genes are redundant and novel functions orexpression patterns can be sampled. There are numerous examples of alteredepigenetic states in recently formed polyploids. Newly formed polyploidsoften show substantial genomic instability, however the exact type ofchanges observed varies in different species. There is evidence for sequenceelimination in wheat and Tragopogon polyploids (Feldman et al., 1997;Shaked et al., 2001; Tate et al., 2006). Chromosomal translocations andtransposon insertions are common in Brassica polyploids (Song et al., 1995)while mainly gene expression changes are observed in Arabidopsis andcotton (Adams, 2007; Adams et al., 2003; Lee and Chen, 2001; Wanget al., 2004).

Many polyploids also exhibit altered DNA methylation patterns uponhybridization of the two paternal genomes (reviewed by Liu and Wendel,2003). The alteration of DNA methylation patterns following polyploidiza-tion suggests that epigenetic changes may be common in newly formedallopolyploids. Indeed, a number of studies have provided evidence thatepigenetic changes cause altered gene expression in newly formed poly-ploids (Comai, 2000; Comai et al., 2000; Kashkush et al., 2002; Lee andChen, 2001; Madlung et al., 2002). Madlung and Comai (2004) proposedthat the formation of a polyploidy causes a high level of genomic stresswhich results in relaxation of epigenetic silencing and expression of nor-mally suppressed sequences. As the epigenetic systems are reestablished,novel epigenetic states are formed in the polyploids relative to the parentalgenomes. This system allows for novel expression states to be sampled andselected in polyploids.

3.1.5. Nucleolar dominanceRibosomal RNA genes in plants are highly repeated and occur in longcontiguous stretches. Interestingly, only a portion of these genes are nor-mally expressed in any cell, and the silenced portion is in a contiguousstretch indicating a mechanism to simultaneously silence megabase segmentsof DNA. Furthermore, when multiple clusters of ribosomal genes areintroduced into an organism by hybridization, the phenomena of nucleolardominance is observed. Nucleolar dominance is achieved during an inter-action of gene clusters on different chromosomes in which an entire clusteron one chromosome is silenced and the other remains active. The mecha-nism underlying this process is still being characterized, but the current stateof knowledge is provided in Preuss and Pikaard (2007).


3.2. Genomic and molecular genetic examplesof epigenetic variation

In addition to the previously described examples of epigenetic inheritance,there are several studies that address the prevalence of epigenetic variationwithin plant species. In general, it is quite difficult to attribute variation toepigenetic mechanisms. However, by studying the mechanisms of epige-netic inheritance, such as DNA methylation, it is possible to identifyepigenetic variation. It is likely that these studies represent only the tip ofthe iceberg and that the application of genomic technologies will provide amore detailed understanding of epigenetic variation.

3.2.1. Natural variation for methylation of repetitive elementsOne approach towards the identification of epigenetic variation within aspecies is to simply monitor DNA methylation levels in populations. EricRichards and colleagues have used Southern blot analysis of DNA methyl-ation at repetitive sequences to monitor variation in Arabidopsis (Riddle andRichards, 2002, 2005; Woo et al., 2007). The methylation of rDNA rangedfrom 20% in some Arabidopsis ecotypes to over 90% in others (Riddle andRichards, 2002). There was a correlation between the methylation level ofribosomal DNA and the copy number for rDNA. A QTL analysis of rDNAmethylation identified two major QTL located at the genomic locations ofthe rDNA that explain �50% of the variation in DNA methylation levels.It is likely that much of the methylation variation contributed by these QTLwas due to inheritance of parental DNA methylation patterns at these loci(Riddle and Richards, 2002). In addition, several trans-acting QTL wereidentified on chromosomes 1, 3, and 5 (Riddle and Richards, 2002).A larger screen of DNA methylation levels in Arabidopsis ecotypes revealedthat the Bor-4 ecotype exhibits low levels of DNA methylation at the180-bp repeats but not at other loci (Woo et al., 2007). This reducedmethylation segregated as a simple Mendelian trait and map-based cloningidentified the VIM1 gene (Woo et al., 2007). There is a large deletionwithin the Bor-4 allele of VIM1 (Woo et al., 2007). The intraspecificvariation for VIM1 function therefore leads to intraspecific variation forcentromeric methylation in Arabidopsis. There is no evidence for functionalsignificance for the variation of DNA methylation levels at centromeres orrDNA in Arabidopsis.

3.2.2. Natural variation for genic epigenetic variationSeveral approaches have been used to identify genes that exhibit variationfor DNA methylation levels within a population. Rangwala et al. (2006)used microarray profiling to identify natural epigenetic variants in Arabi-dopsis. One noncoding transcript that may be derived from a retrotransposonwas characterized in detail. This Sadhu element (At2g10410) is methylated


in most of the accessions tested, but in Col and N13 this sequence is highlyexpressed and hypomethylated (Rangwala et al., 2006). Multiple membersof the Sadhu family of retroelements exhibit natural epigenetic variation(Rangwala et al., 2006). Interestingly, different Sadhu elements are con-trolled through different epigenetic mechanisms (Rangwala et al., 2007) andthese members of this family do not exhibit coordinate regulation(Rangwala et al., 2006).

Vaughn et al. (2007) used chromosome 4 tiling microarrays to charac-terize the DNA methylation patterns in two Arabidopsis accessions. Thisapproach identified numerous examples of variable DNA methylation inLer and Col ecotypes. A limited analysis of the stability of DNAmethylationpatterns found that some polymorphisms were quite stable while othersexhibit high reversion frequencies in F2 families (Vaughn et al., 2007).A survey of sixteen loci in 96 different ecotypes revealed high levels ofmethylation polymorphism within Arabidopsis. Using a different approachZhang et al. (2008) identified high levels of DNA methylation amongArabidopsis ecotypes. Methylation polymorphisms were more commonnear gene ends than within the coding region and were correlated withexpression differences in some examples (Zhang et al., 2008). Anotherrecent study identified widespread epigenetic natural variation for RNAitargets in Arabidopsis (Zhai et al., 2008).

There is also evidence for natural epigenetic variation in maize.Makarevitch et al. (2007) used microarray profiling to identify the targetsof CpHpG methylation in the maize inbreds B73 and Mo17. Over 100genes sensitive to CpHpG methylation were identified by comparingexpression isogenic wild-type inbred and zmet2-m1 mutant lines. Themajority of the genes are sensitive to CpHpG methylation only in one ofthe two inbred lines. In most cases, these genes exhibit different expressionlevels in wild-type B73 and Mo17 and this variation maps to the gene itselfand is controlled by DNA methylation (Makarevitch et al., 2007). A surveyof the methylation and expression levels for several of these genes providedevidence for stable natural epigenetic variation in eight different inbred lines(Makarevitch et al., 2007).

4. Epigenetic Inheritance and Crop Improvement

Heritable transcriptional gene silencing is common in transgenic experi-ments. Transgene silencing reduces the efficiency of the transformationprocess and increases the work necessary to identify good events.

Epigenetics also has a role in somaclonal variation (Kaeppler et al., 2000).Somaclonal variation generally is detrimental to the processes of transfor-mation and clonal propagation, but in some instances can produce usefulphenotypes.


One vastly underexplored area of epigenetics is the potential role ofheritable states of expression as a mechanism of quantitative variation, and asa rapid-response reservoir of variation that can contribute to selectionresponse. In the following section, we will discuss the potential role ofepigenetic inheritance in the process of plant breeding.

4.1. Epigenetics in quantitative inheritanceand selection response

Recent research provides evidence for the following interesting attributes ofepigenetic inheritance.

1. Naturally occurring epialleles have been documented in numerousspecies and are relatively stable, sometimes over many generations.

2. Epialleles have reversion frequency 2–3 orders of magnitude greater thanchanges in the primary sequence. In the case of silenced alleles, reversionis to the active state in contrast to reversion of base sequence to a priorstate which is very rare.

3. Stability and formation of epigenetic states can be influenced by theenvironment.

4. Epigenetic states of expression are not simply on or off, but stableintermediate levels of expression can be established.

5. Introduction of trans-acting alleles in ‘‘chromatin genes’’ can causetransient (one generation) or permanent alteration in state. For example,an epiallele could be stably maintained over many generations, but couldrevert to activity after a single generation interaction in a loss-of-function methyltransferase mutant.

6. Hybridization/heterozygosity is required for establishment of some epi-genetic states such as paramutation, so epiallelic variation is enhanced inoutcrosses and may increase in proportion to diversity.

The molecular basis of allelic variation for quantitative traits in plants is juststarting to be characterized, so it is difficult to provide examples for whichquantitative models based on sequence information would be insufficient toexplain the variation present. In fact, the stability of epialleles would makethem appear no different than any sequence variant within the temporalcontext of most plant breeding experiments.

However, long-term selection experiments indicate an amazing abilityof plants to respond to selection in short periods of time. The Illinois long-term selection program is one unique example of selection in plants(Dudley, 2007). An interesting attribute of this program was the strongresponse to reverse and switchback selection. Especially intriguing is theability of populations selected in the low direction (e.g., for low oil) torespond to selection for increases in the biochemical components. Thereverse selection in the low oil population was initiated at a cycle when it


appeared that selection response had dramatically reduced and that there waslittle genetic variation remaining. While there are various sequence-basedgenetic mechanisms that could cause this result, it is intriguing to speculatethat epiallelic variation may maintain a reservoir of variation that might beselected upon in a situation such as this. In natural populations, such areservoir of variation could allow population responses to rapid changes inenvironment for traits with seemingly little phenotypic variation.

4.2. Epialleles and gene discovery

Substantial effort is currently being devoted in plants to characterizing thesequence variation that underlies quantitative phenotypic variation. This isaccomplished by associating sequence-based haplotypes with phenotypicperformance to determine causal phenotypes. Epigenetic variation thatcontributes to phenotypic variation would complicate this effort.

Epigenetic molecular variation would not be detected by normalsequence-based approaches to haplotype characterization. Methylationmapping, using a technique such as bisulfite sequencing, or chromatinmapping would be required to characterize the epigenetic states of targetgenes. Alternatively, epialleles might be predicted by detection of transcrip-tion states that cannot be explained by sequence haplotype variation.

A further complication of epigenetics in this process is that epialleles mayoccur in multiple lineages in a comparatively short time frame. Whereassequence variants occur in a logical progression with a founder sequencegiving rise to a series of accumulating variants over the course of times,epialleles may appear and disappear at any point within this process, and willlikely occur independently of most sequence polymorphisms. Therefore,epialleles may at best cause noise in the process of searching for causalsequence polymorphisms, and in extreme cases may be more important forspecific genes in determining phenotype than any sequence polymorphism.

Growing documentation of the presence of stable epialleles in numerousspecies suggests that epigenetic variation needs to be considered in theprocess of associating molecular variation with phenotype. Advancingtechnology may allow chromatin patterns to be included with sequence asanother layer of information that can be included in the analysis complextraits in plants.

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Epigenetics: The Second Genetic Codesgpwe.izt.uam.mx/.../11Epigenetica.pdf · Epigenetic Phenomena in Plants 65 3.1. Phenotypic examples of epigenetic inheritance 66 3.2. Genomic