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. Author manuscript; available in PMC: 2013 Aug 29.
Published in final edited form as: New Phytol. 2010 Apr;186(1):46–53. doi: 10.1111/j.1469-8137.2010.03193.x

Heterochromatin, small RNA and post-fertilization dysgenesis in allopolyploid and interploid hybrids of Arabidopsis

Robert A Martienssen 1
PMCID: PMC3756494  NIHMSID: NIHMS506435  PMID: 20409176

Summary

In manylants, including Arabidopsis, hybrids between species and subspecies encounter postfertilization barriers in which hybrid seed fail to develop, or else give rise to infertile progeny. In Arabidopsis, some of these barriers are sensitive to ploidy and to the epigenetic status of donor and recipient genomes. Recently, a role has been proposed for heterochromatin in reprogramming events that occur in reproductive cells, as well as in the embryo and endosperm after fertilization. 21 nt small interfering RNA (siRNA) from activated transposable elements accumulate in pollen, and are translocated from companion vegetative cells into the sperm, while in the maturing seed 24 nt siRNA are primarily maternal in origin. Thus maternal and paternal genomes likely contribute differing small RNA to the zygote and to the endosperm. As heterochromatic sequences also differ radically between, and within, species, small RNA sequences will diverge in hybrids. If transposable elements in the seed are not targeted by small RNA from the pollen, or vice versa, this could lead to hybrid seed failure, in a mechanism reminiscent of hybrid dysgenesis in Drosophila. Heterochromatin also plays a role in apomixis and nucleolar dominance, and may utilize a similar mechanism.

Keywords: allopolyploidy, dosage, hybrid lethality, siRNA, transposon

Introduction

Post-fertilization barriers to hybridization between and within species are widespread in animals and plants, and involve a variety of mechanisms that operate at various stages of development (Bomblies & Weigel, 2007; Li et al., 2009). Typically, these mechanisms result in lethality, either of the zygote, of developing embryonic and extra-embryonic tissues, of somatic tissues and/or of germ cells in the F1 hybrids, causing sterility. In plants, the viability and reproductive fitness of hybrids are also compromised when they are derived from egg and sperm cells that differ in ploidy. Interploid hybrid failure ranges from early seed inviability to infertility. The impact of interploidy varies widely between plant species, but one mechanism common to many angiosperms depends on the balance of maternal (M) and paternal (P) genomes in the endosperm. This balance is normally 2 : 1 because of fusion of the polar nuclei to form the central cell on the maternal side. For example, in maize, while 4 : 2 and 6 : 3 M : P endosperms are fully viable, 4 : 1 or 2 : 2 endosperms are not (Birchler et al., 2007).

Both interspecific and interploidy barriers are under genetic control, and genes required for interspecific and intraspecific hybrid lethality have been identified in Drosophila (Brideau et al., 2006) and Arabidopsis (Bomblies & Weigel, 2007), respectively. Genetic components of interploidy barriers to endosperm development have also been noted in many species, leading to the concept of ‘endosperm balance number’ in which the sum of individual components on the maternal and paternal side is thought to contribute to the optimal ratio of M and P genomes (Johnston & Hanneman, 1982; Carputo et al., 2003; Kinoshita, 2007). These results indicate that the M and P genomes differ in some fundamental property depending on parental origin.

Imprinting and reprogramming

The maternally derived and paternal genomes in the zygote differ owing to imprinting of specific genes in the male and female germline (Garnier et al., 2008; Ikeda & Kinoshita, 2009; Jullien & Berger, 2009). This small set of genes differ in their expression in the seed depending on whether they are inherited from the pollen or seed parent. A similar phenomenon occurs in mammals where imprinted genes are essential, so that parthenogenetic and androgenetic embryos are inviable because of failure of the extra-embryonic and embryonic tissues (respectively) derived from the fertilized zygote (Sha, 2008). While imprinted genes acquire their imprints in the germline, where they are maintained, the majority of the mammalian genome undergoes reprogramming, both before and just after fertilization (Solter et al., 2004; Bartolomei, 2009; Hayashi & Surani, 2009). During this reprogramming genes are thought to lose any memory of their previous expression levels so that the zygote and early embryo adopt a totipotent fate. Reprogramming involves the loss of ‘epigenetic’ information, such as DNA methylation and histone modification, which are perpetuated from cell to cell but do not result from changes in the DNA sequence. DNA methylation, especially, is dramatically lost from much of the mammalian genome in primordial germ cells, and again in the male pronucleus during fertilization, and once again in the pre-implantation embryo after fertilization (Bartolomei, 2009; Hayashi & Surani, 2009). Loss of methylation is associated with altered expresion and localization of the enzymes required for DNA methylation (Branco et al., 2008). Histone modification of key developmental regulatory genes is also reprogrammed in the germline, but in a way that anticipates modifications found in the early embryo that predict differentiation (Hammoud et al., 2009).

In plants, in contrast with animals, maternally and paternally derived haploid embryos are perfectly viable, and doubled haploids are extensively used in plant breeding (Forster et al., 2007). The biological impact of imprinting in plants is therefore mostly confined to the endosperm, which is the product of a separate fertilization (Kinoshita, 2007; Jullien & Berger, 2009). As in animals, imprinted genes in plants also acquire their imprints in the germline, except that the germline is composed of just a few cells, so that maternally imprinted genes, for example, must acquire imprints in the central cell before fertilization (Kinoshita, 2007; Jullien & Berger, 2009). An important component of these imprints is the methylation of histone H3 lysine 27, mediated by the Polycomb Complex PRC2. Many PRC2 components are expressed in the central cell and early endosperm, and some are encoded by genes that are themselves imprinted (Grossniklaus et al., 1998; Garnier et al., 2008; Jullien & Berger, 2009).

Recently, genome reprogramming has also been found to occur in plants. For example, the DNA methyltransferase MET1 is downregulated in the central cell (Jullien et al., 2008) and the chromatin remodeler Decrease in DNA methylation 1 (DDM1) is downregulated in pollen (Slotkin et al., 2009), while the DNA glycosylase/demethylase gene DEMETER is upregulated in the central cell (Gehring et al., 2006; Jullien & Berger, 2009). Unlike in animals, however, reprogramming seems to be largely confined to germline cells that do not contribute to the next generation – so called ‘companion’ cells such as the vegetative pollen tube cell, and the central cell in the seed. Imprinting and eprogramming have thus become candidate mechanisms to explain aspects of interspecific and interploid hybridization failure (Martienssen et al., 2008).

Silencing of transposable elements

Both imprinting and reprogramming have been associated with the activity of transposable elements (TEs). TEs are widespread components of eukaryotic genomes that can influence the expression of neighboring genes. Anecdotal (Kinoshita et al., 2004; Lippman et al., 2004) and genome wide (Gehring et al., 2009; Hsieh et al., 2009) methylation profiling of genes from endosperm has revealed that imprinted genes often lie close to a TE whose methylation is significantly decreased in the endosperm. This has led to the proposition that TEs play a major role in imprinting (Martienssen, 1998). Reprogramming affects TEs profoundly, so that they are highly active in the vegetative nucleus of the pollen grain. Interestingly, this TE activity leads to the production of a specific class of 21 nt siRNA (Slotkin et al., 2009). On the maternal side, a different class of siRNA, namely 24 nt siRNA, appear to accumulate from TEs instead (Mosher et al., 2009). Both classes of small RNA can silence TEs that share the complementary sequence, (Slotkin & Martienssen, 2007), and indeed the 21nt post-transcriptional pathway interacts with the 24nt transcriptional silencing pathway (Eamens et al., 2008). However, neither class of siRNA is required to silence most fully methylated TEs (Lippman et al., 2003; Teixeira et al., 2009). Small RNA thus become important in the germline when DNA methylation is reduced (Jullien et al., 2008; Slotkin et al., 2009).

Postfertilization hybrid barriers in Arabidopsis

In Arabidopsis, both interspecific and interploidy crosses give rise to seed collapse in F1 hybrid endosperm – either because of over-proliferation and failure to cellularize, or else under-proliferation and premature cellularization (Adams et al., 2000; Comai et al., 2000; Bushell et al., 2003). For example, crosses between diploid Arabidopsis thaliana (TT) seed parents and hexaploid (TTTTTT) pollen parents result in hybrid endosperm over-proliferation and shriveled seed (Adams et al., 2000). Collapsed and shriveled seed are inviable, especially after desiccation. By contrast, crosses between diploid and tetraploid parents are viable but differ in size according to the direction of the cross. In reciprocal crosses with diploids, TTTT pollen parents give rise to dramatically larger seed, and TTTT seed parents to slightly smaller seed (Adams et al., 2000).

In Arabidopsis, viability depends critically on genotype. C24 triploid seed are mostly viable (Adams et al., 2000), while Landsberg erecta (Ler) are intermediate, and Columbia (Col) triploid seed are mostly inviable following interploid crosses (Dilkes et al., 2008). Differences in viability between Ler and Col have been mapped to three quantitative trait loci (QTLs) linked to the TRANSPARENT TESTA GLABRA2 (TTG2), IKU1 and IKU2 genes (Dilkes et al., 2008), which impact seed development maternally (ttg2) and in the endosperm (iku), respectively (Garcia et al., 2003, 2005). A weak allele of ttg2 in Ler is thought to be at least in part responsible for this variation, as strong alleles of ttg2 largely rescue the interploidy defects in the Col background (Dilkes et al., 2008).

Interspecific crosses between A. thaliana TT and Arabidopsis arenosa AAAA, also result in shriveled inviable seed (Comai et al., 2000; Bushell et al., 2003). The degree of inviability again depends on genotype, with Col TT parents giving rise to no viable seeds (Bushell et al., 2003; Henry et al., 2005). Viability can be partially restored by using a TTTT seed parent, especially when the C24 or Ler ecotypes are used. Importantly, these viable seed give rise for the most part to flowering and at least partially fertile progeny (Bushell et al., 2003), while the few seed that survive in a Col TTTT × AAAA cross have severe developmental phenotypes, and do not flower for the most part (Josefsson et al., 2006; P. Finigan & R. Martienssen, unpublished).

Imprinting as an epigenetic hybrid barrier

In addition to their similar phenotypic outcomes and genetic regulation, interspecific and interploidy crosses also respond similarly to epigenetic regulation. About 50% of the hybrid seed from C24 TTTT × AAAA are viable, but this viability is reduced to 7% when using TTTT seed parents that have been transformed with an antisense construct targeted to the DNA methyltransferase gene MET1 (Bushell et al., 2003). These surviving plants are severely late flowering. Similarly, almost all seed resulting from C24 TT × TTTT crosses are viable, but this viability is reduced to less than 10% when TT antisense MET1 seed parents are used (Adams et al., 2000). When MET1 knockdown was repeated in the naturally occurring allopolyploid Arabidopsis suecica it also caused severe growth and developmental abnormalities (Chen et al., 2008). Similarly, treatment of synthetic allopolyploids with DNA methylation inhibitors resulted in developmental consequences that were not observed in the parents (Madlung et al., 2002). In each case, transposons and centromeric satellite repeats were upregulated as in met1 mutants (Lippman et al., 2003; May et al., 2005).

Several possible explanations were considered. Genomic shock, the activation of genes and transposons normally quiescent in each genome, was considered as an explanation although it was difficult to account for the influence of dosage on both interspecific and interploidy crosses (Adams et al., 2000). Instead, imprinting defects were proposed to account for endosperm failure (Adams et al., 2000; Dilkes & Comai, 2004). Support for imprinting defects included the fact that the phenotype of TT by TTTT interploidy hybrids resembles, in some respects, the phenotype of mutants in the PRC2 components fis2 and fis1/medea, which result in over-proliferation of the endosperm (Bushell et al., 2003; Dilkes & Comai, 2004). The interploidy phenotype is associated with mis-expression of some imprinted genes (Josefsson et al., 2006) and could be partly rescued by over-expression of MEDEA consistent with the idea that MEDEA silences these same imprinted genes, acting as a ploidy ‘sensor’ (Erilova et al., 2009). MET1 is also known to silence imprinted genes (Xiao et al., 2003, 2006a,b; Kinoshita et al., 2004; Gehring et al., 2006) and knockdowns might be expected to enhance the TT × TTTT phenotype (Adams et al., 2000), especially when both maternal and paternal copies are knocked down via dominant RNAi constructs. However, MET1 is expressed at very low levels in the central cell making imprinting a less satisfactory explanation in this case (Jullien et al., 2006, 2008; FitzGerald et al., 2008).

A role for transposons and RNAi in post-fertilization hybrid barriers

In the light of recent results concerning the role of RNAi in silencing transposons both pre- and post-fertilization, it is time to reconsider the possibility that ‘genome shock’ is at least in part responsible for hybridization barriers in both interspecific and interploidy crosses (Fig. 1a). In pollen, genome reprogramming results in the loss of heterochromatin from the vegetative cell nucleus (VN), accompanied by loss of MET1 and DDM1 proteins from the VN (Slotkin et al., 2009). This in turn results in the massive reactivation of transposable elements, and the production of a novel class of transposon small RNA, 21 nt in length. Unexpectedly, this class of 21 nt siRNA is translocated into the sperm cells, from which they are presumably delivered into the zygote and into the endosperm (Slotkin et al., 2009). The 24 nt siRNAs from some classes of TE also accumulate in the sperm, though in lower numbers (Slotkin et al., 2009).

Fig. 1.

Fig. 1

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A model for postfertilization transposon activation in interspecific and interploid hybrids of Arabidopsis. The dosage of Arabidopsis thaliana (T) and bidopsis arenosa (A) genomes derived from diploid and tetraploid parents is indicated in pollen, egg and central cells. According to the model, the pollen grain (circle) has active transposable elements (TE) that give rise to siRNA (black hammerheads) which can inhibit complementary TE in the sperm. If 24 nt siRNA are also produced from TEs in the central cell (octagon), or from maternal somatic tissues (not shown), they may also contribute to silencing in the egg cell (dotted lines). According to this model, TEs require both 21 nt siRNA from the sperm, and 24 nt siRNA from the maternal side for silencing in the endosperm and embryo after fertilization (TE and siRNA activity is indicated by font and point size). In interspecific allotetraploid hybrids (a), sequence differences between siRNA from the A genome and TE from the T genome (and vice versa) result in TE activation in the endosperm. This effect is enhanced when diploid TT seed parents are used (b), because A. arenosa transposons are in even greater excess. In interploid A. thaliana crosses with a tetraploid seed parent (c), TE are also activated in the endosperm because of maternal TE excess relative to sperm cell siRNA. Conversely, in interploid crosses with a tetraploid pollen parent (d) TEs are activated because of paternal excess relative to maternal siRNA.

On the female side, 24 nt small RNA found in seed coat and endosperm are derived primarily from the maternal genome (Mosher et al., 2009) and may influence silencing in the embryo, related perhaps to the maternal role of MET1 (FitzGerald et al., 2008). Similarly to the VN, the central cell nucleus has aberrant heterochromatin devoid of histone H3 K9me2 (Baroux et al., 2007), and transposon reprogramming has been detected in the endosperm (Gehring et al., 2009; Hsieh et al., 2009) where MET1 is also downregulated (Jullien & Berger, 2009).

The TE sequences in the A and T genomes differ substantially, as do the major classes of centromeric repeats (Josefsson et al., 2006; Chen et al., 2008; Beaulieu et al., 2009). The possibility thus exists that the small RNA sequences derived from one parent differ from the TEs and satellite repeats in the other. If DNA copies of A. thaliana TEs in the central cell do not match A. arenosa siRNA in the pollen this may result in enhanced TE activity in the endosperm (Fig. 1a). Similarly, if siRNA from the endosperm do not match TE from the sperm, this may result in enhanced TE activity. Depending on the dose of paternal and maternal genomes, both the copy number of TE and the copy number of siRNA could determine the outcome of interspecific crosses (Fig. 1b).

In previous studies, individual transposons from A. arenosa (Josefsson et al., 2006) and from A. thaliana (Madlung et al., 2005) have been found to be active in early and late developmental stages of allopolyploid hybrids, respectively, consistent with aspects of this model. Recently, both increases (Chen et al., 2008) and decreases (Ha et al., 2009) in A. thaliana siRNA have been reported in TTTT × AAAA hybrid plants, but unfortunately small RNA matching A. arenosa (the pollen parent genome) were not investigated. It is these siRNA that might play a role in interspecific TE activation (Fig. 1a).

Activation of TEs may account for the role of methylation in enhancing the impact of interspecific hybridity, as silent TE are activated in met1 mutants (Kato et al., 2003; Lippman et al., 2003; Slotkin & Martienssen, 2007). In diploid met1 mutants, TE activation is primarily at the level of transcription, rather than transposition, and does not cause lethality post-fertilization (Kato et al., 2003; Mirouze et al., 2009), although gametic lethality has been observed (Saze et al., 2003). However, TE transposition does occur in double mutants between met1 and RNA polymerase IV (PolIV), which lack 24 nt siRNA and have strong developmental phenotypes (Mirouze et al., 2009). If maternal 24 nt siRNA in hybrid zygotes failed to match incoming TE from A. arenosa pollen, this might result in TE transposition in met1 embryos, just as it does in a met1 polIV double mutant that lacks 24nt siRNA altogether (Mirouze et al., 2009). Activation of TEs in both embryo and endosperm would be expected to enhance seed abortion.

What about interploidy crosses? We might predict that deviations in the ratio between TE DNA in the central cell and 21 nt siRNA from pollen could contribute to TE silencing in the endosperm and consequently to endosperm proliferation. According to this view the increased dosage of heterochromatic sequences in a tetraploid central cell might not be fully countered by lower levels of 21 nt siRNA provided by haploid pollen (Fig. 1c). In the absence of DNA methylation and of RNAi in the fertilized central cell, rampant transposition might inhibit endosperm proliferation promoting cellularization instead. Conversely, excess TE from diploid pollen might not be efficiently silenced by 24 nt siRNA in normal diploid central cells (Fig. 1d). Premature cellularization and over-proliferation are consequences of 4 × 2 and 2 × 4 interploid crosses, respectively (Adams et al., 2000), and may reflect the differing kinetics of maternal and paternal activation of TEs, which differ in sperm cells (Slotkin et al., 2009) and central cells (Gehring et al., 2009).

How can we explain the high levels of variation between maternal TT genomes from different thaliana accessions in accommodating pollen from TTTT and AAAA tetraploids? Genes that maternally affect endosperm development, such as TTG2, could play an important role (Dilkes et al., 2008). Equally, A. thaliana accessions differ markedly in heterochromatin content (Fransz et al., 2006; Tessadori et al., 2009), and in the composition of TEs that are known to be mutagenic (Henk et al., 1999).Thus, heterochromatin may also contribute to variation in the incompatibility of diploid and tetraploid genomes, via TE sequences that are unique to each species and accession.

Heterochromatic small RNA in apomixis and nucleolar dominance

Heterochromatin in both diploid and especially in polyploid species, has been associated, albeit indirectly, with a wide variety of epigenetic phenomena (Fransz et al., 2006; Lamb et al., 2007). Examples include nucleolar dominance, chromosome pairing, flowering time and apomixis. Nucleolar dominance results in silencing of the nucleolar organizer (NOR), which comprises thousands of rRNA genes, by exposure to the NOR from a different species in the same nucleus, and occurs in thaliana × arenosa allotetraploids of Arabidopsis. Genes that affect heterochromatin are required for this process, such as those encoding histone deacetylase and methyl DNA-binding proteins (Pontes et al., 2003, 2007; Earley et al., 2006; Preuss & Pikaard, 2007). Recently, the 24 nt siRNA pathway has also been implicated in NOR silencing (Preuss et al., 2008), but the signal for silencing, which presumably emanates from the A. arenosa NOR, has yet to be identified in allopolyploid hybrids. It is tempting to speculate that this signal involves 21 nt siRNA (Finigan & Martienssen, 2008).

Apomixis refers to the ability of some plants to reproduce without sex, and in the special case of apospory, without meiosis as well. Almost all aposporous apomicts are polyploid (Ozias-Akins & van Dijk, 2007) and give rise to unreduced gametes via differentiation of a ‘competent’ neighboring somatic cell into a functional megaspore. Interestingly, aposporous apomicts are generally hybrids, so that the genetic components responsible for apomixis are heterozygous and segregate in sexual crosses (Ozias-Akins & van Dijk, 2007). Genetic mapping of these factors in several different species has revealed large regions of heterochromatin that have very low levels of recombination (Ozias-Akins & van Dijk, 2007). It is generally assumed that the role of heterochromatin is to inhibit recombination in order to preserve complex linkage groups responsible for apomixis. Of course, the heterochromatin also harbors transposable elements, and TE activity in hybrids might contribute to apomixis. For example, transposition might damage germ cells following meiosis, prohibiting sexual fertilization. Recently, evidence for such a mechanism has been obtained in Arabidopsis, in which TE small RNA is implicated in the fate of somatic cells surrounding the functional megaspore, such that unreduced megaspores develop in RNAi mutants (Olmedo-Monfil et al., 2010). However, the trigger for reproductive differentiation of the surrounding somatic cells remains a mystery.

Hybrid dysgenesis and hybrid lethality in Drosophila

Hybrid lethality in animals has also been linked to heterochromatin. In crosses between Drosophila simulans and Drosophila melanogaster, lethal hybrid progeny are rescued by a combination of several loci that interact with X chromosome dosage compensation (Barbash & Ashburner, 2003). Lhr (lethal hybrid rescue) encodes a protein that binds heterochromatin, and interacts with the rapidly evolving MYB protein Mhr (maternal hybrid rescue) (Brideau et al., 2006). Conversely, the Zhr (zygotic hybrid rescue) locus comprises a heterochromatic block of satellite sequences on the X chromosome (Ferree & Barbash, 2009). The satellite repeat contained within this heterochromatic region prevents separation of X chromatids at mitosis. This leads to lagging chromosomes and hybrid female lethality. By analogy with the fission yeast Schizosaccharomyces pombe, chromosome segregation may depend on small RNA-mediated heterochromatin formation (Zaratiegui et al., 2007). Interestingly, the Zhr satellite repeat gives rise to abundant repeat-associated small interfering RNA (rasi-RNA), and it has been proposed that the presence of the small RNA in maternal cytoplasm accounts for differences in the outcome of reciprocal crosses between the two species (Ferree & Barbash, 2007). It is very tempting to propose a similar mechanism for interspecific hybrid interactions between plant genomes, especially polyploids which frequently display high levels of aneuploidy (Martienssen et al., 2008).

Hybrid dysgenesis in Drosophila provides an excellent model for interspecific TE activation in Arabidopsis (Slotkin & Martienssen, 2007). Active copies of transposable elements give rise to large amounts of small RNA in the egg, which can inactivate incoming elements from the sperm (Blumenstiel & Hartl, 2005; Brennecke et al., 2008). These small RNA provide immunity from rampant transposition, but only when provided by the female parent, a property known as cytotype. This property accounts for the dependence of cytotype and related phenomena on the PIWI-related Argonaute protein Aubergine (Reiss et al., 2004). In the absence of piRNA, hybrids develop normally, but are almost completely sterile owing to transposon activation in the subsequent germline (Malone et al., 2009). Plants are not thought to contain PIWI class Argonaute proteins, or piRNA, but may employ 21 nt siRNA for much the same function (Slotkin et al., 2009).

In conclusion, postfertilization hybrid barriers arise from a wide variety of mechanisms, many of which are sensitive to genome dosage. Heterochromatic repeats, and corresponding small RNA, differ in sequence from species to species. Reprogramming of the genome in endosperm and pollen results in activation of TEs and the production of distinct classes of small RNA. If small RNA from pollen fails to recognize transposons and repeats in the central cell, when TE methylation is reduced, these small RNA could influence TE activity and chromosome behavior after fertilization. Similar activation would result if TE from the pollen are not recognized by 24 nt siRNA on the maternal side. TE activation could contribute to seed failure in interploid as well as in interspecific crosses.

Acknowledgements

I thank my colleagues Keith Slotkin, Jean-Philippe Vielle Calzada, Fred Berger, Jim Birchler and Brian Dilkes for helpful discussions, and Milos Tanurdzic and Patrick Finigan, as well as our collaborators Matt Vaughn, Paul Auer and R. W. Doerge for critical input. Work on polyploidy in the author’s laboratory is supported by grant number DBI-0733857 from the National Science Foundation.

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