Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.

Postzygotic reproductive isolation in response to interploidy hybridizations is a well-known phenomenon in plants that forms a major path for sympatric speciation. A main determinant for the failure of interploidy hybridizations is the endosperm, a nutritious tissue supporting embryo growth, similar to the functional role of the placenta in mammals. Although it has been suggested that deregulated imprinted genes underpin dosage sensitivity of the endosperm, the molecular basis for this phenomenon remained unknown. In a genetic screen for suppressors of triploid seed abortion, we have identified the paternally expressed imprinted gene ADMETOS (ADM). Here, we present evidence that increased dosage of ADM causes triploid seed arrest. A large body of theoretical work predicted that deregulated imprinted genes establish the barrier to interploidy hybridization. Our study thus provides evidence strongly supporting this hypothesis and generates the molecular basis for our understanding of postzygotic hybridization barriers in plants.

Genomic imprinting is an epigenetic phenomenon causing parent-of-origin specific differential expression of maternally and paternally inherited alleles. While many imprinted genes have been identified in plants, the functional roles of most of them are unknown. In this study, we systematically examine the functional requirement of paternally expressed imprinted genes (PEGs) during seed development in Arabidopsis thaliana. While none of the 15 analyzed peg mutants has qualitative or quantitative abnormalities of seed development, we identify three PEGs that establish postzygotic hybridization barriers in the endosperm, revealing that PEGs have a major role as speciation genes in plants. Our work reveals that a subset of PEGs maintains functional roles in the inbreeding plant Arabidopsis that become evident upon deregulated expression.DOI: http://dx.doi.org/10.7554/eLife.10074.001

Genomic imprinting, the differential expression of an autosomal gene that is dependent on its parent of origin, has independently evolved in flowering plants and mammals. In both of these organism classes, imprinting occurs in embryo-nourishing tissues-the placenta and the endosperm, respectively. It has been proposed that some imprinted genes control nutrient flow from the mother to the offspring. Genome-wide analyses of imprinted genes in plants have revealed that many imprinted genes are located in the vicinity of transposon or repeat sequences, implying that transposon insertions are associated with the evolution of imprinted loci. Imprinted expression of a number of genes is conserved between monocots and dicots, suggesting that long-term selection can maintain imprinted expression at some loci. In terms of epigenetic mechanisms, imprinted expression is largely controlled by an antagonistic action of DNA methylation and Polycomb group-mediated histone methylation in the vicinity of imprinted genes, whereby the position of such epigenetic modifications can determine whether a gene will be expressed mainly from either the maternally or paternally inherited alleles.

The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The 'triploid block' is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number. This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism . Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.

Chromosome dosage plays a significant role in reproductive isolation and speciation in both plants and animals, but underlying mechanisms are largely obscure1. Transposable elements can promote hybridity through maternal small RNA2, and have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here, we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of LTR-retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21 to 22-nucleotide small RNA in a dose dependent fashion via RNA polymerase IV. We show that these epigenetically activated small-interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (“the triploid block”), and that natural variation for miR845 may account for “endosperm balance” allowing formation of triploid seeds. Targeting the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

Plants of different ploidy levels are separated by a strong postzygotic hybridization barrier that is established in the endosperm. Deregulated parent-of-origin specific genes cause the response to interploidy hybridizations, revealing an epigenetic basis of this phenomenon. In this study, we present evidence that paternal hypomethylation can bypass the interploidy hybridization barrier by alleviating the requirement for the Polycomb Repressive Complex 2 (PRC2) in the endosperm. PRC2 epigenetically regulates gene expression by applying methylation marks on histone H3. Bypass of the barrier is mediated by suppressed expression of imprinted genes. We show that the hypomethylated pollen genome causes de novo CHG methylation directed to FIS-PRC2 target genes, suggesting that different epigenetic modifications can functionally substitute for each other. Our work presents a method for the generation of viable triploids, providing an impressive example of the potential of epigenome manipulations for plant breeding.

The regulation of parental genome dosage is of fundamental importance in animals and plants, exemplified by X chromosome inactivation and dosage compensation. The “triploid block” is a classical example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number 1,2. This barrier acts in the endosperm, an ephemeral tissue that nurtures the developing embryo and induces the abortion of hybrid seeds through a yet unknown mechanism. Interploidy hybridizations involving diploid (2×) maternal parents and tetraploid (4×) pollen donors cause failure in endosperm cellularization, leading to embryo arrest 3. Here we show that paternal epigenetically activated small interfering RNAs (easiRNAs) are responsible for the establishment of the triploid block-associated seed abortion in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). We propose that excess of paternally derived easiRNAs in diploid pollen prevents establishment of DNA methylation, leading to triploid seed abortion. Our data further suggest that easiRNAs form a quantitative signal for chromosome number and their balanced dosage is required for post-fertilization genome stability and seed viability.