Genome‐wide screen of genes imprinted in sorghum endosperm,and the roles of allelic differential cytosine methylation

Imprinting is an epigenetic phenomenon referring to allele‐biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species‐specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent‐of‐origin expression patterns in the endosperm of a pair of reciprocal F₁ hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum‐specific imprinted genes relative to these three plant species. Allele‐biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty‐six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT–PCR, and the majority of them showed endosperm‐specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5’ upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele‐differential methylation.     

High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm

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.     

Wild tomato endosperm transcriptomes reveal common roles of genomic imprinting in both nuclear and cellular endosperm

Genomic imprinting is a conspicuous feature of the endosperm, a triploid tissue nurturing the embryo and synchronizing angiosperm seed development. An unknown subset of imprinted genes (IGs) is critical for successful seed development and should have highly conserved functions. Recent genome‐wide studies have found limited conservation of IGs among distantly related species, but there is a paucity of data from closely related lineages. Moreover, most studies focused on model plants with nuclear endosperm development, and comparisons with properties of IGs in cellular‐type endosperm development are lacking. Using laser‐assisted microdissection, we characterized parent‐specific expression in the cellular endosperm of three wild tomato lineages (Solanum section Lycopersicon). We identified 1025 candidate IGs and 167 with putative homologs previously identified as imprinted in distantly related taxa with nuclear‐type endosperm. Forty‐two maternally expressed genes (MEGs) and 17 paternally expressed genes (PEGs) exhibited conserved imprinting status across all three lineages, but differences in power to assess imprinted expression imply that the actual degree of conservation might be higher than that directly estimated (20.7% for PEGs and 10.4% for MEGs). Regardless, the level of shared imprinting status was higher for PEGs than for MEGs, indicating dissimilar evolutionary trajectories. Expression‐level data suggest distinct epigenetic modulation of MEGs and PEGs, and gene ontology analyses revealed MEGs and PEGs to be enriched for different functions. Importantly, our data provide evidence that MEGs and PEGs interact in modulating both gene expression and the endosperm cell cycle, and uncovered conserved cellular functions of IGs uniting taxa with cellular‐ and nuclear‐type endosperm.     

Genome-wide analysis of genomic imprinting in the endosperm and allelic variation in flax

Genomic imprinting is an epigenetic phenomenon that causes biased expression of maternally and paternally inherited alleles. In flowering plants, genomic imprinting predominantly occurs in the triploid endosperm and plays a vital role in seed development. In this study, we identified 248 candidate imprinted genes including 114 maternally expressed imprinted genes (MEGs) and 134 paternally expressed imprinted genes (PEGs) in flax (Linum usitatissimum L.) endosperm using deep RNA sequencing. These imprinted genes were neither clustered in specific chromosomal regions nor well conserved among flax and other plant species. MEGs tended to be expressed specifically in the endosperm, whereas the expression of PEGs was not tissue-specific. Imprinted single nucleotide polymorphisms differentiated 200 flax cultivars into the oil flax, oil-fiber dual purpose flax and fiber flax subgroups, suggesting that genomic imprinting contributed to intraspecific variation in flax. The nucleotide diversity of imprinted genes in the oil flax subgroup was significantly higher than that in the fiber flax subgroup, indicating that some imprinted genes underwent positive selection during flax domestication from oil flax to fiber flax. Moreover, imprinted genes that underwent positive selection were related to flax functions. Thirteen imprinted genes related to flax seed size and weight were identified using a candidate gene-based association study. Therefore, our study provides information for further exploration of the function and genomic variation of imprinted genes in the flax population.     

Imprinting in Plants and Its Underlying Mechanisms

Genomic imprinting (or imprinting) refers to an epigenetic phenomenon by which the allelic expression of a gene depends on the parent of origin. It has evolved independently in placental mammals and flowering plants. In plants, imprinting is mainly found in endosperm. Recent genome-wide surveys in Arabidopsis, rice, and maize identified hundreds of imprinted genes in endosperm. Since these genes are of diverse functions, endosperm development is regulated at different regulatory levels. The imprinted expression of only a few genes is conserved between Arabidopsis and monocots, suggesting that imprinting evolved quickly during speciation. In Arabidopsis, DEMETER (DME) mediates hypomethylation in the maternal genome at numerous loci (mainly transposons and repeats) in the central cell and results in many differentially methylated regions between parental genomes in the endosperm, and subsequent imprinted expression of some genes. In addition, histone modification mediated by Polycomb group (PcG) proteins is also involved in regulating imprinting. DME-induced hypomethylated alleles in the central cell are considered to produce small interfering RNAs (siRNAs) which are imported to the egg to reinforce DNA methylation. In parallel, the activity of DME in the vegetative cell of the male gametophyte demethylates many regions which overlap with the demethylated regions in the central cell. siRNAs from the demethylated regions are hypothesized to be also transferred into sperm to reinforce DNA methylation. Imprinting is partly the result of genome-wide epigenetic reprogramming in the central cell and vegetative cell and evolved under different selective pressures.     

Sequential gene activation and gene imprinting during early embryo development in maize

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high‐throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3–13 days after pollination, DAP) to analyze allelic gene expression patterns. Co‐expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty‐four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5–13 DAP (more imprinted genes at 5 DAP). Forty‐one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.     

植物胚乳发育的表观遗传学调控

被子植物的种子发育从双受精开始, 产生二倍体的胚和三倍体的胚乳。在种子发育和萌发过程中, 胚乳向胚组织提供营养物质, 因此胚乳对胚和种子的正常生长发育至关重要。开花植物发生基因组印迹的主要器官是胚乳。印迹基因的表达受表观遗传学机制的调控, 包括DNA甲基化和组蛋白H3K27甲基化修饰以及依赖于PolIV的siRNAs (p4-siRNAs)调控。基因组印迹的表观遗传学调控对胚乳的正常发育和种子育性具有不可或缺的重要作用。最新研究显示, 胚乳的整个基因组DNA甲基化水平降低, 而且去甲基化作用可能源于雌配子体的中央细胞。该文综述了种子发育的表观遗传学调控机制, 包括基因组印迹机制以及胚乳基因组DNA甲基化变化研究的最新进展。     

Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.     

Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5'-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5'-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.     

A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

Adenine methylation in zein genes

This paper reports the novel finding of adenine methylation in higher plants. Comparison of restriction patterns of genomic maize DNA digested with enzymes MboI and Sau3A enabled us to detect the existence of adenine methylation in zein genes. Adenine methylation within or around zein genes turned out to be similar in endosperm (where zeins are actively synthesized) and in seedling tissue (where zein genes are not expressed). Furthermore, adenine methylation patterns were found to be similar both in wild-type and opaque-2 mutant plants. These lines of evidence suggest that adenine methylation is unrelated to the regulation of gene expression.     

Genome-wide survey of imprinted genes

The developmental failure of mammalian parthenogenote has been a mystery for a long time and posed a question as to why bi-parental reproduction is necessary for development to term. In the 1980s, it was proven that this failure was not due to the genetic information itself, but to epigenetic modification of genomic DNA. In the following decade, several studies successfully identified imprinted genes which were differentially expressed in a parent-of-origin-specific manner, and it was shown that the differential expression depended on the pattern of DNA methylation. These facts prompted development of genome-wide systematic screening methods based on DNA methylation and differential gene expression to identify imprinted genes. Recently computational approaches and microarray technology have been introduced to identify imprinted genes/loci, contributing to the expansion of our knowledge. However, it has been shown that the gene silencing derived from genomic imprinting is accomplished by several mechanisms in addition to direct DNA methylation, indicating that novel approaches are further required for comprehensive understanding of genomic imprinting. To unveil the mechanism of developmental failure in mammalian parthenogenote, systematic screenings for imprinted genes/loci have been developed. In this review, we describe genomic imprinting focusing on the history of genome-wide screening.     

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.     

Reproductive barrier and genomic imprinting in the endosperm of flowering plants

In flowering plants, success or failure of seed development is determined by various genetic mechanisms. During sexual reproduction, double fertilization produces the embryo and endosperm, which both contain maternally and paternally derived genomes. In endosperm, a reproductive barrier is often observed in inter-specific crosses. Endosperm is a tissue that provides nourishment for the embryo within the seed, in a similar fashion to the placenta of mammals, and for the young seedling after germination. This review considers the relationship between the reproductive barrier in endosperm and genomic imprinting. Genomic imprinting is an epigenetic mechanism that results in mono-allelic gene expression that is parent-of-origin dependent. In Arabidopsis, recent studies of several imprinted gene loci have identified the epigenetic mechanisms that determine genomic imprinting. A crucial feature of genomic imprinting is that the maternally and paternally derived imprinted genes must carry some form of differential mark, usually DNA methylation and/or histone modification. Although the epigenetic marks should be complementary on maternally and paternally imprinted genes within a single species, it is possible that neither the patterns of epigenetic marks nor expression of imprinted genes are the same in different species. Moreover, in hybrid endosperm, the regulation of expression of imprinted genes can be affected by upstream regulatory mechanisms in the male and female gametophytes. Species-specific variations in epigenetic marks, the copy number of imprinted genes, and the epigenetic regulation of imprinted genes in hybrids might all play a role in the reproductive barriers observed in the endosperm of interspecific and interploidy crosses. These predicted molecular mechanisms might be related to earlier models such as the "endosperm balance number" (EBN) and "polar nuclei activation" (PNA) hypotheses.