Genomic imprinting and endosperm development in flowering plants

Genomic imprinting, the parent-of-origin-specific expression of genes, plays an important role in the seed development of flowering plants. As different sets of genes are imprinted and hence silenced in maternal and paternal gametophyte genomes, the contributions of the parental genomes to the offspring are not equal. Imbalance between paternally and maternally imprinted genes, for instance as a result of interploidy crosses, or in seeds in which imprinting has been manipulated, results in aberrant seed development. It is predominantly the endosperm, and not or to a far lesser extent the embryo, that is affected by such imbalance. Deviation from the normal 2m:1p ratio in the endosperm genome has a severe effect on endosperm development, and often leads to seed abortion. Molecular expression data for imprinted genes suggest that genomic imprinting takes place only in the endosperm of the developing seed. Although far from complete, a picture of how imprinting operates in flowering plants has begun to emerge. Imprinted genes on either the maternal or paternal side are marked and silenced in a process involving DNA methylation and chromatin condensation. In addition, on the maternal side, imprinted genes are most probably under control of the polycomb FIS genes.     

Mutants in the imprinted PICKLE RELATED 2 gene suppress seed abortion of fertilization independent seed class mutants and paternal excess interploidy crosses in Arabidopsis

Endosperm cellularization is essential for embryo development and viable seed formation. Loss of function of the FERTILIZATION INDEPENDENT SEED (FIS) class Polycomb genes, which mediate trimethylation of histone H3 lysine27 (H3K27me3), as well as imbalanced contributions of parental genomes interrupt this process. The causes of the failure of cellularization are poorly understood. In this study we identified PICKLE RELATED 2 (PKR2) mutations which suppress seed abortion in fis1/mea by restoring endosperm cellularization. PKR2, a paternally expressed imprinted gene (PEG), encodes a CHD3 chromatin remodeler. PKR2 is specifically expressed in syncytial endosperm and its maternal copy is repressed by FIS1. Seed abortion in a paternal genome excess interploidy cross was also partly suppressed by pkr2. Simultaneous mutations in PKR2 and another PEG, ADMETOS (ADM), additively rescue the seed abortion in fis1 and in the interploidy cross, suggesting that PKR2 and ADM modulate endosperm cellularization independently and reproductive isolation between plants of different ploidy is established by imprinted genes. Genes upregulated in fis1 and downregulated in the presence of pkr2 are enriched in glycosyl‐hydrolyzing activity, while genes downregulated in fis1 and upregulated in the presence of pkr2 are enriched with microtubule motor activity, consistent with the cellularization patterns in fis1 and the suppressor line. The antagonistic functions of FIS1 and PKR2 in modulating endosperm development are similar to those of PICKLE (PKL) and CURLY LEAF (CLF), which antagonistically regulate root meristem activity. Our results provide further insights into the function of imprinted genes in endosperm development and reproductive isolation.     

Balance between maternal and paternal alleles sets the timing of resource accumulation in the maize endosperm

Key aspects of seed development in flowering plants are held to be under epigenetic control and to have evolved as a result of conflict between the interests of the male and female gametes (kinship theory). Attempts to identify the genes involved have focused on imprinted sequences, although imprinting is only one mechanism by which male or female parental alleles may be exclusively expressed immediately post-fertilization. We have studied the expression of a subset of endosperm gene classes immediately following interploidy crosses in maize and show that departure from the normal 2 : 1 ratio between female and male genomes exerts a dramatic effect on the timing of expression of some, but not all, genes investigated. Paternal genomic excess prolongs the expression of early genes and delays accumulation of reserves, while maternal genomic excess foreshortens the expression period of early genes and dramatically brings forward endosperm maturation. Our data point to a striking interdependence between the phases of endosperm development, and are consonant with previous work from maize showing progression from cell proliferation to endoreduplication is regulated by the balance between maternal and paternal genomes, and from Arabidopsis suggesting that this ‘phasing’ is regulated by maternally expressed imprinted genes. Our findings are discussed in context of the kinship theory.     

Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza

In most eudicot and monocot species, interspecific and interploidy crosses generally display abnormalities in the endosperm that are the major cause of a post‐zygotic hybridization barrier. In some eudicot species, however, this type of hybridization barrier can be overcome by the manipulation of ploidy levels of one parental species, suggesting that the molecular mechanisms underlying the species hybridization barrier can be circumvented by genome dosage. We previously demonstrated that endosperm barriers in interspecific and interploidy crosses in the genus Oryza involve overlapping but different mechanisms. This result contrasts with those in the genus Arabidopsis, which shows similar outcomes in both interploidy and interspecific crosses. Therefore, we postulated that an exploration of pathways for overcoming the species hybridization barrier in Oryza endosperm, by manipulating the ploidy levels in one parental species, might provide novel insights into molecular mechanisms. We showed that fertile hybrid seeds could be produced by an interspecific cross of female tetraploid Oryza sativa and male diploid Oryza longistaminata. Although the rate of nuclear divisions did not return to normal levels in the hybrid endosperm, the timing of cellularization, nucellus degeneration and the accumulation of storage products were close to normal levels. In addition, the expression patterns of the imprinted gene MADS87 and YUCCA11 were changed when the species barrier was overcome. These results suggest that the regulatory machinery for developmental transitions and imprinted gene expression are likely to play a central role in overcoming species hybridization barriers by genome dosage in the genus Oryza.     

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.     

Aberrant endosperm development in interploidy crosses reveals a timer of differentiation

The common assumption that the seed failure in interploidy crosses of flowering plants is due to parental genomic imprinting is based on vague interpretations and needs reevaluation since the general question is involved, how differentiation is timed so that cell progenies, while specializing, pass through proper numbers of amplification divisions before proliferation ceases. As recently confirmed, endosperm differentiation is accelerated or de-accelerated, depending upon whether polyploid females are crossed with diploid males, or vice-versa. Unlike the zygote, the first cell of the endosperm is determined to produce a tissue that successively induces growth of maternal tissues, stimulates and nourishes the embryo, and finally ceases cell cycling. Altered timing of endosperm differentiation, thus, perturbs seed development. During fertilization, only the female genomes contribute cytoplasmic equivalents to endosperm development so that in interploidy crosses, the initial amount of cytoplasm per chromosome set is altered, and due to semi-autonomy of cytoplasmic growth, altered numbers of division cycles are needed to provide the amount of cytoplasmic organelles required for differentiation. Cytoplasmic semi-autonomy and dependence of differentiation on an increase in cytoplasm has been shown in other tissues of plants and animals, thus, revealing a common mechanism for intracellular timing of differentiation. As demonstrated, imprinted genes can alter the extent of cell proliferation by interfering with this mechanism.     

Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.     

Cell cycle progression during endosperm development in Zea mays depends on parental dosage effects

Interploidy crosses in flowering plants often cause seed abortion. Studies in maize have shown that failure of kernel development results from dosage effects among products of imprinted but as-yet-unknown genes in the endosperm, and that the operative stoichiometry is established for a ratio of two maternal genomes to one paternal genome. In this study, we used flow cytometry to monitor cell cycle activities in developing endosperms obtained after reciprocal crosses between diploid and tetraploid maize individuals. Our data show that dosage effects alter critical events involved in the establishment of endoreduplication during maize endosperm development. Particularly, maternal genomic excess (4x x 2x crosses) forces endosperm cells to enter early into endoreduplication while paternal genomic excess (2x x 4x crosses) prevents its establishment. Our results also suggest that altering mechanisms depend on two different sets of cell cycle regulatory genes--one imprinted through the female that is required for mitotic arrest, and another responsible for re-entry into S phase that is imprinted through the male. Further, molecular and physiological analyses should provide insights into the interaction of parental imprinting action and cell cycle regulation during endosperm development.     

When genomes collide: aberrant seed development following maize interploidy crosses

Background and Aims: The results of wide- or interploidy crosses in angiosperms areunpredictable and often lead to seed abortion. The consequencesof reciprocal interploidy crosses have been explored in maizein detail, focusing on alterations to tissue domains in themaize endosperm, and changes in endosperm-specific gene expression. Methods: Following reciprocal interploidy crosses between diploid andtetraploid maize lines, development of endosperm domains wasstudied using GUS reporter lines, and gene expression in resultingkernels was investigated using semi-quantitative RT-PCR on endospermsisolated at different stages of development. Key Results: Reciprocal interploidy crosses result in very small, largelyinfertile seeds with defective endosperms. Seeds with maternalgenomic excess are smaller than those with paternal genomicexcess, their endosperms cellularize earlier and they accumulatesignificant quantities of starch. Endosperms from the reciprocalcross undergo an extended period of cell proliferation, andaccumulate little starch. Analysis of reporter lines and geneexpression studies confirm that functional domains of the endospermare severely disrupted, and are modified differently accordingto the direction of the interploidy cross. Conclusions: Interploidy crosses affect factors which regulate the balancebetween cell proliferation and cell differentiation within theendosperm. In particular, unbalanced crosses in maize affecttransfer cell differentiation, and lead to the temporal deregulationof the ontogenic programme of endosperm development.     

Bypassing reproductive barriers in hybrid seeds using chemically induced epimutagenesis

The triploid block, which prevents interploidy hybridizations in flowering plants, is characterized by a failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed genes (PEGs), which are upregulated in tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in the CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of “epimutagenized” plants, which may allow epialleles involved in the triploid block response to be identified in future studies. Finally, we demonstrate that chemically induced epimutagenesis facilitates hybridization between different Capsella species, thus potentially emerging as a strategy for producing triploids and interspecific hybrids with high agronomic interest.

Genome-wide loss of DNA methylation induced by 5-Azacytidine allows interploidy and interspecific hybridization barriers to be bypassed in Arabidopsis and Capsella.     

The occurrence of phenotypically complementary apomixis-recombinants in crosses between sexual and apomictic dandelions (<Emphasis Type="Italic">Taraxacum officinale</Emphasis>)

Apomictic seed development in dandelion ( Taraxacum officinale) involves (1) restitutional meiosis (diplospory), (2) egg cell parthenogenesis, and (3) autonomous endosperm development. The question is whether these elements of apomixis are controlled by one single gene or by several independent genes. Five triploid non-apomictic hybrids, obtained in diploid sexual × triploid apomict crosses were characterized using cyto-embryological and genetic methods. Nomarski-differential interference contrast microscopy and the transmission of microsatellite markers and ploidy levels indicated that the hybrids combined elements of the apomictic and the sexual developmental pathway. Hybrids form two complementary groups with respect to the presence or absence of parthenogenesis and autonomous endosperm development. The occurrence of complementary apomixis-recombinants suggests that parthenogenesis and autonomous endosperm development in Taraxacum are regulated independently by different genes. This study also indicates that early embryo development is independent of endosperm formation, but that endosperm is essential for later embryo growth.     

Diploid endosperm formation in Tulipa spp. and identification of a 1:1 maternal-to-paternal genome ratio in endosperms of T. gesneriana L.

Most Liliaceae plants have the tetrasporic Fritillaria-type embryo sac and normally form diploid embryos and pentaploid endosperms derived from a 4:1 maternal-to-paternal genome ratio (4m:1p) after double fertilization. Here we characterize embryo sac and endosperm formation in Tulipa spp. of Liliaceae. Chromosome analysis using seeds derived from 2x × 2x crosses of Tulipa gesneriana (2n = 2x = 24) identified diploid chromosome number in the endosperm. Similarly, flow cytometric analysis confirmed diploid endosperm formation in T. gesneriana, T. fosteriana (2n = 2x = 24) and T. greigii (2n = 2x = 24). To further study the possible mechanism of diploid endosperm formation, we made interploidy crosses of triploid (2n = 3x = 36) × diploid in which aneuploid seeds with various chromosome numbers (2n = 25–36) were produced. Again, flow cytometric analysis confirmed the same ploidy level in both embryos and endosperms at all aneuploidy levels, suggesting that only a single haploid polar nucleus contributes to endosperm formation at fertilization. Histological observation further confirmed the physical separation of two polar nuclei by a large vacuole in the Fritillaria-type embryo sac of T. gesneriana that appeared to prevent the fusion of the two polar nuclei that originated at the micropylar and chalazal ends before fertilization. Taken together, these results indicate that diploid endosperms (1m:1p) are normally formed in Tulipa spp. by fusion of the micropylar polar nucleus (n) and a spermatid (n) but not by normal triple fusion. We also show that tulip endosperm partially overcomes the triploid block mechanism that occurs in interploidy crosses. Based on these observations, the possible role of triple nuclear fusion in double fertilization is discussed.     

The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species

The success or failure of interspecific crosses is vital to evolution and to agriculture, but much remains to be learned about the nature of hybridization barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Another explanation is imbalance of paternally and maternally imprinted genes in the endosperm. Interspecific crosses between diploid Arabidopsis thaliana as the seed parent and tetraploid Arabidopsis arenosa as the pollen parent produced seeds that aborted with the same paternal excess endosperm phenotype seen in crosses between diploid and hexaploid A. thaliana. Doubling maternal ploidy restored seed viability and normal endosperm morphology. However, substituting a hypomethylated tetraploid A. thaliana seed parent reestablished the hybridization barrier by causing seed abortion and a lethal paternal excess phenotype. We conclude from these findings that the dominant cause of seed abortion in the diploid A. thaliana x tetraploid A. arenosa cross is parental genomic imbalance. Our results also demonstrate that manipulation of DNA methylation can be sufficient to erect hybridization barriers, offering a potential mechanism for speciation and a means of controlling gene flow between species.     

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.     

<Emphasis Type="Italic">Cdkn1c</Emphasis> (<Emphasis Type="Italic">p57</Emphasis><Superscript><Emphasis Type="Italic">Kip2</Emphasis></Superscript>) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7

Background  

Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.     

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.