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1.  Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa 
BMC Genomics  2010;11:338.
Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots.
We used Affymetrix GeneChip® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each.
Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity pre-synthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination.
PMCID: PMC2895629  PMID: 20507633
2.  Quantitative Genetics Identifies Cryptic Genetic Variation Involved in the Paternal Regulation of Seed Development 
PLoS Genetics  2016;12(1):e1005806.
Embryonic development requires a correct balancing of maternal and paternal genetic information. This balance is mediated by genomic imprinting, an epigenetic mechanism that leads to parent-of-origin-dependent gene expression. The parental conflict (or kinship) theory proposes that imprinting can evolve due to a conflict between maternal and paternal alleles over resource allocation during seed development. One assumption of this theory is that paternal alleles can regulate seed growth; however, paternal effects on seed size are often very low or non-existent. We demonstrate that there is a pool of cryptic genetic variation in the paternal control of Arabidopsis thaliana seed development. Such cryptic variation can be exposed in seeds that maternally inherit a medea mutation, suggesting that MEA acts as a maternal buffer of paternal effects. Genetic mapping using recombinant inbred lines, and a novel method for the mapping of parent-of-origin effects using whole-genome sequencing of segregant bulks, indicate that there are at least six loci with small, paternal effects on seed development. Together, our analyses reveal the existence of a pool of hidden genetic variation on the paternal control of seed development that is likely shaped by parental conflict.
Author Summary
In plants and mammals, embryo development occurs under the protection and nourishment of maternal tissues. In polygamous species, this can lead to competition between siblings for privileged access to maternal nutrients. According to the parental conflict theory—a variation of the kinship theory—the asymmetric genetic relatedness between offspring from multiple fathers may lead to the evolution of parent-of-origin-dependent developmental regulation; paternally inherited alleles would benefit from maximizing embryo growth (at the expense of siblings), whereas maternally inherited alleles would benefit from restraining growth (to equalize sibling resource allocation). The kinship theory assumes that the paternal genome can actually influence embryo development; however, plant seed development is under strong maternal control. Here, we show that there is a hidden pool of variation in paternal effect loci that can be released upon loss of MEDEA, a major maternal regulator of seed development. Our results demonstrate that the maternal genome actively buffers the effects of paternal genomes on seed development, thereby providing strong functional support to the parental conflict theory.
PMCID: PMC4727937  PMID: 26811909
3.  Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins 
PLoS Genetics  2007;3(10):e171.
The female gametophyte of flowering plants, the embryo sac, develops within the diploid (sporophytic) tissue of the ovule. While embryo sac–expressed genes are known to be required at multiple stages of the fertilization process, the set of embryo sac–expressed genes has remained poorly defined. In particular, the set of genes responsible for mediating intracellular communication between the embryo sac and the male gametophyte, the pollen grain, is unknown. We used high-throughput cDNA sequencing and whole-genome tiling arrays to compare gene expression in wild-type ovules to that in dif1 ovules, which entirely lack embryo sacs, and myb98 ovules, which are impaired in pollen tube attraction. We identified nearly 400 genes that are downregulated in dif1 ovules. Seventy-eight percent of these embryo sac–dependent genes were predicted to encode for secreted proteins, and 60% belonged to multigenic families. Our results define a large number of candidate extracellular signaling molecules that may act during embryo sac development or fertilization; less than half of these are represented on the widely used ATH1 expression array. In particular, we found that 37 out of 40 genes encoding Domain of Unknown Function 784 (DUF784) domains require the synergid-specific transcription factor MYB98 for expression. Several DUF784 genes were transcribed in synergid cells of the embryo sac, implicating the DUF784 gene family in mediating late stages of embryo sac development or interactions with pollen tubes. The coexpression of highly similar proteins suggests a high degree of functional redundancy among embryo sac genes.
Author Summary
During the sexual reproduction of flowering plants, a pollen tube delivers sperm cells to a specialized group of cells known as the embryo sac, which contains the egg cell. It is known that embryo sacs are active participants in guiding the growth of pollen tubes, in facilitating fertilization, and in initiating seed development. However, the genes responsible for the complex biology of embryo sacs are poorly understood. The authors use two recently developed technologies, whole-genome tiling microarrays and high-throughput cDNA sequencing, to identify hundreds of genes expressed in embryo sacs of Arabidopsis thaliana. Most embryo sac–dependent genes have no known function, and include entire families of related genes that are only expressed in embryo sacs. Furthermore, most embryo sac–dependent genes encode small proteins that are potentially secreted from their cells of origin, suggesting that they may act as intracellular signals or to modify the extracellular matrix during fertilization or embryo sac development. These results illustrate the extent to which our understanding of plant sexual reproduction is limited and identifies hundreds of candidate genes for future studies investigating the molecular biology of the embryo sac.
PMCID: PMC2014789  PMID: 17937500
4.  Round Spermatid Injection Rescues Female Lethality of a Paternally Inherited Xist Deletion in Mouse 
PLoS Genetics  2016;12(10):e1006358.
In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.
Author Summary
In sexual reproduction, maternal and paternal haploid sets of DNA are combined in one new diploid individual. However, not only DNA, but also epigenetic information, defined by DNA and histone modifications, is transferred to the zygote. Specific inactivation of the paternally inherited X chromosome (Xp) in the preimplantation female mouse embryo is required for embryo survival. This imprinted X chromosome inactivation (iXCI) is initiated by transcription of the Xist gene from Xp. In contrast, the maternal Xist gene is imprinted during oogenesis to remain silent. We have investigated the consequences of elimination of the histone-to-protamine and protamine-to-histone transitions on iXCI, by fertilization through injection of immature round spermatids into oocytes (ROSI). Interestingly, when the round spermatids used for ROSI carried an X chromosome with an Xist deletion (ΔXist), we found that the Xist gene on the maternal X chromosome was activated, which rescued the female lethality of embryos that is invariably observed upon fertilization with mature ΔXist spermatozoa. This striking result is best explained by deregulation of embryonic gene expression, in particular from Xp, when the paternal genome originates from round spermatids rather than spermatozoa. From this, we suggest that the use of round spermatids has unforeseen consequences for embryonic gene expression and its use in human assisted reproduction must be carefully considered.
PMCID: PMC5065126  PMID: 27716834
5.  The impact of plant and flower age on mating patterns 
Annals of Botany  2009;105(1):7-22.
Over a season, plant condition, amount of ongoing reproduction and biotic and abiotic environmental factors vary. As flowers age, flower condition and amount of pollen donated and received also vary. These internal and external changes are significant for fitness if they result in changes in reproduction and mating.
Literature from several fields was reviewed to provide a picture of the changes that occur in plants and flowers that can affect mating over a season. As flowers age, both the entire flower and individual floral whorls show changes in appearance and function. Over a season, changes in mating often appear as alteration in seed production vs. pollen donation. In several species, older, unpollinated flowers are more likely to self. If flowers are receiving pollen, staying open longer may increase the number of mates. In wild radish, for which there is considerable information on seed paternity, older flowers produce fewer seeds and appear to discriminate less among pollen donors. Pollen donor performance can also be linked to maternal plant age. Different pollinators and mates are available across the season. Also in wild radish, maternal plants appear to exert the most control over paternity when they are of intermediate age.
Although much is known about the characters of plants and flowers that can change over a season, there is less information on the effects of age on mating. Several studies document changes in self-pollination over time, but very few, other than those on wild radish, consider more subtle aspects of differential success of pollen donors over time.
PMCID: PMC2794063  PMID: 19875519
Mating patterns; plant age; flower age; wild radish; Raphanus
6.  Parental genetic distance and patterns in nonrandom mating and seed yield in predominately selfing Arabidopsis thaliana 
Plant Reproduction  2013;26(4):317-328.
In this study, we ask two questions: (1) Is reproductive success independent of parental genetic distance in predominately selfing plants? (2) In the absence of early inbreeding depression, is there substantial maternal and/or paternal variation in reproductive success in natural populations? Seed yield in single pollinations and proportion of seeds sired in mixed pollinations were studied in genetically defined accessions of the predominately selfing plant Arabidopsis thaliana by conducting two diallel crosses. The first diallel was a standard, single pollination design that we used to examine variance in seed yield. The second diallel was a mixed pollination design that utilized a standard pollen competitor to examine variance in proportion of seeds sired. We found no correlation between reproductive success and parental genetic distance, and self-pollen does not systematically differ in reproductive success compared to outcross pollen, suggesting that Arabidopsis populations do not experience embryo lethality due to early-acting inbreeding or outbreeding depression. We used these data to partition the contributions to total phenotypic variation from six sources, including maternal contributions, paternal contributions and parental interactions. For seed yield in single pollinations, maternal effects accounted for the most significant source of variance (16.6 %). For proportion of seeds sired in mixed pollinations, the most significant source of variance was paternal effects (17.9 %). Thus, we show that population-level genetic similarities, including selfing, do not correlate with reproductive success, yet there is still significant paternal variance under competition. This suggests two things. First, since these differences are unlikely due to early-acting inbreeding depression or differential pollen viability, this implicates natural variation in pollen germination and tube growth dynamics. Second, this strongly supports a model of fixation of pollen performance genes in populations, offering a focus for future genetic studies in differential reproductive success.
PMCID: PMC3825607  PMID: 23843176
Mate choice; Nonrandom mating; Seed yield; Inbreeding; Diallel; Pollen competition
7.  Pre- and Post-harvest Influences on Seed Dormancy Status of an Australian Goodeniaceae species, Goodenia fascicularis 
Annals of Botany  2008;102(1):93-101.
Background and Aims
The period during which seeds develop on the parent plant has been found to affect many seed characteristics, including dormancy, through interactions with the environment. Goodenia fascicularis (Goodeniaceae) seeds were used to investigate whether seeds of an Australian native forb, harvested from different environments and produced at different stages of the reproductive period, differ in dormancy status.
During the reproductive phase, plants were grown ex situ in warm (39/21 °C) or cool (26/13 °C) conditions, with adequate or limited water availability. The physiological dormancy of resulting seeds was measured in terms of the germination response to warm stratification (34/20 °C, 100 % RH, darkness).
Key Results
Plants in the cool environment were tall and had high above-ground biomass, yet yielded fewer seeds over a shorter, later harvest period when compared with plants in the warm environment. Seeds from the cool environment also had higher viability and greater mass, despite a significant proportion (7 % from the cool-wet environment) containing no obvious embryo. In the warm environment, the reproductive phase was accelerated and plants produced more seeds despite being shorter and having lower above-ground biomass than those in the cool environment. Ten weeks of warm stratification alleviated physiological dormancy in seeds from all treatments resulting in 80–100 % germination. Seeds that developed at warm temperatures were less dormant (i.e. germination percentages were higher) than seeds from the cool environment. Water availability had less effect on plant and seed traits than air temperature, although plants with reduced soil moisture were shorter, had lower biomass and produced fewer, less dormant seeds than plants watered regularly.
Goodenia fascicularis seeds are likely to exhibit physiological dormancy regardless of the maternal environment. However, seeds collected from warm, dry environments are likely to be more responsive to warm stratification than seeds from cooler, wetter environments.
PMCID: PMC2712421  PMID: 18430743
Goodenia fascicularis; Goodeniaceae; Australia; physiological dormancy; seeds; temperature; soil moisture; maternal influence; climate
8.  Effects of pollination timing on seed paternity and seed mass in Silene latifolia (Caryophyllaceae) 
Annals of Botany  2009;104(4):767-773.
Background and Aims
Competition among genetically different pollen donors within one recipient flower may play an important role in plant populations, increasing offspring genetic diversity and vigour. However, under field conditions stochastic pollen arrival times may result in disproportionate fertilization success of the first-arriving pollen, even to the detriment of the recipient plant's and offspring fitness. It is therefore critical to evaluate the relative importance of arrival times of pollen from different donors in determining siring success.
Hand pollinations and genetic markers were used to investigate experimentally the effect of pollination timing on seed paternity, seed mass and stigmatic wilting in the the dioecious plant Silene latifolia. In this species, high prevalence of multiply-sired fruits in natural populations suggests that competition among different donors may often take place (at fertilization or during seed development); however, the role of variation due to pollen arrival times is not known.
Key Results
First-arriving pollen sired significantly more seeds than later-arriving pollen. This advantage was expressed already before the first pollen tubes could reach the ovary. Simultaneously with pollen tube growth, the stigmatic papillae wilted visibly. Individual seeds were heavier in fruits where one donor sired most seeds than in fruits where both donors had more even paternity shares.
In field populations of S. latifolia, fruits are often multiply-sired. Because later-arriving pollen had decreased chances of fertilizing the ovules, this implies that open-pollinated flowers often benefit from pollen carry-over or pollinator visits within short time intervals, which may contribute to increase offspring genetic diversity and fitness.
PMCID: PMC2729624  PMID: 19567418
Reproduction; reproductive success; pollen; siring success; microsatellite DNA; paternity; pollen tube growth; seed mass; Silene alba; stigma wilting
9.  Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis 
PLoS Genetics  2009;5(9):e1000663.
Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues.
Author Summary
Crosses between plants of different ploidy often fail because seed development does not proceed normally and non-viable seeds are produced. It is assumed that abnormalities in growth and structure of the endosperm (the nutritional tissue of the seed) are the cause of triploid seed failure, consistent with the proposed role of the endosperm in reproductive isolation and angiosperm speciation. In many species, the ratio of maternal to paternal genomes in the endosperm is important for normal seed development, giving rise to the hypothesis that parent-of-origin–specific gene expression (imprinting) of regulatory genes in the endosperm is the underlying cause for developmental failure in seeds with deviating parental contributions. We tested this hypothesis using the jason mutant that forms unreduced male gametes and triploid seeds with increased paternal genome dosage. Based on the results of our study, we propose that imprinting of the FIS component MEDEA serves as a dosage sensor for increasing paternal genome contributions, establishing the molecular basis for dosage sensitivity. Our study provides strong evidence supporting the hypothesis that misbalanced expression of imprinted genes is the cause of seed development defects after interploidy crosses and demonstrates that MEDEA imprinting is a major origin of developmental defects caused by increased paternal genome contributions.
PMCID: PMC2738949  PMID: 19779546
10.  Genome-Wide Transcript Profiling of Endosperm without Paternal Contribution Identifies Parent-of-Origin–Dependent Regulation of AGAMOUS-LIKE36 
PLoS Genetics  2011;7(2):e1001303.
Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin–specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin–dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.
Author Summary
Seeds of flowering plants consist of three different organisms that develop in parallel. In contrast to animals, a double fertilization event takes place in plants, producing two fertilization products, the embryo and the endosperm. Imprinting, the parent-of-origin–specific expression of genes, typically takes place in the mammalian placenta and in the plant endosperm. A prevailing hypothesis predicts that a parental tug-of-war on the allocation of available recourses to the developing progeny has led to the evolution of imprinting systems where genes expressed from the mother dampen growth whereas genes expressed from the father are growth enhancers. The number of imprinted genes identified in plants is low compared to mammals, and this precludes the elucidation of the epigenetic mechanisms responsible for this specialized expression system. Here, we have used genome-wide transcript profiling of endosperm without paternal contribution to identify seed regulators and, among these, imprinted genes. We identified a cluster of downregulated MADS-box transcription factors, including AGL36, that was subsequently shown to be imprinted by an epigenetic mechanism involving the DNA methylase MET1 and the glycosylase DME. In addition, the expression of the active AGL36 allele was dampened by the FIS Polycomb Repressive Complex, identifying a novel mode of regulation of imprinted genes.
PMCID: PMC3040660  PMID: 21379330
11.  The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution 
PLoS Genetics  2010;6(11):e1001214.
In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
Author Summary
In mammals, a subset of genes is expressed from only one chromosomal copy, depending on its parental origin. This process, known as genomic imprinting, results from DNA methylation marks deposited in gametes at regulatory sequences called imprinting control regions (ICRs). Most of the DNA methylation controlling imprinting is established in the oocyte, while very few ICRs are methylated in the sperm. We provided insight into the impact and origins of the parental imbalance in genomic imprinting control. We defined the transcriptome-wide effect of imprinting, during the transition period when the embryo becomes dependent upon maternal resources. We found that maternal ICRs have a vital effect on developmental pathways related to the mother-to-fetus exchanges, while paternal ICRs have a dispersed and non-significant effect at that stage. We evidenced that paternal ICRs are lost at a much faster rate than maternal ICRs during mammalian evolution, probably as a mechanistic consequence of different kinetics of the parental germlines. Our results support the notion that two independent evolutionary forces have led to the numerical and functional dominance of maternal ICRs: a selective advantage of parent-specific regulation of genes important for the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
PMCID: PMC2987832  PMID: 21124941
12.  Mitotic fidelity requires transgenerational action of a testis-restricted HP1 
eLife  null;4:e07378.
Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. In this study, we show that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo.
eLife digest
The genetic information of cells is packaged into structures called chromosomes, which are made up of long strands of DNA that are wrapped around proteins to form a structure called chromatin. The cells of most animals contain two copies of every chromosome, but the egg and sperm cells contain only one copy. This means that when an egg fuses with a sperm cell during fertilization, the resulting ‘zygote’ will contain two copies of each chromosome—one inherited from the mother, and one from the father. These chromosomes duplicate and divide many times within the developing embryo in a process known as mitosis.
The first division of the zygote is particularly complicated, as the egg and sperm chromosomes must go through extensive—and yet different—chromatin reorganization processes. For instance, paternal DNA is inherited via sperm, where specialized sperm proteins package the DNA more tightly than in the maternal DNA, which is packaged by histone proteins used throughout development. For paternal DNA to participate in mitosis in the embryo, it must first undergo a transition to a histone-packaged state. Despite these differences, both maternal and paternal chromosomes must undergo mitosis at the same time if the zygote is to successfully divide. Although it is known that the egg cell contributes essential proteins that are incorporated into the sperm chromatin to help it reorganize, the importance of paternal proteins in coordinating this process remains poorly understood.
Many members of a family of proteins called Heterochromatin Protein 1 (or HP1 for short) have previously been shown to control chromatin organization in plants and animals. In 2012, researchers found that several HP1 proteins are found only in the testes of the fruit fly species Drosophila melanogaster. They predicted that these proteins might help control the reorganization of the paternal chromosomes following fertilization.
Levine et al.—including researchers involved in the 2012 study—have now used genetic and cell-based techniques to show that one member of the HP1 family (called HP1E) ensures that the paternal chromosomes are ready for cell division at the same time as the maternal chromosomes. Male flies that are unable to produce this protein do not have any offspring because, while these flies' sperm can fertilize eggs, the resulting zygotes cannot divide as normal.
Further experiments revealed that HP1E is not inherited through the chromatin of mature sperm, but instead influences the structure of the chromosomes during the final stages of the development of the sperm cells in the fly testes.
This study shows that both maternal and paternal proteins are needed to control how the paternal chromosomes reorganize in fruit fly embryos. Although difficult to discover and decipher, this work re-emphasizes the importance of paternal epigenetic contributions—changes that alter how DNA is read, without changing the DNA sequence itself—for ensuring the viability of resulting offspring. Future work will reveal both the molecular mechanism of this epigenetic transfer of information, as well as why certain Drosophila species are able to naturally overcome the loss of the essential HP1E protein.
PMCID: PMC4491702  PMID: 26151671
paternal effect lethal; karyotype evolution; sex chromosomes; heterochromatin protein; spermiogenesis; mitosis; D. melanogaster
13.  In Vitro Fertilization and Multiple Pregnancies 
Executive Summary
The objective of this health technology policy assessment was to determine the clinical effectiveness and cost-effectiveness of IVF for infertility treatment, as well as the role of IVF in reducing the rate of multiple pregnancies.
Clinical Need: Target Population and Condition
Typically defined as a failure to conceive after a year of regular unprotected intercourse, infertility affects 8% to 16% of reproductive age couples. The condition can be caused by disruptions at various steps of the reproductive process. Major causes of infertility include abnormalities of sperm, tubal obstruction, endometriosis, ovulatory disorder, and idiopathic infertility. Depending on the cause and patient characteristics, management options range from pharmacologic treatment to more advanced techniques referred to as assisted reproductive technologies (ART). ART include IVF and IVF-related procedures such as intra-cytoplasmic sperm injection (ICSI) and, according to some definitions, intra-uterine insemination (IUI), also known as artificial insemination. Almost invariably, an initial step in ART is controlled ovarian stimulation (COS), which leads to a significantly higher rate of multiple pregnancies after ART compared with that following natural conception. Multiple pregnancies are associated with a broad range of negative consequences for both mother and fetuses. Maternal complications include increased risk of pregnancy-induced hypertension, pre-eclampsia, polyhydramnios, gestational diabetes, fetal malpresentation requiring Caesarean section, postpartum haemorrhage, and postpartum depression. Babies from multiple pregnancies are at a significantly higher risk of early death, prematurity, and low birth weight, as well as mental and physical disabilities related to prematurity. Increased maternal and fetal morbidity leads to higher perinatal and neonatal costs of multiple pregnancies, as well as subsequent lifelong costs due to disabilities and an increased need for medical and social support.
The Technology Being Reviewed
IVF was first developed as a method to overcome bilateral Fallopian tube obstruction. The procedure includes several steps: (1) the woman’s egg is retrieved from the ovaries; (2) exposed to sperm outside the body and fertilized; (3) the embryo(s) is cultured for 3 to 5 days; and (4) is transferred back to the uterus. IFV is considered to be one of the most effective treatments for infertility today. According to data from the Canadian Assisted Reproductive Technology Registry, the average live birth rate after IVF in Canada is around 30%, but there is considerable variation in the age of the mother and primary cause of infertility.
An important advantage of IVF is that it allows for the control of the number of embryos transferred. An elective single embryo transfer in IVF cycles adopted in many European countries was shown to significantly reduce the risk of multiple pregnancies while maintaining acceptable birth rates. However, when number of embryos transferred is not limited, the rate of IVF-associated multiple pregnancies is similar to that of other treatments involving ovarian stimulation. The practice of multiple embryo transfer in IVF is often the result of pressures to increase success rates due to the high costs of the procedure. The average rate of multiple pregnancies resulting from IVF in Canada is currently around 30%.
An alternative to IVF is IUI. In spite of reported lower success rates of IUI (pregnancy rates per cycle range from 8.7% to 17.1%) it is generally attempted before IVF due to its lower invasiveness and cost.
Two major drawbacks of IUI are that it cannot be used in cases of bilateral tubal obstruction and it does not allow much control over the risk of multiple pregnancies compared with IVF. The rate of multiple pregnancies after IUI with COS is estimated to be about 21% to 29%.
Ontario Health Insurance Plan Coverage
Currently, the Ontario Health Insurance Plan covers the cost of IVF for women with bilaterally blocked Fallopian tubes only, in which case it is funded for 3 cycles, excluding the cost of drugs. The cost of IUI is covered except for preparation of the sperm and drugs used for COS.
Diffusion of Technology
According to Canadian Assisted Reproductive Technology Registry data, in 2004 there were 25 infertility clinics across Canada offering IVF and 7,619 IVF cycles performed. In Ontario, there are 13 infertility clinics with about 4,300 IVF cycles performed annually.
Literature Review
Royal Commission Report on Reproductive Technologies
The 1993 release of the Royal Commission report on reproductive technologies, Proceed With Care, resulted in the withdrawal of most IVF funding in Ontario, where prior to 1994 IVF was fully funded. Recommendations of the Commission to withdraw IVF funding were largely based on findings of the systematic review of randomized controlled trials (RCTs) published before 1990. The review showed IVF effectiveness only in cases of bilateral tubal obstruction. As for nontubal causes of infertility, there was not enough evidence to establish whether IVF was effective or not.
Since the field of reproductive technology is constantly evolving, there have been several changes since the publication of the Royal Commission report. These changes include: increased success rates of IVF; introduction of ICSI in the early 1990’s as a treatment for male factor infertility; and improved embryo implantation rates allowing for the transfer of a single embryo to avoid multiple pregnancies after IVF.
Studies After the Royal Commission Report: Review Strategy
Three separate literature reviews were conducted in the following areas: clinical effectiveness of IVF, cost-effectiveness of IVF, and outcomes of single embryo transfer (SET) in IVF cycles.
Clinical effectiveness of IVF: RCTs or meta-analyses of RCTs that compared live birth rates after IVF versus alternative treatments, where the cause of infertility was clearly stated or it was possible to stratify the outcome by the cause of infertility.
Cost effectiveness of IVF: All relevant economic studies comparing IVF to alternative methods of treatment were reviewed
Outcomes of IVF with SET: RCTs or meta-analyses of RCTs that compared live birth rates and multiple birth rates associated with transfer of single versus double embryos.
OVID MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, Cochrane Library, the International Agency for Health Technology Assessment database, and websites of other health technology assessment agencies were searched using specific subject headings and keywords to identify relevant studies.
Summary of Findings
Comparative Clinical Effectiveness of IVF
Overall, there is a lack of well composed RCTs in this area and considerable diversity in both definition and measurement of outcomes exists between trials. Many studies used fertility or pregnancy rates instead of live birth rates. Moreover, the denominator for rate calculation varied from study to study (e.g. rates were calculated per cycle started, per cycle completed, per couple, etc...).
Nevertheless, few studies of sufficient quality were identified and categorized by the cause of infertility and existing alternatives to IVF. The following are the key findings:
A 2005 meta-analysis demonstrated that, in patients with idiopathic infertility, IVF was clearly superior to expectant management, but there were no statistically significant differences in live birth rates between IVF and IUI, nor between IVF and gamete-intra-Fallopian transfer.
A subset of data from a 2000 study showed no significant differences in pregnancy rates between IVF and IUI for moderate male factor infertility.
In patients with moderate male factor infertility, standard IVF was also compared with ICSI in a 2002 meta-analysis. All studies included in the meta-analysis showed superior fertilization rates with ICSI, and the pooled risk ratio for oocyte fertilization was 1.9 (95% Confidence Interval 1.4-2.5) in favour of ICSI. Two other RCTs in this area published after the 2002 meta-analysis had similar results and further confirmed these findings. There were no RCTs comparing IVF with ICSI in patients with severe male factor infertility, mainly because based on the expert opinion, ICSI might only be an effective treatment for severe male factor infertility.
Cost-Effectiveness of IVF
Five economic evaluations of IVF were found, including one comprehensive systematic review of 57 health economic studies. The studies compared cost-effectiveness of IVF with a number of alternatives such as observation, ovarian stimulation, IUI, tubal surgery, varicocelectomy, etc... The cost-effectiveness of IVF was analyzed separately for different types of infertility. Most of the reviewed studies concluded that due to the high cost, IVF has a less favourable cost-effectiveness profile compared with alternative treatment options. Therefore, IVF was not recommended as the first line of treatment in the majority of cases. The only two exceptions were bilateral tubal obstruction and severe male factor infertility, where an immediate offer of IVF/ICSI might the most cost-effective option.
Clinical Outcomes After Single Versus Double Embryo Transfer Strategies of IVF
Since the SET strategy has been more widely adopted in Europe, all RCT outcomes of SET were conducted in European countries. The major study in this area was a large 2005 meta-analysis, followed by two other published RCTs.
All of these studies reached similar conclusions:
Although a single SET cycle results in lower birth rates than a single double embryo transfer (DET) cycle, the cumulative birth rate after 2 cycles of SET (fresh + frozen-thawed embryos) was comparable to the birth rate after a single DET cycle (~40%).
SET was associated with a significant reduction in multiple births compared with DET (0.8% vs. 33.1% respectively in the largest RCT).
Most trials on SET included women younger than 36 years old with a sufficient number of embryos available for transfer that allowed for selection of the top quality embryo(s). A 2006 RCT, however, compared SET and DET strategies in an unselected group of patients without restrictions on the woman’s age or embryo quality. This study demonstrated that SET could be applied to older women.
Estimate of the Target Population
Based on results of the literature review and consultations with experts, four categories of infertile patients who may benefit from increased access to IVF/ICSI were identified:
Patients with severe male factor infertility, where IVF should be offered in conjunction with ICSI;
Infertile women with serious medical contraindications to multiple pregnancy, who should be offered IVF-SET;
Infertile patients who want to avoid the risk of multiple pregnancy and thus opt for IVF-SET; and
Patients who failed treatment with IUI and wish to try IVF.
Since, however, the latter indication does not reflect any new advances in IVF technology that would alter existing policy, it was not considered in this analysis.
Economic Analysis
Economic Review: Cost–Effectiveness of SET Versus DET
Conclusions of published studies on cost-effectiveness of SET versus DET were not consistent. While some studies found that SET strategy is more cost-effective due to avoidance of multiple pregnancies, other studies either did not find any significant differences in cost per birth between SET and DET, or favoured DET as a more cost-effective option.
Ontario-Based Economic Analysis
An Ontario-based economic analysis compared cost per birth using three treatment strategies: IUI, IVF-SET, and IVF-DET. A decision-tree model assumed three cycles for each treatment option. Two separate models were considered; the first included only fresh cycles of IVF, while the second had a combination of fresh and frozen cycles. Even after accounting for cost-savings due to avoidance of multiple pregnancies (only short-term complications), IVF-SET was still associated with a highest cost per birth. The approximate budget impact to cover the first three indications for IVF listed above (severe male factor infertility, women with medical contraindications to multiple pregnancy, and couples who wish to avoid the risk of multiple pregnancy) is estimated at $9.8 to $12.8 million (Cdn). Coverage of only first two indications, namely, ICSI in patients with severe male factor infertility and infertile women with serious medical contraindications to multiple pregnancy, is estimated at $3.8 to $5.5 million Cdn.
Other Considerations
International data shows that both IVF utilization and the average number of embryos transferred in IVF cycles are influenced by IVF funding policy. The success of the SET strategy in European countries is largely due to the fact that IVF treatment is subsidized by governments.
Surveys of patients with infertility demonstrated that a significant proportion (~40%) of patients not only do not mind having multiple babies, but consider twins being an ideal outcome of infertility treatment.
A women’s age may impose some restrictions on the implementation of a SET strategy.
Conclusions and Recommendations
A review of published studies has demonstrated that IVF-SET is an effective treatment for infertility that avoids multiple pregnancies.
However, results of an Ontario-based economic analysis shows that cost savings associated with a reduction in multiple pregnancies after IVF-SET does not justify the cost of universal IVF-SET coverage by the province. Moreover, the province currently funds IUI, which has been shown to be as effective as IVF for certain types of infertility and is significantly less expensive.
In patients with severe male factor infertility, IVF in conjunction with ICSI may be the only effective treatment.
Thus, 2 indications where additional IVF access should be considered include:
IVF/ICSI for patients with severe male factor infertility
IVF-SET in infertile women with serious medical contraindications to multiple pregnancy
PMCID: PMC3379537  PMID: 23074488
14.  Parental Genome Imbalance Causes Post-Zygotic Seed Lethality and Deregulates Imprinting in Rice 
Rice  2016;9(1):43.
Reproductive isolation between rice of different ploidy levels is manifested as endosperm and embryo abortion in seeds produced by interploidy crosses. Genomic imprinting is considered to be the underlying mechanism establishing the post-zygotic hybridization barrier. We characterized disrupted seed development in reciprocal crosses between a diploid Japonica rice and a tetraploid Indica rice.
Triploid seeds from these crosses had aborted development and could not germinate in soil but could be rescued in culture medium with significantly more seeds developing to seedlings in the 4n × 2n (♀-♂) cross with excess maternal genomes than in the 2n × 4n cross with excess paternal genome. Consistent with previous findings, precocious endosperm cellularization and bigger embryos were observed in the seeds from the maternal excess cross, whereas absence of cellularization and arrested globular embryos were found in the seeds from the paternal excess cross, supporting the idea that endosperm cellularization is an important transition for embryo development. Moreover, we found that starch granules were persistently deposited in the pericarp parenchyma cells of the paternal excess cross, while pericarp starch gradually decreased and relocated to the developing endosperm in balanced and maternal excess crosses in which cellularization and starch deposition occur in endosperm, suggesting that parental genome balance influences pericarp starch relocation via cellularization and starch deposition. Loss of imprinting, or altered expression of imprinted genes and epigenetic regulators, OsFIE2 and OsMET1b were observed, implying the potential role of imprinting and epigenetic mechanisms in regulating the differential parental genome dosage effects on endosperm development.
Our results support the hypothesis that the maternal genome dosage promotes endosperm cellularization and the paternal genome dosage delays or inhibits cellularization via contributing different sets of imprinted genes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12284-016-0115-4) contains supplementary material, which is available to authorized users.
PMCID: PMC5002275  PMID: 27568375
Polyploid; Endosperm; Embryo; Cellularization; Reproduction; Polycomb; DNA methylation; Imprinting; Triploid block
15.  Differential selection on pollen and pistil traits in relation to pollen competition in the context of a sexual conflict over timing of stigma receptivity 
AoB Plants  2016;8:plw061.
Sexual conflict and its evolutionary consequences are understudied in plants, but the theory of sexual conflict may help explain how selection generates and maintains variability in both plants and animals. Here, we show that pollen and pistil traits involved in a sexual conflict over timing of stigma receptivity are differentially advantageous during pollen competition depending on stage of floral development and varying pollen deposition schedules. Variation in success of these traits over floral development time may result from sexually antagonistic selection.
Sexual conflict and its evolutionary consequences are understudied in plants, but the theory of sexual conflict may help explain how selection generates and maintains variability. Here, we investigated selection on pollen and pistil traits when pollen arrives sequentially to partially receptive pistils in relation to pollen competition and a sexual conflict over timing of stigma receptivity in the mixed-mating annual Collinsia heterophylla (Plantaginaceae). In this species the conflict is generated by early fertilizing pollen that reduces seed production, which is counteracted by delaying receptivity in the recipient. We performed sequential two-donor pollinations at early floral developmental stages involving two pollen deposition schedules (with or without a time lag of 1 day), using only outcross or self and outcross pollen. We investigated pollen and pistil traits in relation to siring success (male fitness) and seed production (female fitness). In contrast to previous findings in receptive pistils in C. heterophylla and in other species, last arriving pollen donors showed highest siring success in partially receptive pistils. The last male advantage was weaker when self pollen was the first arriving donor. Two measures of germination rate (early and late) and pollen tube growth rate of first arriving donors were important for siring success in crosses with a time lag, while only late germination rate had an effect in contemporary crosses. Curiously, late stigma receptivity was negatively related to seed production in our contemporary crosses, which was opposite to expectation. Our results in combination with previous studies suggest that pollen and pistil traits in C. heterophylla are differentially advantageous depending on stage of floral development and varying pollen deposition schedules. Variation in success of these traits over floral development time may result from sexually antagonistic selection.
PMCID: PMC5063087  PMID: 27562796
Collinsia heterophylla; cryptic self-incompatibility; mixed mating; pollen competition; pollen deposition schedules; sexual conflict; sexual selection; timing of stigma receptivity
16.  An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination 
PLoS Genetics  2015;11(12):e1005708.
Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.
Author Summary
Seeds are remarkable plant structures that appeared late during land plant evolution. Indeed, within seeds plant embryos lie in a metabolic inert and highly resistant state. Seeds allow plants to disperse and find a favorable living environment. Remarkably as well, the “near-dead” embryo is able to germinate and turn into a fragile young seedling. The fragility of this transition is betrayed by the existence of control mechanisms that block germination in response to harmful environmental conditions. Seeds therefore transform plants into time and space travellers and largely explain land plant colonization by flowering plants.
The key to this success lies in the seed’s physiological feats, a major yet unresolved question in plant biology. We show that mature seeds of the model plant Arabidopsis contain an earlier land plant evolutionary innovation: the cuticle, a waxy film covering the aerial parts of the plant preventing excessive transpiration. The seed cuticle, which contains cutin, a major lipid polymer component of the leaf cuticle, encloses all the living tissues within the seed. Seeds with cutin defects are highly oxidized and have low seed viability and dormancy. They are also unable to control their germination. Thus, land plants co-opted an ancient innovation to achieve the remarkable physiology of seeds.
PMCID: PMC4683086  PMID: 26681322
17.  A Genome-Wide Survey of Imprinted Genes in Rice Seeds Reveals Imprinting Primarily Occurs in the Endosperm 
PLoS Genetics  2011;7(6):e1002125.
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.
Author Summary
The expression of maternal or paternal alleles in either a preferentially or exclusively uniparental manner, termed imprinting, is prevalent in the transient endosperm of seeds in the model plant Arabidopsis. Cereals form seeds where both the embryo and endosperm are present at seed maturity. They are an important world food source. To date, very few imprinted genes have been identified in cereal seeds. How parental gene expression biases contribute to rice seed development has not yet been studied in detail. The deep resolution of transcript sequencing platforms was used to identify loci expressed in a parentally biased manner in the embryo and endosperm of Indica and Japonica rice at a genome-wide level. We identified 262 candidate imprinted loci expressed in the endosperm, experimentally verified 56 of these, and found novel features pertaining to their expression. Only one gene was found to be imprinted in the rice embryo. Imprinting in Arabidopsis and rice seeds is confined primarily to the endosperm, but the identified loci do not share extensive sequence conservation. Imprinting thus appears to have evolved independently in these plant species.
PMCID: PMC3121744  PMID: 21731498
18.  Heterochromatin, small RNA and post-fertilization dysgenesis in allopolyploid and interploid hybrids of Arabidopsis 
The New phytologist  2010;186(1):46-53.
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.
PMCID: PMC3756494  PMID: 20409176
allopolyploidy; dosage; hybrid lethality; siRNA; transposon
19.  Increased Maternal Genome Dosage Bypasses the Requirement of the FIS Polycomb Repressive Complex 2 in Arabidopsis Seed Development 
PLoS Genetics  2013;9(1):e1003163.
Seed development in flowering plants is initiated after a double fertilization event with two sperm cells fertilizing two female gametes, the egg cell and the central cell, leading to the formation of embryo and endosperm, respectively. In most species the endosperm is a polyploid tissue inheriting two maternal genomes and one paternal genome. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal to paternal contributions strongly impact on endosperm development. The FERTILIZATION INDEPENDENT SEED (FIS) Polycomb Repressive Complex 2 (PRC2) is essential for endosperm development; however, the underlying forces that led to the evolution of the FIS-PRC2 remained unknown. Here, we show that the functional requirement of the FIS-PRC2 can be bypassed by increasing the ratio of maternal to paternal genomes in the endosperm, suggesting that the main functional requirement of the FIS-PRC2 is to balance parental genome contributions and to reduce genetic conflict. We furthermore reveal that the AGAMOUS LIKE (AGL) gene AGL62 acts as a dosage-sensitive seed size regulator and that reduced expression of AGL62 might be responsible for reduced size of seeds with increased maternal genome dosage.
Author Summary
Flowering plants reproduce by forming seeds that contain an embryo surrounded by a nourishing endosperm tissue that, similar to the mammalian placenta, supports embryo growth. Normal endosperm development requires the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb Repressive Complex2 (PRC2). In most flowering plants the endosperm is a polyploid tissue containing two maternal and one paternal genome copies. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal and paternal genome copies have drastic effects on endosperm development. Here we investigated the consequences of increased maternal genome dosage on endosperm and seed development. We found that increased maternal genome dosage alleviates the need for the FIS-PRC2 in the endosperm. While in fis mutant seeds with normal maternal genome dosage the endosperm fails to cellularize and embryos arrest, in fis mutant seeds with increased maternal genome dosage the endosperm cellularizes and viable embryos develop. Our study suggests a functional role of the FIS-PRC2 in balancing parental genome dosage in the endosperm. We propose that the FIS-PRC2 evolved to reduce genetic conflict that arose as a consequence of unbalanced genome contributions in the endosperm.
PMCID: PMC3542072  PMID: 23326241
20.  Embryonal Control of Yellow Seed Coat Locus ECY1 Is Related to Alanine and Phenylalanine Metabolism in the Seed Embryo of Brassica napus 
G3: Genes|Genomes|Genetics  2016;6(4):1073-1081.
Seed coat color is determined by the type of pigment deposited in the seed coat cells. It is related to important agronomic traits of seeds such as seed dormancy, longevity, oil content, protein content and fiber content. In Brassica napus, inheritance of seed coat color is related to maternal effects and pollen effects (xenia effects). In this research we isolated a mutation of yellow seeded B. napus controlled by a single Mendelian locus, which is named Embryonal Control of Yellow seed coat 1 (Ecy1). Microscopy of transverse sections of the mature seed show that pigment is deposited only in the outer layer of the seed coat. Using Illumina Hisequation 2000 sequencing technology, a total of 12 GB clean data, 116× coverage of coding sequences of B. napus, was achieved from seeds 26 d after pollination (DAP). It was assembled into 172,238 independent transcripts, and 55,637 unigenes. A total of 139 orthologous genes of Arabidopsis transparent testa (TT) genes were mapped in silico to 19 chromosomes of B. napus. Only 49 of the TT orthologous genes are transcribed in seeds. However transcription of all orthologs was independent of embryonal control of seed coat color. Only 55 genes were found to be differentially expressed between brown seeds and the yellow mutant. Of these 55, 50 were upregulated and five were downregulated in yellow seeds as compared to their brown counterparts. By KEGG classification, 14 metabolic pathways were significantly enriched. Of these, five pathways: phenylpropanoid biosynthesis, cyanoamino acid metabolism, plant hormone signal transduction, metabolic pathways, and biosynthesis of secondary metabolites, were related with seed coat pigmentation. Free amino acid quantification showed that Ala and Phe were present at higher levels in the embryos of yellow seeds as compared to those of brown seeds. This increase was not observed in the seed coat. Moreover, the excess amount of free Ala was exactly twice that of Phe in the embryo. The pigment substrate chalcone is synthesized from two molecules of Ala and one molecule of Phe. The correlation between accumulation of Ala and Phe, and disappearance of pigment in the yellow seeded mutant, suggests that embryonal control of seed coat color is related with Phe and Ala metabolism in the embryo of B. napus.
PMCID: PMC4825642  PMID: 26896439
Brassica napus; yellow seed coat; xenia effect; free amino acids; transcriptome sequencing
21.  Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana 
eLife  null;4:e10074.
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.
eLife digest
When plants and animals reproduce sexually, their offspring inherit two copies of every gene, one from each parent, which are arranged in two sets of structures called chromosomes. In some tissues, one gene copy may be switched off—through a process called ‘genomic imprinting’—while the other copy remains active. In plants, genomic imprinting is vital for seeds to develop normally. It is particularly important in the tissue that provides nutrients for the growing embryo (the endosperm), in which one of the copies of many genes are switched off. Genes inherited from the male parent that have been imprinted are known as paternally expressed imprinted genes (PEGs).
Unlike most animals, it is common for plants to have more than two sets of chromosomes. When plants with different numbers of chromosome sets cross-fertilize each other, their offspring may have three copies of every gene instead of two. These ‘triploid’ seeds often die because their endosperm fails to develop normally. This is due to the increased activity of imprinted genes, which causes changes in the activity of many other genes in the endosperm. Although it is known that genomic imprinting in the endosperm helps to establish this reproductive barrier, it is not clear what specific roles many of the imprinted genes play.
Here, Wolff et al. switched off several different PEGs in the plant Arabidopsis to investigate how they affect seed development. The experiments show that in seeds that have the normal two copies of every gene, inactivating these imprinted genes does not affect seed development. However, in triploid seeds, inactivating three of the imprinted genes rescues seeds that would normally die. These genes encode proteins that activate pathways in the endosperm that promote the formation of cell walls, which is a crucial stage in seed development.
Wolff et al.'s findings reveal how imprinted genes in the endosperm establish a barrier to reproduction by preventing seeds produced from crosses between plants with different numbers of chromosome sets from being able to survive. Reproductive barriers are a major obstacle in plant breeding, so understanding how these barriers form may open new avenues for developing new plant varieties.
PMCID: PMC4589659  PMID: 26344545
genomic imprinting; hybridization barriers; seed development; arabidopsis
22.  The Female Gametophyte 
The angiosperm female gametophyte is critical for plant reproduction. It contains the egg cell and central cell that become fertilized and give rise to the embryo and endosperm of the seed, respectively. Female gametophyte development begins early in ovule development with the formation of a diploid megaspore mother cell that undergoes meiosis. One resulting haploid megaspore then develops into the female gametophyte. Genetic and epigenetic processes mediate specification of megaspore mother cell identity and limit megaspore mother cell formation to a single cell per ovule. Auxin gradients influence female gametophyte polarity and a battery of transcription factors mediate female gametophyte cell specification and differentiation. The mature female gametophyte secretes peptides that guide the pollen tube to the embryo sac and contains protein complexes that prevent seed development before fertilization. Post-fertilization, the female gametophyte influences seed development through maternal-effect genes and by regulating parental contributions. Female gametophytes can form by an asexual process called gametophytic apomixis, which involves formation of a diploid female gametophyte and fertilization-independent development of the egg into the embryo. These functions collectively underscore the important role of the female gametophyte in seed and food production.
PMCID: PMC3268550  PMID: 22303279
23.  Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development 
Time-resolved transcriptome analysis of early pou5f1 mutant zebrafish embryos identified groups of developmental regulators, including SoxB1 genes, that depend on Pou5f1 activity, and a large cluster of differentiation genes which are prematurely expressed.Pou5f1 represses differentiation genes indirectly via activation of germlayer-specific transcriptional repressor genes, including her3, which may mediate in part Pou5f1-dependent repression of neural genes.A dynamic mathematical model is established for Pou5f1 and SoxB1 activity-dependent temporal behaviour of downstream transcriptional regulatory networks. The model predicts that Pou5f1-dependent increase in SoxB1 activity significantly contributes to developmental timing in the early gastrula.Comparison to mouse Pou5f1/Oct4 reveals evolutionary conserved targets. We show that Pou5f1 developmental function is also conserved by demonstrating rescue of Pou5f1 mutant zebrafish embryos by mouse POU5F1/OCT4.
The transcription factor Pou5f1/Oct4 controls pluripotency of mouse embryonic inner cell mass cells (Nichols et al, 1998), and of mouse and human ES cell lines (Boiani and Scholer, 2005). Although Pou5f1/Oct4-dependent pluripotency transcriptional circuits and many transcriptional targets have been characterized, little is known about the mechanisms by which Pou5f1/Oct4 controls early developmental events. A detailed understanding of Pou5f1/Oct4 functions during mammalian blastocyst and gastrula development as well as studies of the temporal changes in the Pou5f1/Oct4-regulated networks are precluded by the early lineage defects in pou5f1/oct4 mutant mice. To investigate Pou5f1-dependent transcriptional circuits in developmental control, we used the zebrafish (Danio rerio) as a genetic and experimental model representing an earlier state of vertebrate evolution. Zebrafish have one pou5f1/pou2 gene (Takeda et al, 1994) orthologous to the mammalian gene (Niwa et al, 2008; Frankenberg et al, 2009). Both fish and mammalian orthologs are expressed broadly in tissues giving rise to the embryo proper during blastula and early gastrula stages, as well as in the neural plate (Belting et al, 2001; Reim and Brand, 2002; Downs, 2008).
Zebrafish pou5f1 loss-of-function mutant embryos, MZspg (abbreviated ‘MZ'), are completely devoid of maternal and zygotic Pou5f1 activity (Lunde et al, 2004; Reim et al, 2004). MZ embryos have gastrulation abnormalities (Lachnit et al, 2008), dorsoventral patterning defects (Reim and Brand, 2006), and do not develop endoderm (Lunde et al, 2004; Reim et al, 2004). In contrast to Pou5f1/Oct4 mutant mice, which are blocked in development due to loss of inner cell mass, MZ mutant embryos are neither blocked in development nor display a general delay. Therefore, zebrafish present a good model system to identify specific transcriptional targets of Pou5f1 during development.
Our study aims to understand the structure, regulatory logic, and developmental temporal changes in the Pou5f1-dependent transcriptional network in the context of an intact embryo. Therefore, we investigated transcriptome changes in MZ compared with WT zebrafish by microarray analysis at 10 distinct time-points during development, from ovaries to late gastrulation. We identified changes in Pou5f1 target gene expression both with respect to their expression level and temporal behavior. We used correlation analysis to identify clusters of target genes enriched for genes with developmentally regulated expression profiles. This correlation analysis revealed a cluster of genes, which were not activated or were significantly delayed in MZ. Interestingly, there was also a large gene cluster with premature onset of expression in MZ.
Several targets activated by Pou5f1 encode known repressors of differentiation (RODs), of which we analyzed her3 in detail. Pou5f1 also activates several SoxB1 group transcription factors, which are known to act together with Pou5f1 in mammalian systems. Among the large group of genes prematurely activated in MZ, many genes encode developmental regulators of differentiation normally acting during organogenesis (promoters of differentiation—PODs). Our analysis of potential direct transcriptional interactions by suppression of translation of intermediate zygotic Pou5f1 or SoxB1 targets, enabled us to distinguish Sox-dependent and independent subgroups of the Pou5f1 transcriptional network. Interestingly, tissue-specific expression of Pou5f1 targets correlated with their regulation by Sox2, with Sox-dependent targets being mostly localized to ectoderm and neuroectoderm, whereas Sox-independent targets localized to mesendoderm of the developing zebrafish embryo. Further, SoxB1 independent Pou5f1 targets (for example foxD3) differ from SoxB1-dependent targets (e.g her3) in temporal dynamics of expression. Most Sox-independent direct Pou5f1 targets in WT reach maximal expression levels soon after midblastula transition (MBT) at 3–4 h postfertilization (hpf). In contrast, genes depending both on Sox2 and Pou5f1 tend to have a biphasic temporal expression curve or are activated with >2 h delay after MBT to reach maximum levels at 6–7 hpf only.
To better understand the impact of our findings on Pou5f1/SoxB1-dependent versus Pou5f1-only regulation on developmental mechanisms, we built a small dynamic network model that links the temporal control of target genes to regulatory principles exerted by Pou5f1 and SoxB1 proteins (Figure 6A). The model is based on ordinary differential equations, and parameters were determined by a fit to the WT and MZ gene expression data. The optimized model highlights two qualitatively different temporal expression modes of Pou5f1 downstream targets: monophasic for targets depending only on Pou5f1 (foxd3), and biphasic for Pou5f1- and SoxB1-dependent targets (sox2 and her3; Figure 6B). To test whether the model is also able to correctly predict a different genetic condition, we simulated the M mutant, which is lacking maternal Pou5f1, but gradually rescued by the paternal pou5f1 contribution after MBT (Figure 6B, blue, dashed curve). The model predicts an overall shift in the developmental program. Most importantly, the sox2 and her3 expression is rescued with a delay of about 2 h. The model predictions were checked experimentally by quantitative RT–PCR (Figure 6B, blue dots). Most predictions are in good agreement with the experimental data, for example the delayed rescue of the sox2 and her3 temporal expression profile. With respect to the ‘POD' nr2f1, the model correctly predicts the efficient downregulation by zygotic targets of Pou5f1 (Figure 6B).
We identified an evolutionary conserved core set of Pou5f1 targets, by comparing our gene list with the lists of mouse Pou5f1/Oct4 targets (Loh et al, 2006; Sharov et al, 2008). The evolutionary conservation suggests equivalent Pou5f1 functions during the pregastrulation and gastrulation period of vertebrate embryogenesis. Therefore, we tested whether mouse Pou5f1/Oct4 was able to rescue MZ embryos. Injection of mRNA encoding mouse Pou5f1/Oct4 into MZ embryos (Figure 8A) was able to restore normal zebrafish development to an extent comparable with zebrafish pou5f1/pou2 mRNA (Figure 8B and C). The significant overlap between zebrafish and mammalian Pou5f1 targets together with the ability of mouse Pou5f1/Oct4 to functionally replace the zebrafish Pou5f1/Pou2 (Figure 8A–C), suggests that the mammalian network may have evolved from a basal situation similar to what is observed in teleosts. We propose models that emphasize the evolution of Pou5f1-dependent transcriptional networks during development of the zebrafish (Figure 8D) and mammals (Figure 8E). Our representation highlights the evolutionary ancient germlayer-specific subnetworks downstream of Pou5f1, which are presumably used for controlling the timing of differentiation during gastrulation in all vertebrates (Figure 8D and E, black arrows). As the Pou5f1 downstream regulatory nodes revealed in our zebrafish model are likely conserved across vertebrates, we envision that their knowledge will contribute to the effort of directing differentiation of pluripotent stem cells to defined cell fates.
The transcription factor POU5f1/OCT4 controls pluripotency in mammalian ES cells, but little is known about its functions in the early embryo. We used time-resolved transcriptome analysis of zebrafish pou5f1 MZspg mutant embryos to identify genes regulated by Pou5f1. Comparison to mammalian systems defines evolutionary conserved Pou5f1 targets. Time-series data reveal many Pou5f1 targets with delayed or advanced onset of expression. We identify two Pou5f1-dependent mechanisms controlling developmental timing. First, several Pou5f1 targets are transcriptional repressors, mediating repression of differentiation genes in distinct embryonic compartments. We analyze her3 gene regulation as example for a repressor in the neural anlagen. Second, the dynamics of SoxB1 group gene expression and Pou5f1-dependent regulation of her3 and foxD3 uncovers differential requirements for SoxB1 activity to control temporal dynamics of activation, and spatial distribution of targets in the embryo. We establish a mathematical model of the early Pou5f1 and SoxB1 gene network to demonstrate regulatory characteristics important for developmental timing. The temporospatial structure of the zebrafish Pou5f1 target networks may explain aspects of the evolution of the mammalian stem cell networks.
PMCID: PMC2858445  PMID: 20212526
developmental timing; mathematical modeling; Oct4; transcriptional networks
24.  The effect of temperature on reproduction in the summer and winter annual Arabidopsis thaliana ecotypes Bur and Cvi 
Annals of Botany  2014;113(6):921-929.
Background and Aims
Seed yield and dormancy status are key components of species fitness that are influenced by the maternal environment, in particular temperature. Responses to environmental conditions can differ between ecotypes of the same species. Therefore, to investigate the effect of maternal environment on seed production, this study compared two contrasting Arabidopsis thaliana ecotypes, Cape Verdi Isle (Cvi) and Burren (Bur). Cvi is adapted to a hot dry climate and Bur to a cool damp climate, and they exhibit winter and summer annual phenotypes, respectively.
Bur and Cvi plants were grown in reciprocal controlled environments that simulated their native environments. Reproductive development, seed production and subsequent germination behaviour were investigated. Measurements included: pollen viability, the development of floral structure, and germination at 10 and 25 °C in the light to determine dormancy status. Floral development was further investigated by applying gibberellins (GAs) to alter the pistil:stamen ratio.
Key Results
Temperature during seed development determined seed dormancy status. In addition, seed yield was greatly reduced by higher temperature, especially in Bur (>90 %) compared with Cvi (approx. 50 %). The reproductive organs (i.e. stamens) of Bur plants were very sensitive to high temperature during early flowering. Viability of pollen was unaffected, but limited filament extension relative to that of the pistils resulted in failure to pollinate. Thus GA applied to flowers to enhance filament extension largely overcame the effect of high temperature on yield.
High temperature in the maternal environment reduced dormancy and negatively affected the final seed yield of both ecotypes; however, the extent of these responses differed, demonstrating natural variation. Reduced seed yield in Bur resulted from altered floral development not reduced pollen viability. Future higher temperatures will impact on seed performance, but the consequences may differ significantly between ecotypes of the same species.
PMCID: PMC3997633  PMID: 24573642
Maternal temperature; seed yield; seed dormancy; flower development; Arabidopsis thaliana ecotypes; winter annual; summer annual; Cvi; Burren; climate change
25.  Floral longevity and autonomous selfing are altered by pollination and water availability in Collinsia heterophylla 
Annals of Botany  2013;112(5):821-828.
Background and Aims
A plant investing in reproduction partitions resources between flowering and seed production. Under resource limitation, altered allocations may result in floral trait variations, leading to compromised fecundity. Floral longevity and timing of selfing are often the traits most likely to be affected. The duration of corolla retention determines whether fecundity results from outcrossing or by delayed selfing-mediated reproductive assurance. In this study, the role of pollination schedules and soil water availability on floral longevity and seed production is tested in Collinsia heterophylla (Plantaginaceae).
Using three different watering regimes and pollination schedules, effects on floral longevity and seed production were studied in this protandrous, flowering annual.
Key Results
The results reveal that soil water status and pollination together influence floral longevity with low soil water and hand-pollinations early in the floral lifespan reducing longevity. However, early pollinations under excess water did not extend longevity, implying that resource surplus does not lengthen the outcrossing period. The results also indicate that pollen receipt, a reliable cue for fecundity, accelerates flower drop. Early corolla abscission under drought stress could potentially exacerbate sexual conflict in this protandrous, hermaphroditic species by ensuring self-pollen paternity and enabling male control of floral longevity. While pollination schedules did not affect fecundity, water stress reduced per-capita seed numbers. Unmanipulated flowers underwent delayed autonomous selfing, producing very few seeds, suggesting that inbreeding depression may limit benefits of selfing.
In plants where herkogamy and dichogamy facilitate outcrossing, floral longevity determines reproductive success and mating system. Reduction in longevity under drought suggests a strong environmental effect that could potentially alter the preferred breeding mode in this mixed-mated species. Extrapolating the findings to unpredictable global drought cycles, it is suggested that in addition to reducing yield, water stress may influence the evolutionary trajectory of plant mating system.
PMCID: PMC3747799  PMID: 23884393
Autonomous selfing; Collinsia heterophylla; drought stress; floral longevity; herkogamy; Plantaginaceae; reproductive success; resource allocation; sexual conflict

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